# Patent

## Title
Differentiation of human embryonic stem cells

## Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/183,656 filed Jul. 31, 2008, which claims priority to provisional Application No. 60/953,178, filed Jul. 31, 2007. The complete disclosures of the aforementioned related patent applications are hereby incorporated herein by reference for all purposes.


FIELD OF THE INVENTION
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.
BACKGROUND
Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent stem cells, such as, for example, embryonic stem cells.
In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF-3beta, GATA4, Mixl1, CXCR4 and Sox-17.
Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, Pdx1. In the absence of Pdx1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, Pdx1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.
Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.
In one example, Hori et al. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled β cells.
In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4.
Micallef et al. reports that retinoic acid can regulate the commitment of embryonic stem cells to form Pdx1 positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdx1 expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).
Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdx1. Their results show that exogenous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, P48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).
Skoudy et al. reports that activin A (a member of the TGFβ superfamily) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouse embryonic stem cells. The maximal effect was observed using 1 nM activin A. They also observed that the expression level of insulin and Pdx1 mRNA was not affected by retinoic acid; however, 3 nM FGF7 treatment resulted in an increased level of the transcript for Pdx1 (Biochem. J. 379: 749, 2004).
Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into Pdx1 positive cells. They observed that TGFβ2 reproducibly yielded a higher proportion of Pdx1 positive cells (Genes Cells. 2005 June; 10(6): 503-16.).
Gordon et al. demonstrated the induction of brachyury+/HNF-3beta+ endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A1).
Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the anterior primitive streak”.
However, the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.
Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).
D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into Pdx1 positive cells after addition of FGF-10 (US 2005/0266554A1).
D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states: “We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones”.
In another example, Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of sodium butyrate and activin A. The cells were then cultured with TGFβ antagonists such as Noggin in combination with EGF or betacellulin to generate Pdx1 positive cells. The terminal differentiation was induced by nicotinamide.
In one example, Benvenistry et al. states: “We conclude that over-expression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo” (Benvenistry et al, Stem Cells 2006; 24:1923-1930).
Therefore, there still remains a significant need to develop conditions for establishing pluripotent stem cell lines that can be expanded to address the current clinical needs, while retaining the potential to differentiate into pancreatic endocrine cells, pancreatic hormone expressing cells, or pancreatic hormone secreting cells. We have taken an alternative approach to improve the efficiency of differentiating human embryonic stem cells toward pancreatic endocrine cells.
SUMMARY
In one embodiment, the present invention provides a method for differentiating pluripotent stem cells, comprising the steps of:

    a. Culturing the pluripotent stem cells,
    b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,
    c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and
    d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.


In one embodiment, cells expressing markers characteristic of the definitive endoderm lineage are differentiated from pluripotent stem cells by treating pluripotent stem cells by any one of the following methods:

    a. Culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration, or
    b. Culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration, or
    c. Culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum, or
    d. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and culturing the pluripotent stem cells with activin A and a Wnt ligand, or
    e. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum, then culturing the pluripotent stem cells with activin A in a second culture medium containing serum, or
    f. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a second culture medium containing serum of a different concentration.


In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are differentiated from cells expressing markers characteristic of the definitive endoderm lineage by treating cells expressing markers characteristic of the definitive endoderm lineage by any one of the following methods:

    a. Treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and a hedgehog signaling pathway inhibitor, then removing the medium containing the fibroblast growth factor and the hedgehog signaling pathway inhibitor and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and the hedgehog signaling pathway inhibitor, or
    b. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor, or
    c. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid, then removing the retinoic acid and subsequently treating the cells with at least one fibroblast growth factor.


In one embodiment, cells expressing markers characteristic of the pancreatic endocrine lineage are differentiated from cells expressing markers characteristic of the pancreatic endoderm lineage by treating cells expressing markers characteristic of the pancreatic endoderm lineage by any one of the following methods:

    a. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF, or
    b. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF, or
    c. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAFT and exendin 4, or
    d. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, or
    e. Treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, or
    f. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing from about 10 mM to about 20 mM glucose and exendin 4.


In one embodiment, the present invention provides a method for treating a patient suffering from diabetes, comprising the steps of:

    a. Culturing pluripotent stem cells,
    b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,
    c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage,
    d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells of a β-cell lineage, and
    e. Implanting the cells of a β-cell lineage into the patient.





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 panel a shows the expression of the definitive endoderm markers CXCR4, GATA4, HNF-3beta, Mixl1, Sox-17 in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for two, five and eight days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. Panel b shows the expression of the anterior endoderm markers Cerberus, Otx-1 and Hex genes in the human embryonic stem cell line H9 following treatment with with 100 ng/ml activin A for three and five days.
FIG. 2 shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for five days. Expression of the definitive endoderm markers was detected by immunohistochemistry. Panel (a) shows Sox-17 expression. Panel (b) shows HNF-3beta expression. Panel (c) shows Oct3/4 expression.
FIG. 3 shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. Panel (a) shows GATA4 expression. Panel (b) shows Sox-17 expression. Panel (c) shows HNF-3beta expression. Panel (d) shows Mixl1 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
FIG. 4 shows the expression of extra-embryonic endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the extraembryonic endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. Panel (a) shows the effect of 100 ng/ml activin A on AFP expression. Panel (b) shows the effect of 100 ng/ml activin A on Sox7 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
FIG. 5 shows the expression of mesoderm and ectoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the mesoderm and ectoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. Panel (a) shows the effect of 100 ng/ml activin A on Brachyury expression. Panel (b) shows the effect of 100 ng/ml activin A on Zic1 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
FIG. 6 shows the expression of the definitive endoderm markers Brachyury (panel a) CXCR4 (panel b), Mixl1 (panel c), Sox17 (panel d), HNF-3beta (panel e), Oct4 (panel f) in the human embryonic stem cell line H7 following treatment with 100 ng/ml activin A for one, three, five and seven days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
FIG. 7 shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the definitive endoderm markers was detected by immunohistochemistry. Panels (a) and (b) show Sox-17 expression. Panels (c) and (d) show HNF-3beta expression. Panels (e) and (f) show GATA4 expression. Panels (b), (d) and (f) show counter staining of the nuclei with DAPI. The columns marked ‘treated’ denote activin A treatment (100 ng/ml) for five days. The columns marked ‘untreated’ denote untreated controls.
FIG. 8 shows the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. Panel (a) shows Pdx1 expression. Panel (b) shows GLUT-2 expression. Panel (c) shows PTF1a expression.
FIG. 9 shows the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was detected by immunohistochemistry. Panel (a) shows Pdx1 expression in the untreated control, and panel (b) shows Pdx1 expression in the culture treated by the stepwise differentiation protocol.
FIG. 10 shows the expression of pancreatic endocrine markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the pancreatic endocrine markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. Panel (a) shows NeuroD1 expression. Panel (b) shows Ngn3 expression. Panel (c) shows insulin expression. Panel (d) shows Hes-1 expression, the expression level is normalized to pancreatic endoderm cells.
FIG. 11 shows the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. Panel (a) shows Nkx2.2 expression. Panel (b) shows Pdx1 expression.
FIG. 12 shows the expression of PDX-1 in cells with each passage (P0, P1 and P2) in culture. Expression of the PDX-1 was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells H9.
FIG. 13 shows the expression of hepatocyte markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the hepatocyte markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. Panel (a) shows AFP expression. Panel (b) shows albumin expression.
FIG. 14 shows the expression of markers of pluripotency in the human embryonic stem cell line H9. Expression of the markers of pluripotency was assayed by immunohistochemistry. Panel (a) shows Oct-4 expression. Panel (b) shows alkaline phosphatase expression.
FIG. 15 shows the karyotype of the human embryonic cell line H9. The Karyotype was determined on cells at passage number P36 that were cultured on mouse embryonic fibroblast feeder cells.
FIG. 16 depicts the outline of a differentiation protocol in this invention, where human embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
FIG. 17 depicts the FACS profile of human embryonic stem cell line H9 at passage number 44, cultured on varying concentrations of MATRIGEL and exposed to (0.5-2%) low serum and high activin A (100 ng/ml) for 5 days. The expression of definite endoderm marker CXCR4 (CD184) is shown on the Y-axis and the expression of ES marker CD9 is shown on the X-axis.
FIG. 18 shows the real-time PCR results for markers of definitive endoderm, from cultures of the human embryonic stem cell line H9 at passage 44 cultured on a 1:10 dilution of MATRIGEL (▪), a 1:20 dilution of MATRIGEL (), or a 1:30 dilution of MATRIGEL (□) and exposed to the differentiation protocol disclosed in Example 14. The fold induction is relative to undifferentiated cells of the human embryonic stem cell line H9, at passage number 44, cultured in medium conditioned using mouse embryonic fibroblasts.
FIG. 19 shows the scatter plots for global gene expression in undifferentiated pluripotent stem cells and definitive endoderm cells obtained from differentiating pluripotent stem cells. Data shown is from cultures of the human embryonic stem cell line H9 cell line at passage 44 cultured on mouse embryonic fibroblasts (right panel) and passage 83 cultured on MATRIGEL (left panel).
FIG. 20 depicts the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 (panel a), the human embryonic stem cell line H7 (panel b), and the human embryonic stem cell line H9 (panel c) cultured on mouse embryonic fibroblast feeder cells exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
FIG. 21 shows the real-time PCR results of expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 (panel a) and the human embryonic stem cell line H9 (panel b) cultured on mouse embryonic fibroblast feeder cells. Results are expressed as fold increase over undifferentiated cells.
FIG. 22 depicts the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 (panel a), the human embryonic stem cell line H7 (panel b), and the human embryonic stem cell line H9 (panel c) cultured on MATRIGEL (1:30 dilution) and exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
FIG. 23 shows the real-time PCR results of the expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 (panel a) and the human embryonic stem cell line H9 (panel b) and the human embryonic stem cell line H1 (panel c). Results are expressed as fold increase over undifferentiated cells. Cells were treated according to the methods disclosed in Example 4.
FIG. 24 depicts phase contrast images of cultures of the human embryonic stem cell line H9 at passage 46 in the presence of 100 ng/ml of activin A (panel a) or 100 ng/ml of activin A+20 ng/ml Wnt-3a (panel b). Cells were treated for five days.
FIG. 25 depicts the expression of CXCR4 by FACS in cultures of the human embryonic stem cell line H7 at passage 44 (panels a & b) and H9 at passage 46 (panels c & d), following treatment according to the methods disclosed in Example 4. Panels b and d show the effect of 20 ng/ml of Wnt-3a on CXCR4 expression. Panels a and c show CXCR4expression in the absence of Wnt-3a. Results were obtained 5 days post treatment.
FIG. 26 displays the real-time PCR data for expression of the genes indicated in cultures of the human embryonic stem cell line H7 (panel a) and H9 (panel b). Cultures were treated with the differentiation protocol disclosed in Example 4. The effects of Wnt agonists Wnt-3a (20 ng/ml), Wnt-5a (20 ng/ml) and Wnt-7a (20 ng/ml) were also tested, as indicated in the panels. Cells were treated for 5 days. Results are expressed as fold increase over undifferentiated cells.
FIG. 27 depicts the expression of CXCR4 in cultures of the human embryonic stem cell line H9 at passage 46, by FACS at five days post treatment. Panel (a) depicts CXCR4 expression in the absence of Wnt-3a. Panel (b) depicts CXCR4 expression following treatment with 10 ng/ml Wnt-3a. Panel (c) depicts CXCR4 expression following treatment with 20 ng/ml Wnt-3a, and panel (d) depicts CXCR4 expression following treatment with 50 ng/ml Wnt-3a.
FIG. 28 depicts the expression of definitive markers indicated in cultures of the human embryonic stem cell line H9 after 5 days of treatment. Results are shown as fold increase in expression over untreated cells, as determined by real-time PCR. Panel (a) shows the effect of 10, 20 and 50 ng/ml Wnt-3a on the expression of definitive endoderm marker genes indicated. Panel (b) shows the effect of 1, 5 or 10 ng/ml Wnt-3a (x-axis labels: 10, 5, 1) on the expression on goosecoid (▪) and CXCR4 (□) expression, at 2 (2d) and 5 (5 d) days post treatment. Panel (c) shows the effect of 1, 5 or 10 ng/ml Wnt-3a on cell number, at 2 days (▪) or 5 days (□).
FIG. 29 depicts the expression of CXCR4 in cultures of the human embryonic stem cell line H9 by FACS, following a 5 day treatment with the differentiation protocol disclosed in Example 4. Cells were cultured in the absence of Wnt-3a or GSK-3B inhibitor (panel a), 20 ng/ml Wnt-3a for the entire 5 day period (panel b), 1000 nM GSK-3B inhibitor IX for the entire 5 day period (panel c), 500 nM GSK-3B inhibitor IX for the entire 5 day period (panel d), 100 nM GSK-3B inhibitor IX for the entire 5 day period (panel e), 10 nM GSK-3B inhibitor IX for the entire 5 day period (panel f), 100 nM GSK-3B inhibitor IX for days 1-2 (panel g), 10 nM GSK-3B inhibitor IX for days 1-2 (panel h).
FIG. 30 depicts the gene expression of definitive endoderm markers by real-time PCR. Results are expressed as fold increase over untreated cells. Panel (a) shows data obtained from the human embryonic cell line H9 at passage number 48, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated. Panel (b) shows data obtained from the human embryonic cell line H9 at passage number 46, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated.
FIG. 31 depicts the expression of CXCR4 by FACS for embryonic stem cell lines used in the present invention. Panels (a-d) show data obtained from the human embryonic stem cell line H9 at passage number 49. Panels (e-f) show data obtained from the human embryonic stem cell line H1 at passage number 46. Data was obtained 5 days post treatment. Cells were treated with the following conditions: Panel (a): 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; panel (b): 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; panel (c): 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days; panel (d): 10 ng/ml activin A for all five days plus 100 nM GSK-3B IX inhibitor for the first two days, panel (e): 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days, and panel (f):10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days.
FIG. 32 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
FIG. 33 depicts the expression of CXCR4 by FACS for the embryonic stem cell line H9 at passage 53. Data was obtained 5 days post treatment. Cells were treated with the following conditions: Panel (a): 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days and 25 ng/ml BMP-4 for days 3-5; panel (b): 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; panel (c): 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days; panel (d): 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days; panel (e): 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a+100 nm GSK-3B inhibitor IX for the first two days, and panel (f): 100 ng/ml activin A+25 ng/ml BMP-4 for all five days. For all the panels, the X-axis represents expression of CD9 and the Y-axis represents expression of CXCR4 (CD184).
FIG. 34 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and panel (b): SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells.
FIG. 35 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
FIG. 36 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 53, treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4, for five days: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and panel (b): SOX-17, HNF-3B and GATA4.
FIG. 37 depicts the percentage of CXCR4 expression, determined by FACS, in cultures of the human embryonic stem cell line H9, treated with the conditions listed in Example 22.
FIG. 38 depicts the expression of definitive endoderm markers as determined by FACS in cultures of the human embryonic stem cell line H9, cultured on fibronectin (panel a) or MATRIGELTM (panel b).
FIG. 39 depicts the expression of definitive endoderm markers as determined by real-time PCR in cultures of the human embryonic stem cell line H9, cultured on fibronectin (□) or a 1:10 dilution of growth factor reduced MATRIGEL (▪).
FIG. 40 depicts the effect of various concentrations of MATRIGEL in the presence of low serum, 100 ng/ml of activin A and 20 ng/ml of Wnt-3a on differentiating human embryonic stem cells into definitive endoderm. Cells were treated according to the methods disclosed in Example 4. Results shown are the expression levels of the genes indicated, as determined by real-time PCR.
FIG. 41 depicts the role of Wnt-3a in definitive endoderm formation by human embryonic stem cells maintained on MATRIGEL, but differentiated on mouse embryonic fibroblasts. Panels (a-d) show real-time PCR data for the genes indicated. Panels (e-g) show FACS data for the conditions indicated.
FIG. 42 shows the differentiation of human embryonic stem cells cultured on tissue culture substrate coated with MATRIGELTM to definitive endoderm following treatment with the Wnt Inhibitor DKK-1. Results shown are the expression of the genes indicated, as determined by real-time PCR in H9 cells treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3A plus 100 ng/ml of DKK1 (DE+DKK1), or in the absence of DKK1 (DE).
FIG. 43 shows immunofluorescence staining of definitive endoderm markers in cultures of the human embryonic stem cell line H9 cultured on tissue culture substrate coated with MATRIGEL and differentiated in low serum plus 100 ng/ml of activin-A without (panel a), or with (panel b) 20 ng/ml of Wnt-3a. Ecad=E-cadherin, NCAM=N-cadherin,
FIG. 44 shows the differentiation of the human embryonic stem cell line SA002 at passage 38 into definitive endoderm. Cells were treated for five days with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in the panels.
FIG. 45 shows the expression of CXCR4 by FACS in the human embryonic stem cell line SA002 at passage 38, following treatment with 100 ng/ml activin A treatment (panel a), 100 ng/ml activin A+20 ng/ml Wnt-3a (panel b), or 100 ng/ml activin A+100 nM GSK-3B inhibitor IX (panel c). Cells were treated for five days.
FIG. 46 shows the differentiation of the human embryonic stem cell line H1 at passage 55 into definitive endoderm on tissue culture substrate coated with human serum. Cells were treated with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in the panels.
FIG. 47 shows the differentiation of cultures of the human embryonic stem cell line H1 at P54, on tissue culture substrate coated with MATRIGELTM to definitive endoderm. The effects of various GSK-B inhibitors were tested following a five-day DE protocol. The following GSK-3B inhibitors were evaluated at 100 nM for the first two days of treatment: GSK-3B VIII, IX, XI, and XII.
FIG. 48 shows the expression of AFP (panel a), Pdx-1 (panel b), Cdx-2 and Glut-2 (panel c) and HNF-3beta, HNF-6 and somatostatin (panel d) in cultures of the human embryonic stem cell line H9 at passage 49, cultured and treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3a for the first two days of treatment. Following the treatment, the cells were treated for three additional days with 2% FBS plus 1 μM retinoic acid, 0.1 to 1 μM TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid), or 0.1-10 μM AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid). The cells were next treated for three additional days in 2% FBS plus 20 ng/ml of bFGF.
FIG. 49 shows the real-time PCR results of the expression of the definitive endoderm markers indicated in panels a and b. in cultures of the human embryonic stem cell line H1 treated with activin A and Wnt-1 for the times and concentrations indicated.
FIG. 50 depicts insulin (panel a) and glucagon (panel b) mRNA expression in cultures of pancreatic endocrine cells, formed from the treatment of pancreatic endoderm cells in DMEM/F12 or DMEM-low glucose. Data shown are results observed from two separate experiments.
FIG. 51 depicts insulin expression as determined by immunocytochemistry in cells treated in DMDM-low glucose (panel a), DMEM/F12 (panel b). Panel c shows the co-staining of PDX-1 and insulin.
FIG. 52 shows the effect of glucose concentration on gene expression in pancreatic endocrine cells derived from the human embryonic stem cell line H9. Genes are identified in the panels.
FIG. 53 shows c-peptide release from pancreatic endocrine cells formed in 2, 10 and 20 mM glucose. Cells were stimulated with IBMX or 20 mM glucose.



DETAILED DESCRIPTION
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.
Definitions
Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (selfrenewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
Various terms are used to describe cells in culture. “Maintenance” refers generally to cells placed in a growth medium under conditions that facilitate cell growth and/or division, which may or may not result in a larger population of the cells. “Passaging” refers to the process of removing the cells from one culture vessel and placing them in a second culture vessel under conditions that facilitate cell growth and/or division.
A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passages depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging.
“β-cell lineage” refer to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Is1-1, HNF-3 beta, MAFA, Pax4, and Pax6. Cells expressing markers characteristic of the β cell lineage include β cells.
“Cells expressing markers characteristic of the definitive endoderm lineage” as used herein refer to cells expressing at least one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mixlike homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
“Cells expressing markers characteristic of the pancreatic endoderm lineage” as used herein refer to cells expressing at least one of the following markers: PDX-1, HNF-1beta, HNF-3beta, PTF-1 alpha, HNF-6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.
Cells expressing markers characteristic of the pancreatic endocrine lineage” as used herein refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, or PTF-1 alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.
“Definitive endoderm” as used herein refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: CXCR4, HNF-3 beta, GATA-4, SOX-17, Cerberus, OTX2, goosecoid, c-Kit, CD99, and Mixl1.
“Extraembryonic endoderm” as used herein refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, and SPARC.
“Markers” as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
“Mesendoderm cell” as used herein refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta, GSC, FGF17, GATA-6.
“Pancreatic endocrine cell” or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin.
“Pancreatic hormone secreting cell” as used herein refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
“Pre-primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Nodal, or FGF8.
“Primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
Isolation, Expansion and Culture of Pluripotent Stem Cells
Characterization of Pluripotent Stem Cells
Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
Sources of Pluripotent Stem Cells
The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BG01v (BresaGen, Athens, Ga.).
In one embodiment, human embryonic stem cells are prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).
Culture of Pluripotent Stem Cells
In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways. Alternatively, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation. The growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type. Alternatively, the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
For example, Reubinoff et al (Nature Biotechnology 18: 399-404 (2000)) and Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391, pp. 1145-1147) disclose the culture of pluripotent stem cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer.
Richards et al, (Stem Cells 21: 546-556, 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture. Richards et al, states: “human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripotent”.
US20020072117 discloses cell lines that produce media that support the growth of primate pluripotent stem cells in feeder-free culture. The cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. US20020072117 also discloses the use of the cell lines as a primary feeder cell layer.
In another example, Wang et at (Stem Cells 23: 1221-1227, 2005) discloses methods for the long-term growth of human pluripotent stem cells on feeder cell layers derived from human embryonic stem cells.
In another example, Stojkovic et at (Stem Cells 2005 23: 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic stem cells.
In a further example, Miyamoto et at (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta.
Amit et at (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder cell layer derived from human foreskin.
In another example, Inzunza et at (Stem Cells 23: 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts.
U.S. Pat. No. 6,642,048 discloses media that support the growth of primate pluripotent stem (pPS) cells in feeder-free culture, and cell lines useful for production of such media. U.S. Pat. No. 6,642,048 states: “This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure.”
In another example, WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. WO2005014799 states: “The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte).”
In another example, Xu et at (Stem Cells 22: 972-980, 2004) discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase.
In another example, US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells.
An alternative culture system employs serum-free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells. For example, Cheon et al (BioReprod DOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose a feeder-free, serum-free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.
In another example, Levenstein et at (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF.
In another example, US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.”
In another example, US20050244962 states: “In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor.”
In a further example, WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of bFGF sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGFβ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
The pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a the suitable culture substrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
Other extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
Differentiation of Pluripotent Stem Cells in to Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line SA002 (Cellartis, Sweden). Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cells: ABCG2, cripto, CD9, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, Tral-81.
Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX-17, GATA4, Hnf-3beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of Pdx1, HNF-1beta, PTF1a, HNF-6, HB9 and PROX1. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1, Pax-4, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.
In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdx1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Is1-1, HNF-3 beta, MAFA, Pax4, and Pax6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.
Formation of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention.
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534-1541 (2005).
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651-1662 (2004).
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29-38 (2007).
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration. An example of this method is disclosed in Nature Biotechnology 23, 1534-1541 (2005).
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2005.
For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
In one aspect of the present invention, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for a period of time, and then culturing the pluripotent stem cells with activin A in a second culture medium containing a greater concentration of serum for about another period of time.
The concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days. The concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
In an alternate embodiment of the present invention, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for about a period of time, and then culturing the pluripotent stem cells with activin A and a Wnt ligand in a second culture medium containing a greater concentration of serum for another period of time.
The concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days. The concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
In one embodiment, the present invention provides a method for differentiating pluripotent stem cells expressing markers characteristic of the definitive endoderm lineage, comprising the steps of:

    a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and
    b. Culturing the pluripotent stem cells with activin A and a Wnt ligand.


Culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed in a single culture medium. Alternatively, culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed separately or together in more than one culture media. In one embodiment, culturing the pluripotent stem cells with activin A and a Wnt ligand is performed in two culture media.
Extracellular Matrix
In one aspect of the present invention, the pluripotent stem cells are cultured and differentiated on a tissue culture substrate coated with an extracellular matrix. The extracellular matrix may be a solubilized basement membrane preparation extracted from mouse sarcoma cells (which is sold by BD Biosciences under the trade name MATRIGEL). Alternatively, the extracellular matrix may be growth factor-reduced MATRIGEL. Alternatively, the extracellular matrix may fibronectin. In an alternate embodiment, the pluripotent stem cells are cultured and differentiated on tissue culture substrate coated with human serum.
The extracellular matrix may be diluted prior to coating the tissue culture substrate. Examples of suitable methods for diluting the extracellular matrix and for coating the tissue culture substrate may be found in Kleinman, H. K., et al., Biochemistry 25:312 (1986), and Hadley, M. A., et al., J. Cell. Biol. 101:1511 (1985).
In one embodiment, the extracellular matrix is MATRIGEL. In one embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:60 dilution.
In one embodiment, the extracellular matrix is growth factor-reduced MATRIGEL. In one embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:60 dilution.
Differentiation of Pluripotent Stem Cells into Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage on an Extracellular Matrix, Using a Single Culture Medium
When a single culture medium is used, it should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells to definitive endoderm, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et at (Exp Cell Res. 1999 Feb. 25; 247(1): 241-8.).
The culture medium may have a serum concentration in the range of about 0% to about 10%. In an alternate embodiment, the concentration may be in the range of about 0% to about 5%. In an alternate embodiment, the concentration may be in the range of about 0% to about 2%. In an alternate embodiment, the concentration may be about 2%.
The time of culturing with activin A and a Wnt ligand may range from about 1 day to about 7 days. In an alternate embodiment, the time of culturing may range from about 1 day to about 3 days. In an alternate embodiment, the time of culturing may be about 3 days.
Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 ng/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
The choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process. The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
The single culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
The single culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the ells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
The at least one additional factor may be, for example, nicotinamide, members of the TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and —BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine 1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241), and primary or transformed endothelial cells.
Differentiation of Pluripotent Stem Cells into Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage on an Extracellular Matrix, Using Two Culture Media
Differentiation of pluripotent stem cells into cells of a definitive endoderm lineage may be accomplished by culturing the pluripotent stem cells with activin A and a Wnt ligand using two culture media. Thus, the differentiation of the pluripotent stem cells may be accomplished as follows:

    a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix,
    b. Culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium, and
    c. Culturing the pluripotent stem cells with activin A in a second culture medium.


The first culture medium may contain serum at a low concentration, and the second culture medium may contain serum at a higher concentration than the first culture medium.
The second culture medium may contain a Wnt ligand.
First Culture Medium:
The first culture medium should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
In the first culture medium there may be a lower concentration of serum, relative to the second culture medium. Increasing the serum concentration in the second culture medium increases the survival of the cells, or, alternatively, may enhance the proliferation of the cells. The serum concentration of the first medium may be in the range of about 0% to about 10%. Alternatively, the serum concentration of the first medium may be in the range of about 0% to about 2%. Alternatively, the serum concentration of the first medium may be in the range of about 0% to about 1%. Alternatively, the serum concentration of the first medium may be about 0.5%.
When culturing the pluripotent stem cells with activin A and a Wnt ligand using at least two culture media, the time of culturing in the first culture medium may range from about 1 day to about 3 days.
Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 ng/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
The choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process. The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
The first culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
The first culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and —BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
Second Culture Medium:
The second culture medium should contain certain factors, such as, for example, insulin and IGF (as disclosed in WO2006020919), at a sufficient concentration to promote the survival of the cultured cells. This may be achieved by increasing the serum concentration, or, alternatively, by using chemically defined media where the concentrations of insulin and IGF are increased relative to the first culture medium. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
In a second culture medium having higher concentrations of serum, the serum concentration of the second culture medium may be in the range about 0.5% to about 10%. Alternatively, the serum concentration of the second culture medium may be in the range of about 0.5% to about 5%. Alternatively, the serum concentration of the second culture medium may be in the range of about 0.5% to about 2%. Alternatively, the serum concentration of the second culture medium may be about 2%. When culturing pluripotent stem cells with the second culture medium, the time of culturing may range from about 1 day to about 4 days.
Similar to the first culture medium, Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 μg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
The second culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
Similar to the first culture medium, the second culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
Differentiation of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
For example, characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, Tral-81.
After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage.
Formation of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention.
For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time. That period of time may be from about one to about six days.
In an alternate aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells with retinoic acid for a period of time. That period of time maybe from about one to about three days. The retinoic acid is subsequently removed and the cells are treated with at least one fibroblast growth factor for another period of time. That period of time may be from about one to about three days.
In one embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:

    a. Culturing cells expressing markers characteristic of the definitive endoderm lineage, and
    b. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor.


Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about one to about six days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about six days.
The at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.\
In an alternate embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:

    a. Culturing cells expressing markers characteristic of the definitive endoderm lineage,
    b. Treating the cells expressing markers characteristic of the definitive endoderm lineage treating the cells with retinoic acid, and
    c. Removing the retinoic acid and subsequently treating the cells with at least one fibroblast growth factor.


Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about three days.
The at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method. In one embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid. Alternatively, the cells expressing markers characteristic of the definitive endoderm lineage are treated with FGF-2, or alternatively FGF-4, or alternatively FGF-10. In an alternate embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one of the following factors: retinoic acid, FGF-2, FGF-4 or FGF-10. In an alternate embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and at least one of the following fibroblast growth factors: FGF-2, FGF-4 or FGF-10. In one embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-2. In another embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-4. In a further embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-10.
Retinoic acid may be used at a concentration from about 1 nM to about 1 mM. In one embodiment, retinoic acid is used at a concentration of 1 μM.
FGF-2 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-2 is used at a concentration of 50 ng/ml.
FGF-4 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-4 is used at a concentration of 50 ng/ml.
FGF-10 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-10 is used at a concentration of 50 ng/ml.
Cells expressing markers characteristic of the definitive endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endoderm lineage. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endoderm lineage. Pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1beta.
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
Formation of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method disclosed in this invention.
For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway. The factor that inhibits the Notch signaling pathway may be an antagonist for the Notch extracellular receptor. Alternatively, the factor may inhibit the biological activity of the Notch receptor. Alternatively, the factor may inhibit or be an antagonist of an element in the Notch signal transduction pathway within a cell.
In one embodiment the factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl]carbamic Acid tert-butyl Ester, also known as L-685,458.
L-685,458 may be used at a concentration from about 0.1 μM to about 100 μM. In one embodiment, L-685,458 is used at a concentration of about 90 μM. In one embodiment, L-685,458 is used at a concentration of about 80 μM. In one embodiment, L-685,458 is used at a concentration of about 70 μM. In one embodiment, L-685,458 is used at a concentration of about 60 μM. In one embodiment, L-685,458 is used at a concentration of about 50 μM. In one embodiment, L-685,458 is used at a concentration of about 40 μM. In one embodiment, L-685,458 is used at a concentration of about 30 μM. In one embodiment, L-685,458 is used at a concentration of about 20 μM. In one embodiment, L-685,458 is used at a concentration of about 10 μM.
In one embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

    a. Culturing cells expressing markers characteristic of the pancreatic endoderm lineage, and
    b. Treating the cells with a factor that inhibits the Notch signaling pathway.


Any cell expressing markers characteristic of the pancreatic endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endocrine lineage using this method.
In one embodiment, factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl]carbamic Acid tert-butyl Ester, also known as L-685,458.
The cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about one to about five days. Alternatively, the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about three to about five days. Alternatively, the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about five days.
In one embodiment, factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl]carbamic Acid tert-butyl Ester, also known as L-685,458.
L-685,458 may be used at a concentration from about 0.1 μM to about 100 μM. In one embodiment, L-685,458 is used at a concentration of about 90 μM. In one embodiment, L-685,458 is used at a concentration of about 80 μM. In one embodiment, L-685,458 is used at a concentration of about 70 μM. In one embodiment, L-685,458 is used at a concentration of about 60 μM. In one embodiment, L-685,458 is used at a concentration of about 50 μM. In one embodiment, L-685,458 is used at a concentration of about 40 μM. In one embodiment, L-685,458 is used at a concentration of about 30 μM. In one embodiment, L-685,458 is used at a concentration of about 20 μM. In one embodiment, L-685,458 is used at a concentration of about 10 μM.
Cells expressing markers characteristic of the pancreatic endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
In one embodiment, the present invention provides an improved method for differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

    a. Culturing cells expressing markers characteristic of the pancreatic endoderm lineage, and
    b. Treating the cells with a factor capable of differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, in medium containing glucose at a concentration from about 10 mM to about 20 mM.


Any cell expressing markers characteristic of the pancreatic endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endocrine lineage using this method.
Any method capable of differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage is suitable for the improvement of the present invention.
In one embodiment, the cells expressing markers characteristic of the pancreatic endoderm lineage are treated in a medium containing glucose at a concentration of about 10 mM. In an alternate embodiment, the cells are treated in a medium containing glucose at a concentration of about 20 mM.
Cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 2 to about 30 days. In one embodiment cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 2 to about 20 days. In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 2 to about 10 days. In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 10 days. In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 4 days. In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are treated for about 2 days.
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage. Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN-3, NeuroD, Islet-1.
Markers characteristic of cells of the β cell lineage are well known to those skilled in the art, and additional markers characteristic of the β cell lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the β-cell lineage. β cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, Pdx1 (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Isl1, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3beta, and MafA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the β cell lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
Therapies
In one aspect, the present invention provides a method for treating a patient suffering from, or at risk of developing, Type1 diabetes. This method involves culturing pluripotent stem cells, differentiating the pluripotent stem cells in vitro into a β-cell lineage, and implanting the cells of a β-cell lineage into a patient.
In yet another aspect, this invention provides a method for treating a patient suffering from, or at risk of developing, Type 2 diabetes. This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a β-cell lineage, and implanting the cells of a β-cell lineage into the patient.
If appropriate, the patient can be further treated with pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells. These agents may include, for example, insulin, members of the TGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-tderived growth factor-AA, and —BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -7, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others. Other pharmaceutical compounds can include, for example, nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2 mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
The pluripotent stem cells may be differentiated into an insulin-producing cell prior to transplantation into a recipient. In a specific embodiment, the pluripotent stem cells are fully differentiated into β-cells, prior to transplantation into a recipient. Alternatively, the pluripotent stem cells may be transplanted into a recipient in an undifferentiated or partially differentiated state. Further differentiation may take place in the recipient.
Definitive endoderm cells or, alternatively, pancreatic endoderm cells, or, alternatively, 0 cells, may be implanted as dispersed cells or formed into clusters that may be infused into the hepatic portal vein. Alternatively, cells may be provided in biocompatible degradable polymeric supports, porous non-degradable devices or encapsulated to protect from host immune response. Cells may be implanted into an appropriate site in a recipient. The implantation sites include, for example, the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach, or a subcutaneous pocket.
To enhance further differentiation, survival or activity of the implanted cells, additional factors, such as growth factors, antioxidants or anti-inflammatory agents, can be administered before, simultaneously with, or after the administration of the cells. In certain embodiments, growth factors are utilized to differentiate the administered cells in vivo. These factors can be secreted by endogenous cells and exposed to the administered cells in situ. Implanted cells can be induced to differentiate by any combination of endogenous and exogenously administered growth factors known in the art.
The amount of cells used in implantation depends on a number of various factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art.
In one aspect, this invention provides a method for treating a patient suffering from, or at risk of developing diabetes. This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a β-cell lineage, and incorporating the cells into a three-dimensional support. The cells can be maintained in vitro on this support prior to implantation into the patient. Alternatively, the support containing the cells can be directly implanted in the patient without additional in vitro culturing. The support can optionally be incorporated with at least one pharmaceutical agent that facilitates the survival and function of the transplanted cells.
Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures, which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No. 5,759,830, U.S. Pat. No. 6,626,950, U.S. Pat. No. 6,534,084, U.S. Pat. No. 6,306,424, U.S. Pat. No. 6,365,149, U.S. Pat. No. 6,599,323, U.S. Pat. No. 6,656,488, U.S. Published Application 2004/0062753 A1, U.S. Pat. No. 4,557,264 and U.S. Pat. No. 6,333,029.
To form a support incorporated with a pharmaceutical agent, the pharmaceutical agent can be mixed with the polymer solution prior to forming the support. Alternatively, a pharmaceutical agent could be coated onto a fabricated support, preferably in the presence of a pharmaceutical carrier. The pharmaceutical agent may be present as a liquid, a finely divided solid, or any other appropriate physical form. Alternatively, excipients may be added to the support to alter the release rate of the pharmaceutical agent. In an alternate embodiment, the support is incorporated with at least one pharmaceutical compound that is an anti-inflammatory compound, such as, for example compounds disclosed in U.S. Pat. No. 6,509,369.
The support may be incorporated with at least one pharmaceutical compound that is an anti-apoptotic compound, such as, for example, compounds disclosed in U.S. Pat. No. 6,793,945.
The support may also be incorporated with at least one pharmaceutical compound that is an inhibitor of fibrosis, such as, for example, compounds disclosed in U.S. Pat. No. 6,331,298.
The support may also be incorporated with at least one pharmaceutical compound that is capable of enhancing angiogenesis, such as, for example, compounds disclosed in U.S. Published Application 2004/0220393 and U.S. Published Application 2004/0209901.
The support may also be incorporated with at least one pharmaceutical compound that is an immunosuppressive compound, such as, for example, compounds disclosed in U.S. Published Application 2004/0171623.
The support may also be incorporated with at least one pharmaceutical compound that is a growth factor, such as, for example, members of the TGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others. Other pharmaceutical compounds can include, for example, nicotinamide, hypoxia inducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 and GLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptides, cathelicidins, defensins, laminin, biological peptides containing cell- and heparin-binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
The incorporation of the cells of the present invention into a scaffold can be achieved by the simple depositing of cells onto the scaffold. Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developed to enhance the efficiency of cell seeding. For example, spinner flasks have been used in seeding of chondrocytes onto polyglycolic acid scaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approach for seeding cells is the use of centrifugation, which yields minimum stress to the seeded cells and enhances seeding efficiency. For example, Yang et al. developed a cell seeding method (J. Biomed. Mater. Res. 55(3): 379-86 (2001)), referred to as Centrifugational Cell Immobilization (CCI).
The present invention is further illustrated, but not limited by, the following examples.
EXAMPLES
Example 1
Human Embryonic Stem Cell Culture
The human embryonic stem cell lines H1, H7 and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM betamercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C (Sigma, MO) for 3 h to arrest cell division, then trypsinized and plated at 2×104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO2 within a humidified tissue culture incubator. When confluent (approximately 5-7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surface using a 5-ml pipette. Cells were spun at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells in fresh culture medium.
Example 2
Formation of Definitive Endoderm Cells
The effects of activin A on the expression of markers of definitive endoderm were examined. Activin A (100 ng/ml) was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts. Cells were cultured continuously in the presence of activin A and harvested at the times indicated. The level of expression of definitive endoderm markers was examined by PCR (FIG. 1), FACS (results summarized in Table II), and immunohistochemistry (FIG. 2).
Activin A evoked a time-dependent increase in the expression of CXCR4, GATA4, HNF-3beta, Mixl1 and Sox-17 mRNA in the H9 line (FIG. 1, panel a). A significant up regulation of anterior endoderm markers, Cerberus, Otx-1 and Hex genes was also observed (FIG. 1, panel b). An increase in CXCR4 protein was observed by FACS analysis following activin A treatment. The expression of E-cadherin and N-cadherin did not change following activin A treatment (Table HA). CXCR4 positive cells were also highly positive for C-kit, EPCAM, CD99, and negative for CD9. The expression pattern for these markers was consistent among all three hES cell lines examined (Table IIB for H7 and Table IIC for H1). Immunocytochemistry conducted on cells treated with activin A for five days revealed that 30-40% cells in the treated culture were positive for Sox17 and HNF-3beta. In parallel, almost 100% of the differentiated cells were still Oct4 positive (FIG. 2). With the decrease in expression of surface markers of pluripotency, combined with an increase in the expression of definitive endoderm markers, these data suggest that activin A promotes the differentiation of human embryonic stem cells to definitive endoderm.
Example 3
Formation of Pancreatic Endoderm Cells
Growth factors known to induce the differentiation of human embryonic stem cells to pancreatic endoderm were added to cell cultures. In particular, activin A, bFGF, and retinoic acid, known to induce the formation of pancreatic endoderm, were added to cell cultures.
In a first series of experiments, activin A, was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts for up to seven days in DMEM/F12 supplemented with 0% to 2% serum and Activin A (100 ng/ml). Cells were harvested at the time points indicated in FIG. 3 and assayed by PCR for the expression of genes shown (FIGS. 3, 4 and 5). In FIG. 3, PCR analysis indicated that activin treated cells expressed a broad spectrum of genes associated with endoderm development, including GATA4 (FIG. 3, panel a), Sox-17 (FIG. 3, panel b), HNF-3beta (FIG. 3, panel c), and Mix1-1 (FIG. 3, panel d). However, no Pdx1 gene expression was observed. The same expression pattern of endoderm lineage markers was observed in Activin A treated H7 cells (FIG. 6, panels a to f). At this stage, there was no significant decrease of Oct4 expression.
Activin A evoked a time-dependent decrease in the expression of the extraembryonic endoderm markers Sox7 (FIG. 4, panel a) and AFP (FIG. 4, panel b). Activin A decreased the expression of Brachyury (FIG. 5, panel a) but had no effect on expression of the neuronal marker Zic1 (FIG. 5, panel b).
Taken together, these data suggest that the increased expression of Sox-17, Mixl1, Gata4, and HNF-3beta together with the up regulation of anterior endoderm markers Otx1, Cer1 and Hex genes, corresponds to the formation of definitive endoderm in response to activin A treatment. Analysis of definitive endoderm markers by immunocytochemistry revealed that protein expression for these genes also reflected the trends observed in mRNA expression. Levels of expression for HNF-3beta, Sox-17, and GATA4 were low in untreated cells, approximately 10 to 20% of all cells. Activin A (100 ng/ml) treatment for five days increased the expression of HNF-3beta, Sox-17, and GATA4 to approximately 50% to 90% of all cells (FIG. 7).
In a second series of experiments, cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After the cells were 70-80% confluent, the medium was changed to DMEM/F12 with 0 to 2% FBS with addition of activin A at 100 ng/ml and cultured in the presence of activin A for either three, five, or seven days. After this time interval, the cells were then further treated for five to six days with combinations of retinoic acid and bFGF as shown in FIG. 8. Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
Gene expression analysis revealed that activin A or retinoic acid alone did not induce the expression of Pdx1. Similar results were observed in cultures of cells treated with retinoic acid in combination with FGF and in the presence of activin A (FIG. 8, panel a). However, treatment of cells with retinoic acid and FGF in the absence of activin A increased the expression of Pdx1 still further (FIG. 8, panel a). Cells treated for three days with activin A, then treated for 5 days with 1 μM retinoic acid and 50 ng/ml bFGF (also known as FGF-2) in the absence of activin A showed a level of Pdx1 expression that was approximately 3500-fold higher than that observed in samples with activin A treatment alone for 5 days (FIG. 8, panel a). Immunocytochemistry showed that 5 to 20% of all cells expressed Pdx1 (FIG. 9).
Treatment with 1 μM retinoic acid and bFGF in the absence of activin A also caused an increase in the expression of GLUT-2 and PTF1a (FIG. 8, panel c) that was not observed in cells treated in the presence of activin A alone. The largest increase in expression of GLUT-2 and PTF1a was observed in cells treated with 1 μM retinoic acid and 50 ng/ml bFGF. Taken together, these data suggest that the formation of pancreatic endoderm is further enhanced by removal of activin A from cell cultures after definitive endoderm has been formed.
Example 4
Formation of Pancreatic Endocrine Cells
Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 3-4 days according to the methods described in Example 1. After the cells were 50-60% confluent, the medium was changed to DMEM/F12 without FBS, containing activin A at 100 ng/ml, and the cells were cultured in this medium for one day. Following the one day culture, the medium was removed and replaced with medium containing 0.5% FBS with 100 ng/ml activin A, and the cells were cultured for one day. Following the second one-day culture, the medium was removed and replaced with medium containing 2% FBS with 100 ng/ml activin A, and the cells were cultured for one day. After this time interval, the cells were then treated for six days with combinations of retinoic acid and FGF as outlined in Example 2, then the culture medium was removed and replaced with medium comprising DMEM/F12 with 2% FBS, containing the γ-secretase inhibitors L-685,458 at 10 μM for three days. Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
Gene expression analysis revealed that activin A alone or in combination with retinoic acid and FGFs did not induce the expression of Ngn3 or insulin (FIG. 10, panel a, c). A decrease in the expression of Hes-1 was also observed following treatment with L-685,458. The maximal inhibition was observed on day three post treatment (FIG. 10, panel d). However, treatment of cells with L-685,458 induced the expression of Ngn3 to a level approximately 50-fold higher than that observed in samples treated with activin A alone or retinoic acid with FGFs in combination. A 70-fold increase of insulin expression was observed in samples treated with the γ-secretase inhibitor. NeuroD1 expression was also increased further by the L-685,458 treatment (FIG. 10, panel a). Taken together, these data suggest that the formation of endocrine cells is further enhanced by removal of retinoic acid and FGFs from cell culture and the addition of γ-secretase inhibitors after pancreatic endoderm has been formed.
Example 5
Formation of Pancreatic Endocrine Cells Expressing Nkx2.2
Definitive endoderm cells obtained according to the methods outlined in Example 2 were treated as follows: Cells were cultured in basal medium, comprising DMEM/F12 with 2% FBS plus 50 ng/ml activin A, 50 ng/ml basic FGF and 1 μM of Retinoic Acid for 3 to 5 days. Cells were continuously cultured for another 3 to 5 days in basal medium with retinoic acid at 1 μM, alone or with bFGF. RNA samples were harvested from cells at various time points along this process to help evaluate the directed differentiation of the cells. Furthermore, culture medium and factors were regularly removed and replenished throughout the differentiation protocol. Addition of activin A showed an increase of Nkx2.2 expression about 35-fold compared to samples without activin A. Samples treated with activin A for the first three days of culture maintained Pdx1 expression at a level similar to samples containing no activin A (FIG. 11). Taken together, these data suggest that the expression of the pancreatic endocrine marker Nkx2.2 is further enhanced by adding Activin A to the first three days of retinoic acid and bFGF treatment.
Example 6
Passage and Expansion of Pancreatic Endoderm Cells in Culture
This example demonstrates that pancreatic endoderm cells derived from human embryonic stem cells herein can be maintained in cell culture and passaged without further differentiation. Pancreatic endoderm cells were differentiated in the presence of 100 ng/ml activin A in low serum DMEM/F12. The low serum DMEM/F12 contained 0% (v/v) fetal bovine serum (FBS) on day 1, 0.5% (v/v) FBS on day two and 2% (v/v) FBS on each day thereafter. After four days of differentiation, the cells were cultured in low serum DMEM/F12 contained 2% (v/v) FBS, 1 μM retinoic acid and 50 ng/ml bFGF for a total of six more days. After the six days of differentiation, the cells were maintained in culture in low serum DMEM/F12 contained 2% (v/v) FBS in the presence of 50 ng/ml FGF10 for a total of 6 days. During the six-day culture period, the pancreatic endoderm cells were passaged twice and cell population-doubling time is about 36 to 48 hours during this 6-day culture. On days 0, 3, and 6 of culture, Q-PCR was used to measure the expression of marker genes indicative of pancreatic endoderm. FIG. 12 shows that cells grown in the presence of 50 ng/ml FGF10 maintained expression of the pancreatic endoderm marker Pdx1 during the 6 day culture period subsequent to their derivation.
Example 7
Derivation of Hepatocytes from Human Embryonic Stem Cells
Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After cells were 70-80% confluent, the medium was changed to DMEM/F12 with 2% FBS containing activin A at 100 ng/ml, and cells were cultured in the presence of activin A for seven days. After 7 days treatment with activin A, the cells were then treated for five days with the conditions shown in FIG. 13. After this time, the cells were harvested, and samples of mRNA were collected for analysis.
An increase in the expression of α-fetoprotein (AFP) and albumin was observed (FIG. 13, panel a) for cells cultured in the absence of activin A. This was further increased by retinoic acid and FGF-4 (FIG. 13, panel b). Taken together, these data suggest that cultures of human embryonic stem cells are capable of expressing hepatocyte markers following the treatment described above. Furthermore, human embryonic stem cells are capable of being differentiated into cells expressing markers that are characteristic of hepatocytes.
Example 8
Characterization of the H9 Human Embryonic Stem Cell Line
The quality of H9 cells was monitored over time by evaluating expression of several markers expressed by undifferentiated ES cells (Carpenter et al., 2001; Reubinoff et al., 2000; Thomson et al., 1998a). H9 cells exhibited reciprocal expression of stage-specific embryonic antigens (Table III). H9 cells play strong immunoreactivity for SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, AP and CD9 antigens, all of which are characteristic of undifferentiated human embryonic stem cells.
Real-Time PCR was performed to assess the expression of genes characteristic of embryonic stem cells, such as, for example, OCT3/4, SOX-2, UTF-1, REX-1, Cx43, Cx45, ABCG-2 and TERT, confirming that the cells grown in this example appeared similar to previously described undifferentiated embryonic stem cells (Table III). OCT3/4 protein expression and Alkaline Phosphatase activity (Chemicon) were confirmed by immunostaining A majority of H9 cells were positive for OCT3/4 and AP (FIG. 14). Overall, these results demonstrate that the H9 cells used in this example were not significantly different in morphology, antigen immunostaining, or pluripotency marker expression when compared to reports from other laboratories.
Example 9
Fluorescence-Activated Cell Sorting (FACS) Analysis
Adhered cells were removed from culture plates by five-minute incubation with TrypLETM Express solution (Invitrogen, CA). Released cells were resuspended in human embryonic stem cell culture medium and recovered by centrifugation, followed by washing and resuspending the cells in a staining buffer consisting of 2% BSA, 0.05% sodium azide in PBS (Sigma, Mo.). As appropriate, the cells were Fc-receptor blocked for 15 minutes using a 0.1% γ-globulin (Sigma) solution. Aliquots (approximately 105 cells) were incubated with either phycoerythirin (PE) or allophycocyanin (APC) conjugated monoclonal antibodies (5 μl antibody per 106 cells), as indicated in Table I, or with an unconjugated primary antibody. Controls included appropriate isotype matched antibodies, unstained cells, and cells stained only with secondary conjugated antibody. All incubations with antibodies were performed for 30 mins at 4° C. after which the cells were washed with the staining buffer. Samples that were stained with unconjugated primary antibodies were incubated for an additional 30 mins at 4° C. with secondary conjugated PE or -APC labeled antibodies. See Table I for a list of secondary antibodies used. Washed cells were pelleted and resuspended in the staining buffer, and the cell surface molecules were identified using a FACS Array (BD Biosciences) instrument, collecting at least 10,000 events.
Example 10
Immunocytochemistry
Cells seeded on 0.1% Matrigel (BD) coated dishes were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were blocked for 1 h at room temperature with PBS/0.1% BSA/10% normal chick serum/0.5% Triton X-100 and then incubated overnight with primary antibodies in PBS/0.1% BSA/10% normal chick serum at 4° C. The list of primary antibodies and their working dilutions are shown in Table IB. After three washes in PBS/0.1% BSA, fluorescent secondary antibodies at a 1:100 dilution in PBS were incubated with cells for 1 h at room temperature to allow binding. Control samples included reactions where the primary antibody was omitted or where the primary antibody was replaced with corresponding matched negative control immunoglobulins at the same concentration as the primary antibodies. Stained samples were rinsed; a drop of PROLONG® (Invitrogen, CA) containing diamidino-2-phenylindole, dihydrochloride (DAPI) was added to each sample to counter-stain the nucleus and to function as an anti-fade reagent. Images were acquired using a Nikon Confocal Eclipse C-1 inverted microscope (Nikon, Japan) and a 10-60× objective.
Example 11
PCR Analysis of Undifferentiated Cells
RNA Extraction, Purification, and cDNA Synthesis:
RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an ABI (ABI, CA) high capacity cDNA archive kit.
Real-Time PCR Amplification and Quantitative Analysis:
Unless otherwise stated, all reagents were purchased from Applied Biosystems. Real-time PCR reactions were performed using the ABI PRISM® 7900 Sequence Detection System. TAQMAN® UNIVERSAL PCR MASTER MIX® (ABI, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μl. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN® probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by Applied Biosystem. Primer and probe sets are listed as follows: Oct3/4 (Hs00742896), SOX-2 (Hs00602736), UTF-1 (Hs00747497), Rex-1 (Hs00399279), Connexin 43 (Hs00748445), Connexin 45 (Hs00271416), ABCG2 (Hs00184979), Tert (Hs00162669), HNF3P (Hs00232764), GATA-4 (Hs00171403), Mixl1 (Hs00430824), Sox7 (Hs00846731), AFP (Hs00173490), Brachyury (Hs00610080), GSC (Hs00418279_m1), Pdx-1 (Hs00426216), PTF1a (Hs00603586), Ngn3 (Hs00360700), NeuroD1 (Hs00159598), Insulin (Hs00355773) and Glu2 (Hs00165775). Sox17 primers were designed using the PRIMERS program (ABI, CA) and were the following sequences: Sox17: TGGCGCAGCAGATACCA (SEQ ID NO:1), AGCGCCTTCCACGACTTG (SEQ ID NO:2) and CCAGCATCTTGCTCAACTCGGCG (SEQ ID NO:3). After an initial incubation at 50° C. for 2 min followed by 95° C. for 10 min, samples were cycled 40 times in two stages—a denaturation step at 95° C. for 15 sec followed by an annealing/extension step at 60° C. for 1 min. Data analysis was carried out using GENEAMP®7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ΔCt). The normalized amount of target was calculated as 2-ΔCt, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.
Example 12
Karyotype Analysis
The karyotype of H9 cells was determined by standard G-banding karyotype analysis. A total of 100 metaphase spreads were evaluated (Applied Genetics Laboratories, Inc.). No chromosome aberrations were found in 100 cells analyzed. Cytogenetic analysis showed that the cells had a normal number of autosomes and a modal chromosome number of 46. FIG. 15 depicts a typical karyotype obtained from the human embryonic stem cell line H9.
Example 13
Human Embryonic Stem Cell Culture on Tissue Culture Substrate Coated with Extracellular Matrix
The human embryonic stem cell lines H1, H7, and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM betamercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C (Sigma, MO) for 3 h to arrest cell division, then trypsinized and plated at 2×104/cm2 on 0.1% bovine gelatin coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO2 within a humidified tissue culture incubator. When confluent (approximately 5 to 7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5 to 10 min and then gently scraped off the surface using a 5 ml glass pipette. Cells were centrifuged at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells on plates coated with a 1:30 dilution of growth factor reduced MATRIGELTM (BD Biosciences). Cells were subsequently cultured in MEF-conditioned media supplemented with 8 ng/ml bFGF and collagenase passaged on MATRIGEL coated plates for at least five passages. The cells cultured on MATRIGELTM were routinely passaged with collagenase IV (Invitrogen/GIBCO), Dispase (BD Biosciences) or Liberase enzyme (Roche, Ind.).
Example 14
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix to Definitive Endoderm
Differentiation of embryonic stem cells to definitive endoderm was carried out as previously described in Nature Biotechnology 23, 1534-1541 (December 2005). Briefly, H9 cultures at approximately 60 to 70% confluency were exposed to DMEM:/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days, followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with growth factor reduced MATRIGEL at a 1:30 to 1:10 dilution or on regular MATRIGEL at a1:30 to 1:10 dilution The plates were coated with MATRIGEL for 1 hr at room temperature.
At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9, and N-cadherin expression and by real time PCR for SOX-17, SOX-7, Alphafetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceral endoderm markers, while GATA4, HNF-3 beta and SOX-17 represent definite endoderm markers, and GSC, Bry, and CXCR4 represent markers of primitive streak. FIG. 17 depicts the expression of CXCR4 by FACS. There was a significant increase in expression of CXCR4 by cells cultured on plates coated with MATRIGEL at a 1:10 dilution as compared to lower concentrations of MATRIGEL. Furthermore, growth factor reduced MATRIGEL was not as effective in formation of definitive endoderm cells as compared to regular MATRIGEL.
FIG. 18 shows the real-time PCR results verifying that cells cultured on plates coated with a 1:10 dilution of MATRIGEL showed a significant up regulation of definitive endoderm markers as compared to cells cultured on a 1:30 dilution of MATRIGEL.
Example 15
Microarray Analysis of Changes in Gene Expression in Human Embryonic Stem Cells Following Formation of Definitive Endoderm
Total RNA was isolated from the following human embryonic stem cell cultures using an RNeasy mini kit (Qiagen): H9P83 cells cultured on MATRIGEL-coated plates and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA) for an additional three days; H9P44 cells cultured on MEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A for an additional three days. Controls for each group included cells plated on MATRIGEL-coated dishes and cultured in MEF-conditioned medium or cells plated on MEFs and cultured in ES medium.
Sample preparation, hybridization, and image analysis were performed according to the Affymetrix Human Genome U133 Plus 2.0 Array. Following normalization and a log transformation, data analysis was performed using OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). The variability within each treatment and among the different treatments was compared using the Pearson correlation coefficient. Variance in gene expression profiles between the different treatments along with the correlation coefficient between the lines are depicted in FIG. 19. Significant differences in gene expression between the samples were evaluated using analysis of variance and an F-test with adjusted P-value (Benjamini and Hochberg correction) of less-than or equal to 0.05. Only genes with a present call were included in the analysis. Table IV lists the genes that are differentially expressed with a difference at least 5-fold between the various samples. The normalized intensity value of the genes that are significantly expressed along with the standard error of the mean (SEM) for each gene are listed.
Example 16
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm
Differentiation of embryonic stem cells to definitive endoderm was carried out as previously described in Nature Biotechnology 23, 1534-1541 (December 2005). Briefly, H9, H7, or H1 cells seeded on growth factor reduced MATRIGELTM (1:30 dilution) cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A (R&D Systems, MN)) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. In all subsequent examples unless otherwise noted, this treatment regimen will be referred to as the definite endoderm (DE) protocol.
In parallel, H9, H7, or H1 cells cultured on MEF feeders were also exposed to the same DE protocol outlined above.
At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9, CD99, and N-cadherin (CD56) expression and by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak.
H-lines cultured on mouse feeders and exposed to the DE protocol resulted in a robust expression of DE markers and expression of CXCR4 by FACS (FIG. 20). There was also a significant decrease in expression of E-cadherin following treatment with the DE protocol. Lastly, the CXCR4+ population also stained positive for CD117. FIG. 21 shows a significant up regulation of definitive endoderm markers as compared to untreated H7 (FIG. 21, panel a) and H9 cells (FIG. 21, panel b).
Unlike H-lines cultured on MEF feeders, H-lines cultured on MATRIGELTM (1:30 dilution) and treated with the definitive endoderm protocol failed to show robust expression of definitive endoderm markers. In particular, the expression of CXCR4 by FACS and by real-time PCR was significantly lower for cells cultured on MATRIGELTM as compared to cells cultured on mouse embryonic fibroblasts. Expression of definitive endoderm markers follows a general response pattern with H1 being greater than H9, which is greater than H7 (FIGS. 22 and 23). From FIG. 22, H1 cells showed a significant increase in CXCR4 expression as compared to H7 and H9 lines. Note that in all cases, the expression of CXCR4 was lower for cells cultured on MATRIGELTM (1:30 dilution) as compared to cells cultured on mouse embryonic fibroblasts. FIG. 23 (panels a-c) shows the real-time PCR results showing that there was modest increase in up regulation of definitive endoderm markers in H7 (FIG. 23, panel a) and H9 (FIG. 23, panel b) lines. However, H1 (FIG. 23, panel c) line showed a more robust up regulation of definitive endoderm markers as compared to H7 and H9 lines.
Example 17
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Role of Wnt Ligands
H7P44 and H9P46 embryonic stem cells were cultured on MATRIGELTM (1:10 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. In some of the cultures 20 ng/ml Wnt-3a (Catalog#1324-WN-002, R&D Systems, MN), 20 ng/ml Wnt-5a (Catalog#654-WN-010, R&D Systems, MN), 25 ng/ml Wnt-7a (Catalog#3008-WN-025, R&D Systems, MN), or 25 ng/ml Wnt-5b (Catalog#3006-WN-025, R&D Systems, MN) was added throughout the five day treatment. FIG. 24 depicts phase contrast images of H9P46 definitive endoderm culture in the presence of high concentration of (a) AA or (b) AA+20 ng/ml Wnt-3a. FIG. 25 depicts the expression of CXCR4 by FACS at day 5 for H7P44, and H9P46 lines cultured on MATRIGELTM (1:30 dilution) and exposed to the DE protocol+Wnt-3a (FIG. 25, panels b and d) and −Wnt-3a (FIG. 25, panels a and c). Presence of Wnt-3a in DE cultures resulted in robust expression of CXCR4 (CD184) as compared to DE cultures treated with low serum plus high concentration of AA. FIG. 26 displays the real-time PCR data for a) H7 and b) H9 cultures treated with low serum+AA+/−Wnt ligands. For both H lines, addition of WNT-3a resulted in significant up regulation of definitive endoderm markers. In contrast, Wnt 5a, Wnt-5b and Wnt-7a had minimal impact on expression of definitive endoderm markers.
Example 18
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Effective Dose of Wnt-3a
H9P46 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml Activin A (AA), and 10-50 ng/ml WNt-3a (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 10-50 ng/ml Wnt-3a for an additional three days. Control cultures were not treated with Wnt-3a. FIG. 27, panel a depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a, b) 10 ng/ml Wnt-3a, c) 20 ng/ml Wnt-3a and d) 50 ng/ml Wnt-3a. In the absence of Wnt-3a the expression of CXCR4 was very low. In contrast, addition of 10-50 ng/ml of Wnt-3a significantly increased the number of CXCR4 positive cells. Furthermore, addition of 10 ng/ml of Wnt-3a was as effective as addition of 50 ng/ml of Wnt-3a. Real-time PCR results (FIG. 28, panel a) also confirm this finding.
In a separate study, H9p52 cells were plated on 1:30 low growth factor MATRIGELTM. For the first 2 days of the DE protocol a range of Wnt-3a doses was used: 10 ng/ml, 5 ng/ml and 1 ng/ml. FIG. 28, panel b shows PCR analysis of the DE markers after 5 days of treatment. The number of cells at the end of the experiment is noted in FIG. 28, panel c. This indicates that cells are proliferating when higher doses of Wnt-3a are used. Extension to 5 days of Wnt-3a treatment (5D) had little effect on DE markers by PCR and did not significantly increase cell numbers (FIG. 28, panel c). These data indicate that 10 ng/ml Wnt3a for 2 days is sufficient to reach optimal cell expansion and definitive endoderm differentiation.
Example 19
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Effect of GSK-3B Inhibitors
In order to confirm that the effect of Wnt-3a was through the Wnt pathway, a GSK-3 inhibitor was used to activate the downstream targets of Wnt, such as beta catenin. H9P46-P48 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin-A (AA), and 10-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 0-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for an additional three days. Control cultures were treated with low serum plus high dose of activin A+/−Wnt-3a. FIG. 29, panel a depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a or GSK-3B inhibitor, b) +20 ng/ml Wnt-3a, c) +1000 nM GSK-3B inhibitor IX, d) +500 nM GSK-3B inhibitor IX, e) +100 nM GSK-3B inhibitor IX, f) +10 nM GSK-3B inhibitor IX, g) +100 nM GSK-3B inhibitor IX for days 1-2, and h) +10 nM GSK-3B inhibitor IX for days 1-2.
In the absence of Wnt-3a or at 10 nm GSK-3B inhibitor the expression of CXCR4 was very low. In contrast, addition of 20 ng/ml of Wnt-3a or 100-1000 nM GSK-3B inhibitor significantly increased the number of CXCR4 positive cells. Furthermore, addition of 100 nM GSK-3B inhibitor for days 1-2 was as effective as addition of 100 nM GSK-3B inhibitor for the entire five day period. FIG. 30 depicts the gene expression of definitive endoderm markers for (panel a) H9P48 cells and (panel b) H9P46 cells.
FIG. 16 depicts the outline of a differentiation protocol in this invention, where embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
Example 20
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Effective Dose of Activin A in the Presence of a GSK-3B Inhibitor or Wnt-3a
H9P49 and H1P46 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 10-100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) or 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus 100 ng/ml of activin A. FIG. 31 depicts the expression of CXCR4 by FACS for H9P49 and H1P46 at day 5 with a) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 10 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, and f) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days. FIG. 31 panels a-d is for H9P49 cells and panels e-f is for H1P46 cells. FIG. 32 depicts the gene expression of definitive endoderm markers for H9P49 cultures treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a: panel a: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel b: SOX-17 and GATA4. It appears that robust expression of definitive endoderm markers can be obtained by using 50 ng/ml of AA+20 ng/ml of Wnt-3A or 100 nM GSK-3B inhibitor IX. Lower doses of activin A lead to formation of extraembryonic endoderm.
Example 16
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Combination of Wnt-3a and GSK-3B Inhibitors
H9P53 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) +/−20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin-A (AA) for an additional three days. In parallel, H9P53 cultures were treated with 25 ng/ml BMP-4 (Catalog#314-BP-010, R&D Systems, MN) +/−20 ng/ml Wnt-3A+/−100 ng/ml activin A. Control cultures were treated with low serum plus 100 ng/ml of activin A. FIG. 33 depicts the expression of CXCR4 by FACS at day 5 with a) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days and 25 ng/ml BMP-4 for days 3-5, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A+100 nm GSK-3B inhibitor IX for the first two days, and f) 100 ng/ml activin A+25 ng/ml BMP-4 for all five days. FIG. 34 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and panel (b): SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells. FIG. 35 depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4. Results are expressed as fold increase over untreated cells. FIG. 36 depicts the gene expression of definitive endoderm markers for H9P53 culture treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4: a) expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and b) SOX-17, HNF-3B and GATA4. Addition of BMP-4 to the DE protocol appears to induce formation of mesoderm marker BRY and combination of Wnt-3A and GSK-4B inhibitor did not lead to significant up regulation of definitive endoderm markers as compared to addition of each agent by itself in the presence of activin A.
Example 22
Differentiation of Human Embryonic Stem Cells Cultured on MEFs to Definitive Endoderm—Combination of Wnt-3a, Activin A, Wnt-5a, BMP-2, BMP-4, BMP-6, BMP-7, IL-4, and SDF-1 in Low Serum
H9P44 cells were plated onto 6 well plates previously coated with mitomycin treated mouse embryonic fibroblasts (MEF). Cells were grown until 70 to 80% confluency in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine (all from Invitrogen/GIBCO) and 8 ng/ml human basic fibroblast growth factor (bFGF) (R&D Systems).
For DE formation, cells were treated in the presence or absence of Activin A (100 ng/ml) in addition to other growth factors detailed below. Growth factors were added to increasing concentration of FBS in a stepwise manner as indicated in the following regimen:
Day 0: 0% FBS in DMEM/F12
Day 1: 0.5% FBS in DMEM/F12
Day 2: 2% FBS in DMEM/F12.
Day 3: Cells were harvested for FACS analysis and RT-PCR.
All growth factors were purchased from R&D Systems, MN. A detailed description and concentration of growth factors for each treatment group is shown below.
1. Control—No growth factor added
2. Activin A (100 ng/ml)
3. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)
4. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)+BMP2 (100 ng/ml)
5. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)
6. Activin A (100 ng/ml)+BMP-6 (100 ng/ml)
7. Activin A (100 ng/ml)+BMP-7 (100 ng/ml)
8. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
9. IL-4 (10 ng/ml)
10. SDF1a (20 ng/ml)
11. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)
12. BMP2 (100 ng/ml)+BMP-4 (100 ng·ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
13. Activin A (100 ng/ml) BMP-2 (100 ng/ml)+BMP-4 (100 ng·ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
14. Activin A (100 ng/ml)+IL-4 (10 ng/ml)
15. Activin A (100 ng/ml)+(SDF1a (20 ng/ml)
16. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt-5a (10 ng/ml)+Wnt-7a (10 ng/ml)
17. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)+BMP-4 (100 ng/ml)
Results:
Cells were harvested on Day 3 of DE protocol treatment. For analysis, an aliquot of treated cells was used for RNA preparation for RT-PCR and the rest of cells used for FACS analysis. The frequency (%) of CXCR4 is shown in FIG. 37. Addition of the above growth factor(s) did not enhance expression of CXCR4 above treatment with 100 ng/ml AA in low serum.
For RT-PCR analysis, cells were analyzed for expression of selected panel of definitive endoderm markers. Results shown were calibrated against cells grown in the base medium but not treated with Activin A or any of the other growth factors. In agreement with the FACS data, Table V shows that there was no significant up regulation of definitive endoderm markers by addition of growth factors, such as Wnt-3a to cultures treated with a high dose of activin A in low serum. This is in contrast to the previous examples showing a significant increase in DE markers for ES cells cultured on feeder-free conditions in the presence of activin A, WNT3A, and low serum.
Example 23
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL or Human Fibronectin to Definitive Endoderm
H9P55 cells were grown and differentiated on human fibronectin or regular growth factor MATRIGELTM (BD Biosciences). 1 ml of DMEM/F12 (Invitrogen/GIBCO) containing 1 ug/ml of human fibronectin (R&D systems, MN) was added to each well of 6 well tissue culture treated dish. Alternatively, regular growth factor MATRIGELTM was diluted 1:10 in DMEM/F12 and 1 ml of diluted MATRIGELTM was added to each well of 6 well tissue culture treated dish. Cells were passed with collagenase. After cells reached 80% confluency, there were treated as follows: 2 days 0.5% FBS containing 10 ng/ml mouse recombinant Wnt3a (R&D) and 100 ng/ml Activin A (R&D). This was followed by 3 days 2% FBS plus 100 ng/ml Activin A. FIG. 38, panels a-b depict the expression of CXCR4 by embryonic stem cells cultured on fibronectin and MATRIGEL, respectively. Real-time PCR results (FIG. 39) confirm that formation of definitive endoderm was equivalent on fibronectin and MATRIGELTM coated plates.
Example 24
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Varying Concentrations of MATRIGEL to Definitive Endoderm
H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with regular MATRIGEL at a 1:60 to 1:10 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
Real time PCR results are shown in FIG. 40. Treatment of human embryonic stem cells with low serum, Activin A and Wnt-3a led to the expression of CXCR4, GATA4, Goosecoid, HNF-3beta, and SOX-17 genes, suggesting that the cells were differentiating to the definitive endoderm stage. However, it does not appear that the in the presence of Wnt-3a concentration of the MATRIGELTM coating plays an important role in differentiation.
Example 25
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix and Subsequently Cultured on MEFs to Definitive Endoderm—Role of Wnt-3a
Cells from the human embryonic stem cell line H9 cultured on MATRIGELTM for at least five passages were seeded onto MEF feeders in ES media. When the cells reached 60 to 70% confluency they were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. Additional treatment groups include Wnt-3a at 20 ng/ml for all five days+10-100 ng/ml of activin A.
At day 3 and 5, the cultures were analyzed by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak. hTERT and Oct-4 are markers for self renewal and pluripotency respectively. Real time-PCR results are shown in FIG. 41, panels a-d. FACS analysis was also performed at day 3 and 5. Expression levels of CXCR-4, and CD9 were analyzed and reported in FIG. 41, panel e.
In the absence of Wnt-3a, AFP expression levels of cells cultured in 100 ng/ml Activin A are similar to those seen in untreated controls. However, with the addition of Wnt-3a to cells cultured in 100 ng/ml activin A, there is an increase in the expression of AFP that increases over time. When a lower concentration of Activin A is used, AFP expression is very high, regardless of the presence of Wnt3a (FIG. 41, panel a). This suggests that a high concentration of Activin A is necessary to keep the cells from differentiating to extra-embryonic tissues.
By FACS analysis, CXCR4 positive cells ranged from 32-42% of the population in samples treated with a high concentration of Activin A but not treated with Wnt-3a as compared to 23-33% of the population in samples treated with a high concentration of Activin A and Wnt3a at day 3 (FIG. 41, panel e). By day 5 of treatment, 28-32% of cells treated with a high concentration of activin A but not treated with Wnt-3a expressed CXCR4 as compared to 43-51% of cells treated with a high concentration of Activin A and Wnt-3a (FIG. 41, panel f). In cells treated with a low concentration of Activin A, there were more CXCR4 positive cells in the treatment group without Wnt-3a (11 to 20%) as compared to the Wnt-3a treated group (3 to 4%) (FIG. 41, panel g). Overall, Wnt-3a does not appear to play a significant role in differentiation of human embryonic stem cells, cultured on MEFs, to definitive endoderm. This suggests that the feeder layer is probably secreting sufficient Wnt-3a or analogous ligand to enhance activin A induced definitive-endoderm formation.
Example 26
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix to Definitive Endoderm Following Treatment with the Wnt Inhibitor DKK-1
To determine if the addition of Wnt-3a was causing the increase in differentiation, an inhibitor of Wnt-3 signaling was added to the cultures. H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt3a, 100 ng/ml Dikkopf-1 (DKK-1) and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with Growth Factor Reduced MATRIGEL at a 1:30 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
At day 5, the cultures were analyzed by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak. hTERT and Oct-4 are markers for self renewal and pluripotency respectively. Results are shown in FIG. 42.
In the presence of Wnt-3a, cells express CXCR4, GATA4, HNF-3beta and SOX17, all markers of definitive endoderm. Markers of primitive streak formation such as goosecoid were also detected at levels higher than that detected in untreated controls. With the addition of DKK1, the expression level of the aforementioned differentiation markers dramatically decrease to levels similar to that of untreated cells.
Example 27
Immunofluorescence Staining of DE Markers for H9 Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL and Differentiated in Low Serum Plus Activin A and ±Wnt-3a
Day 5 DE cultures of H9 cells were stained according to Example 10 for SOX-17, HNF-3B, GATA-4, N-cadherin, and E-cadherin. All nuclei were counter stained with DAPI. 20 ng/ml Wnt-3a resulted in significantly larger number of nuclei stained positive for SOX-17, HNF-3beta. and GATA-4 as compared to cultures differentiated in the absence of Wnt-3a. Furthermore, addition of Wnt-3a resulted in significant loss of expression of e-cadherin and enhanced expression of N-cadherin (FIG. 43, panel a and FIG. 43, panel b).
Example 28
Microarray Analysis of Changes in Gene Expression in Embryonic Stem Cells Following Formation of Definitive Endoderm on MEFS or MATRIGEL
Total RNA was isolated from the following embryonic stem cell cultures using an RNeasy mini kit (Qiagen): A) H9P33 cells cultured on MATRIGELTM-coated plates (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days; B) H9P44 cells cultured on MEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml Activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A for an additional three days, and C) H9P48 cells cultured on MATRIGELTM-coated plates (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A plus 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA) for an additional three days. Controls for each group included cells plated on MATRIGEL-coated dishes and cultured in MEF-conditioned medium or cells plated on MEFs and cultured in ES medium. All groups contained three biological replicates and each biological replicate was repeated on two separate gene chips.
Sample preparation, hybridization, and image analysis were performed according to the Affymetrix Human Genome U133 Plus 2.0 Array. Following normalization and a log transformation, data analysis was performed using OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). Significant differences in gene expression between the samples were evaluated using analysis of variance and an F-test with adjusted P-value (Benjamini and Hochberg correction) of less-than or equal to 0.05. Only genes with a present call in at least one group were included in the analysis. Table VI lists the mean normalized log transformed signal intensity of genes showing at least 5-fold difference between group A, group B, and group C along with the adjusted P-value for each gene.
Example 29
Differentiation of the SA002 ES Line Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm
SA002 P38 cells (Cellartis, Sweden) previously cultured for at least three passages on MATRIGEL-coated plates (1:30 dilution) in MEF-CM supplemented with 8 ng/ml of bFGF were exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN)+/−20 ng/ml of Wnt-3a or 100 nm GSK-3B IX inhibitor for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. Real time PCR results are shown in FIG. 44, panels a & b. Similar to H1, H7, and H9 lines, SA002 line also required addition of Wnt-3A for robust expression of DE markers. Expression of CXCR4 is depicted in FIG. 45: a) AA treatment b) AA+Wnt-3a c) AA+GSK-3B inhibitor.
Example 25
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Human Serum to Definitive Endoderm
Cultures of the human embryonic stem cell line H1 at passage 55 were grown and differentiated on human serum (Sigma, #H1388, MO) coated plates. 0.5 ml of human serum was added to each well of 6 well tissue culture treated dish, incubated for 1 hr at room temperature, and aspirated before adding human embryonic stem cells. After cells reached 80% confluency, they were treated as follows: 2 days 0.5% FBS containing 10 ng/ml mouse recombinant Wnt3a (R&D) or 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) and 100 ng/ml Activin A (R&D). This was followed by 3 days 2% FBS plus 100 ng/ml Activin A. Cultures were then analyzed by real-time PCR (FIG. 46, panels a & b). Robust expression of definitive endoderm markers were noted for cells treated with activin A+GSK-3B inhibitor or Wnt-3A as compared to cells treated with activin A only. These findings parallel our findings for human embryonic stem cells cultured on MATRIGELTM or human fibronectin coated plates.
Example 31
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm—Evaluation of Various GSK-3B Inhibitors
The effectiveness of a number of commercially available GSK-3B inhibitors was evaluated in formation of DE from human embryonic stem cells. The following GSK-3B inhibitors were evaluated at 100 nM: GSK-3B inhibitor VIII (Catalog#361549, Calbiochem, CA), GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA), GSK-3B inhibitor XI (Catalog#361553, Calbiochem, CA), GSK-3B inhibitor XII (Catalog#361554, Calbiochem, CA). H1P54 ES cells were cultured on MATRIGELTM coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA) +/− various GSK-3B inhibitors for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus high dose of AA. FIG. 47, panels a and b depicts the gene expression of definitive endoderm markers at day 5. GSK-3B inhibitor IX and XI were both effective in inducing DE formation as compared to GSK-3B inhibitor VIII and XII.
Example 32
Formation of Pancreatic Endoderm by Human Embryonic Stem Cells Cultured Under Feeder-Free Conditions—Evaluation of Retinoic Acid Analogues
H9P49 embryonic stem cells were cultured on MATRIGELTM (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a (Catalog#1324-WN-002, R&D Systems, MN), and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. At day 5, cells were collected for evaluation by FACS and real-time PCR. As indicated in previous examples, this protocol resulted in robust up regulation of definitive endoderm markers, such as CXCR4 and SOX-17. The resulting definitive endoderm cells at day 5 were exposed to the following media conditions to induce pancreatic endoderm formation: culturing in DMEM/F12 media supplemented with 2% FBS and 1 μM all-trans retinoic acid (RA) (Catalog#R2625, Sigma, Mo.), or 0.1-10 μM AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid, Catalog#A8843, Sigma, Mo.), or 0.1-1 μM TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid, Catalog#T3757, Sigma, Mo.) for 3 days. AM-580 and TTNPB are retinoic acid analogs with affinity for retinoic acid receptors. RA treatment was followed by additional three day treatment in DMEM/F12 media supplemented with 2% FBS and 20-50 ng/ml bFGF (Catalog#F0291, Sigma, Mo.). Cultures were harvested and samples of mRNA were collected for analysis.
Gene expression analysis revealed that (FIG. 48, panels a-d) addition of 1 μM RA followed by exposure to bFGF significantly upregulates pancreatic endoderm markers, such as PDX-1. Furthermore, this protocol resulted in robust expression of foregut endoderm markers, such as CDX-2 and AFP. At 1 μM concentration, addition of RA analogs resulted in equivalent pancreatic endoderm and foregut markers. However, addition of 1 μM RA analogs resulted in more robust expression of AFP as compared to all-trans retinoic acid. However, addition of 10 μM AM-580 suppressed AFP and CDX-2 expression while maintaining a high expression of PDX-1.
Example 34
The Effect of Wnt-3a Treatment on Cytokine Expression in Human Embryonic Stem Cells
The effect that Wnt-3a treatment has on cytokine expression was analyzed using a protein array. Cells of the human embryonic stem cell line H9 were cultured according to the methods described in Example 15. At passage 54, cells were differentiated in the presence of 100 ng/ml ActivinA+/−10 ng/ml Wnt3a for 2 days in 0.5% FBS DMEM/F12. Cells were subsequently cultured for an additional three days in 100 ng/ml Activin A and 2% FBS DMEM/F12. At the end of the 5th day, CXCR4 expression was determined by FACS for each treatment group. Cells treated with Activin A only had 1% of cells expressing CXCR4. Cells treated with Activin A and Wnt3a had 73% of cells positive for CXCR4 expression.
Cell lysates were prepared from cells of each treatment group, with a mammalian cell lysis kit (Sigma-Aldrich, MO). Conditioned media from each treatment group was collected and concentrated. Cytokine array analysis was completed using Cytokine Array panels provided by RayBiotech, GA (http://www.raybiotech.com/). Table VII lists cytokine, growth factor, and receptor expression following normalization of the data and background subtraction. For each panel, positive and negative controls are also included. The data shown are two independent samples per cell treatment group (1,2).
Noticeable upregulation of Angiogenin, IGFBP-1 and EGF are seen in the Wnt-3a treated cell conditioned media. Numerous proteins are upregulated in the Wnt-3a treated cell lysates including IGFBP-1, TGFbeta-1 and TGFbeta-3. These upregulated proteins can be added back into the differentiation media to replace or enhance Wnt-3a effects on definitive endoderm formation.
Example 35
Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL to Definitive Endoderm: Role of Wnt1
H1P55 ES cells were cultured on MATRIGELTM (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A +/−10-20 ng/ml of WNT-1 (PeproTech, NJ, Catalogue#120-17) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) and +/−10 or 20 ng/ml of WNT-1 for an additional three days. The following combinations of WNT1+AA were tested:
a) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for day three, b) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for days 3-5, c) 10 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DMF12+100 ng/ml AA for day three, d) 10 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for days 3-5, e) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for day three, f) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for days 3-5. FIG. 49, panels a and b displays the real-time PCR data for definitive endoderm markers following treatment of the H1 cells with low serum, AA and Wnt-1. Addition of 20 ng/ml of Wnt1 in the presence of 100 ng/ml of AA resulted in significant up regulation of definitive endoderm markers (Bry, CXCR4, GSC, SOX17, HNF-3B, and GATA-4).
Example 36
The Effect of Glucose on Pancreatic Endocrine Differentiation
The efficiency of differentiating pancreatic endoderm cells into pancreatic endocrine cells depends on many factors, including, for example, the choice of basal media, or the concentration of glucose. The effect of glucose concentration on the differentiation of pancreatic endoderm cells, derived from embryonic stem cells, into pancreatic endocrine cells was examined.
Alteration of Glucose Concentration by Changing the Basal Media:
Cultures of undifferentiated human embryonic stem cells (H1 and H9) were cultured according to the methods described in Example 1., prior to differentiation into pancreatic endoderm cells. Embryonic stem cells were differentiated into pancreatic endoderm cells by culturing the embryonic stem cells in RPMI containing activin A at 100 ng/ml in the absence of serum for one day. After this time, the cells were cultured in RPMI containing activin A at 100 ng/ml and 0.2% FBS for an additional two days. Following this treatment, the medium was replaced with RPMI containing 2% FBS, FGF10 (50 ng/ml) and KAAD-cyclopamine (250 nM). Cells were cultured in this medium for four days. After this time, the medium was replaced with medium supplemented with 1×B27, containing all-trans retinoic acid (2 μM), FGF10 (50 ng/ml) and KAAD-cyclopamine (0.25 μM) for four days to induce the formation of pancreatic endoderm cells. The yield of pancreatic endoderm cells was not significantly different in cultures treated with low-glucose DMEM or DMEM/F12.
Pancreatic endoderm cells were differentiated into pancreatic endocrine cells by treating the cells with Exendin 4 and HGF. Excendin 4 (50 ng/ml) and HGF (50 ng/ml) were added for ten days in either low-glucose DMEM or DMEM/F12 for 10 days. Both media were supplemented with 1×B27. Cultures were harvested and samples of mRNA were collected for analysis. Samples were normalized to pancreatic endoderm obtained according to the methods disclosed in Nature Biotechnology 24, 1392-1401 (2006).
Insulin expression was analyzed by real-time PCR. As shown in FIG. 50, panels a and b, both insulin and glucagon gene expression were strongly increased in cells treated DMEM/F12, compared to cells treated in DMEM-low glucose. Insulin expression was also analyzed by immunohistochemistry (FIG. 51). Treatment in DMEM/F12 resulted in a larger percentage of insulin positive cells, compared to DMEM-low glucose (FIG. 51, panels a and b). Insulin positive cells were also positive for PDX-1 (panel c).
Alteration of Glucose Concentration:
Cultures of undifferentiated human embryonic stem cells (H1 and H9) were cultured according to the methods described in Example 1, prior to differentiation into pancreatic endoderm cells. Embryonic stem cells were differentiated into pancreatic endoderm cells by culturing the embryonic stem cells in RPMI containing activin A at 100 ng/ml in the absence of serum for one day. After this time, the cells were cultured in RPMI containing activin A at 100 ng/ml and 0.2% FBS for an additional two days. Following this treatment, the medium was replaced with RPMI containing 2% FBS, FGF10 (50 ng/ml) and KAAD-cyclopamine (250 nM). Cells were cultured in this medium for four days. After this time, the medium was replaced with CMRL supplemented with 1×B27, containing all trans retinoic acid (2 μM), FGF10 (50 ng/ml) and KAAD-cyclopamine (0.25 μM) for four days to induce the formation of pancreatic endoderm cells. The media was supplemented with 5, 10 or 20 mM glucose. The yield of pancreatic endoderm cells was not significantly different in cultures derived from H9 embryonic stem cells treated with 5, 10 or 20 mM glucose (FIG. 52, panel a).
Pancreatic endoderm cells were differentiated into pancreatic endocrine cells by treating the cells with CMRL supplemented with 1×B27, Exendin 4 (50 ng/ml) and HGF (50 ng/ml) for two, four or 10 days in 5, 10 or 20 mM glucose. Cultures were harvested and samples of mRNA were collected for analysis. Samples were normalized to pancreatic endoderm obtained according to the methods disclosed in Nature Biotechnology 24, 1392-1401 (2006).
FIG. 52, panels b-g show the effect of glucose on the expression of Ngn-3, NeuroD-1, Nkx2.2, Pax-4, insulin and glucagon, in cells derived from the human embryonic stem cell line H9. Ngn3 is the first transcription factor involved in determining the pancreatic endocrine fate and NeuroD1 is a direct target of Ngn3. Glucose stimulates a does-dependent increase in both Ngn3 and NeuroD1 mRNA levels. Another two critical pancreatic markers, Nkx2.2 and Pax4, also showed the similar expression pattern (FIG. 52, panels d and e). Optimal insulin and glucagon expression was observed in cells treated with 10 mM glucose for 10 days (FIG. 52, panels f and g).
Similar results for Ngn-3, NeuroD-1, Nkx2.2, Pax-4 were observed in cultures derived from the human embryonic stem cell line H1 (Table VIII). However, optimal insulin and synaptophysin expression was observed in cells treated with 20 mM glucose for 10 days (Table VIII).
C-Peptide Release from Insulin Expressing Cells Formed by the Methods of the Present Invention:
Glucose-mediated c-peptide release was monitored in insulin positive cells derived from H1 cells, that were treated in 2, 10 or 20 mM glucose. To evoke c-peptide release, cells were first incubated with Krebs-Ringer solution with bicarbonate and HEPES (KRBH; 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 10 mM HEPES, 0.1% BSA), for 1 hr. The medium was discarded and replaced with Krebs-Ringer solution containing 2 mM D-glucose. Cells were stimulated with either 20 mM glucose or 0.5 mM IBMX for 1 hr (all purchased from Sigma). The fold stimulation was calculated for each culture by dividing the C-peptide concentration in the simulation supernatant by the C-peptide concentration in the basal supernatant.
BMX stimulated C-peptide release 1.2 to 3 fold (FIG. 53). 20 mM glucose did not stimulate C-peptide release. There was no significant difference in C-peptide secretion observed between insulin positive cells formed in 2, 10 and 20 mM glucose.
Taken together, our data suggest that glucose induces the dose-dependant up regulation of the endocrine markers, Ngn3 and NeuroD1, suggesting that glucose induces the dose-dependent differentiation of human embryonic cells into pancreatic endocrine cells. The expression of insulin is also regulated by glucose in a dose-dependant manner.
Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law.







TABLE IA







LIST OF PRIMARY ANTIBODIES USED FOR FACS


AND IMMUNOSTAINININGANALYSIS.












Antibody
Supplier
Isotype
Clone







SSEA-1
Chemicon (CA)
Mouse IgM
MC-480



SSEA-3
Chemicon (CA)
Mouse IgG3
MC-631



SSEA-4
Chemicon (CA)
Rat IgM
MC-813-70



TRA 1-60
Chemicon (CA)
Mouse IgM
TRA 1-60



TRA 1-81
Chemicon (CA)
Mouse IgM
TRA 1-81



TRA 1-85
Chemicon (CA)
Mouse IgG1
TRA 1-85



AP
R&D Systems
Mouse IgG1
B4-78



HNF3β
R&D Systems
Goat IgG



PDX1
Santa Cruz
Goat IgG
A-17




Biotechnology,




INC



GATA4
R&D Systems
Goat IgG



Sox 17
R&D Systems
Goat IgG



CD 9
BD
Mouse IgG1
M-L13

















TABLE IB







LIST OF SECONDARY CONJUGATED ANTIBODIES


USED FOR FACS AND IMMUNOSTAINININGANALYSIS.











Secondary Conjugated





Antibody
Supplier
Dilution







Goat Anti-Mouse IgG
Jackson
1:200



APC conjugated
ImmunoResearch (PA)



Goat Anti-Mouse IgG
Jackson
1:200



PE conjugated
ImmunoResearch (PA)



Donkey anti-rabbit PE
Jackson
1:200



or -APC conjugated
ImmunoResearch (PA)



Donkey anti-goat PE or -
Jackson
1:200



APC conjugated
ImmunoResearch (PA)



Goat anti-mouse IgM
SouthernBiotech (AL)
1:200



PE



Goat anti-Rat IgM PE
SouthernBiotech (AL)
1:200



Goat anti-mouse IgG3
SouthernBiotech (AL)
1:200



PE

















TABLE IIA







CHANGES IN PROTEIN EXPRESSION IN


HUMAN EMBRYONIC STEM CELLS WITH


TIME, FOLLOWING ACTIVIN A TREATMENT.












0-DAY
2-DAY
5-DAY
8-DAY

















SSEA-3
98.67%
92.14%
 42.9%
22.05%



CD9
92.64%
29.42%
 7.27%
 4.1%



ECAM
61.23%
20.87%
14.17%
 1.02%



NCAM
7.33%
 5.04%
 21.1%
 8.86%



CXCR4
8.53%
 20.2%
55.26%
56.92%

















TABLE IIB







CHANGES IN PROTEIN EXPRESSION IN HUMAN EMBRYONIC STEM


CELLS WITH TIME, FOLLOWING ACTIVIN A TREATMENT.











1-day
3-day
5-day















AA

AA

AA



Untreated
100 ng/ml
Untreated
100 ng/ml
Untreated
100 ng/ml

















CXCR4+
 13%
  6%
 7.6%
  38%
3%
65.5%


CXCR4+
5.32%
2.97%
 2.9%
31.56%
3%
55.21% 


C-Kit+


CXCR4+
11.5%
14.58% 
5.26%
36.67%
3%
54.5%


EPCAM+


CXCR4+
12.27% 
8.13%
2.72%
24.11%
3%
 2.1%


CD9+
















TABLE IIC







CHANGES IN PROTEIN EXPRESSION IN HUMAN


EMBRYONIC STEM CELLS WITH TIME,


FOLLOWING ACTIVIN A TREATMENT.









5-day AA treatment














CXCR4+
92.78%



CXCR4+/C-kit+
92.90%



CXCR4+/EPCAM
87.99%



CXCR4+/CD99+
88.78%



CXCR4+/CD9+
7.03%

















TABLE III







EXPRESSION PROFILE OF PLURIPOTENCY


MARKERS FOR THE EMBRYONIC STEM CELL


USED IN THE PRESENT INVENTION.









H9












Marker
FACS
RT-PCR
Staining







OCT3/4

+
+



SOX-2

+



UTF-1

+



REX-1

+



hTERT

+



Cx 43

+



Cx 45

+



ABCG-2

+



SSEA-1
± (36.35%) 



SSEA-3
+ (94.38%)

+



SSEA-4
+ (98.77%)

+



TRA-1-81
+ (85.85%)

+



TRA-1-60
+ (78.14%)

+



TRA-1-85
+ (95.96%)



CD9
+ (92.02%)



AP
+ (99%)  

+

















TABLE IV







DIFFERENTIAL EXPRESSION OF GENES BETWEEN UNDIFFERENTIATED EMBRYONIC


STEM CELLS AND DEFINITIVE ENDODERM STAGE CELLS CULTURED ON EITHER MATRIGELTM


OR MOUSE EMBRYONIC FIBROBLASTS AFTER 5 DAYS OF TREATMENT.






















H9P83 on



H9P44 on



Gene


H9P83 on

Matrigel-

H9P44 on

MEFs-


Identifier
Gene Title
Gene ID
Matrigel
SEM
DE stage
SEM
MEFs
SEM
DE stage
SEM




















D87811
Homo sapiens
GATA6
−2.12
0.19
2.82
2.20
−2.56
0.46
5.34
1.79



mRNA for GATA-6,



complete cds.



/PROD=GATA-6/



FL=gb:U66075.1



gb:NM_005257.1



gb:D87811.1


AW157548
insulin-like
IGFBP5
−3.28
0.17
3.31
2.11
−3.78
0.36
5.35
2.00



growth factor



binding protein 5



/FL=gb:M65062.1



gb:M62782.1



gb:NM_000599.1



gb:AF055033.1


NM_001898
Homo sapiens
CST4
−2.15
1.26
2.54
1.95
−2.71
0.98
4.64
1.63



cystatin SN



(CST1), mRNA.



/PROD=cystatin SN



/FL=gb:J03870.1



gb:NM_001898.1


AK000680
Homo sapiens
PAG1
−2.87
0.91
1.61
0.22
−4.08
0.50
1.68
0.10



cDNA FLJ20673 fis,



clone KAIA4464.



/FL=gb:AF240634.1



gb:NM_018440.1


NM_022642
Homo sapiens
—
−2.24
0.12
2.97
0.42
−3.78
0.07
2.51
0.44



chorionic



somatomammotropin



hormone 1



(placental lactogen)



(CSH1),



transcript variant 4,



mRNA.



/PROD=chorionic



somatomammotropin



hormone 1,



isoform4



/FL=gb:NM_022642.1


NM_001317
Homo sapiens
CSH1
−2.95
0.57
2.69
0.36
−4.04
0.51
1.86
0.68



chorionic



somatomammotropin



hormone 1



(placental lactogen)



(CSH1),



transcript variant 1,



mRNA.



/PROD=chorionic



somatomammotropin



hormone 1,



isoform 1precursor



/FL=gb:NM_001317.2



gb:J00118.1


BC005921
Homo sapiens,
CSH1
−2.26
0.09
3.26
0.23
−2.96
0.37
2.58
0.45



chorionic



somatomammotropin



hormone 1



(placental lactogen),



clone



MGC:14518, mRNA,



complete cds.



/PROD=chorionic



somatomammotropin



hormone 1



(placentallactogen)



/FL=gb:BC005921.1


AI796169
GATA-binding
GATA3
−4.45
0.10
0.24
1.30
−4.72
0.13
0.80
2.05



protein 3



/FL=gb:NM_002051.1



gb:M69106.1



gb:BC003070.1


NM_020991
Homo sapiens
CSH1
−1.27
0.48
3.19
0.23
−2.91
0.35
2.62
0.54



chorionic



somatomammotropin



hormone 2 (CSH2),



transcript



variant 1, mRNA.



/PROD=chorionic



somatomammotropin



hormone 2, isoform



1precursor



/FL=gb:NM_020991.2



gb:BC002717.1


NM_021827
Homo sapiens
CCDC81
−0.37
0.35
3.16
2.05
−2.02
1.27
5.25
1.98



hypothetical



protein FLJ23514



(FLJ23514), mRNA.



/PROD=hypothetical



protein FLJ23514



/FL=gb:NM_021827.1


AB028021
Cluster Incl.
FOXA2
−2.97
0.25
0.59
3.25
−3.43
0.57
4.12
2.57



AB028021:



Homo sapiens



HNF-3beta mRNA for



hepatocyte nuclear



factor-3 beta,



complete cds



/cds=(196,1569)



/gb=AB028021



/gi=4958949



/ug=Hs.155651



/len=1944


NM_002521
Homo sapiens
NPPB
1.54
0.11
5.47
1.17
−0.15
0.38
6.24
1.23



natriuretic peptide



precursor B (NPPB),



mRNA.



/PROD=natriuretic



peptide precursor B



/FL=gb:NM_002521.1



gb:M25296.1


AA352113
ESTs
ST8SIA4
−4.01
1.24
−0.99
2.04
−4.79
1.00
1.05
1.62


BM128432
Homo sapiens full
IGFBP5
−2.73
1.11
2.31
2.30
−3.48
0.56
4.45
2.02



length insert



cDNA clone



YA81B05


NM_002770
Homo sapiens
PRSS1
−2.77
0.33
1.59
2.68
−3.13
0.48
3.88
2.95



protease, serine, 2



(trypsin 2)



(PRSS2), mRNA.



/PROD=protease,



serine, 2 (trypsin 2)



/FL=gb:M27602.1



gb:NM_002770.1


NM_022579
Homo sapiens
CSH1
−1.58
0.91
2.48
0.38
−3.33
0.13
1.77
0.49



chorionic



somatomammotropin



hormone-like 1



(CSHL1), transcript



variant 3, mRNA.



/PROD=chorionic



somatomammotropin



hormone-like 1,



isoform 3 precursor



/FL=gb:NM_022579.1


NM_005454
Homo sapiens
CER1
2.82
0.09
5.78
1.04
1.48
0.05
6.74
1.18



cerberus 1



(Xenopus laevis)



homolog (cysteine



knot superfamily)



(CER1), mRNA.



/PROD=cerberus 1



/FL=gb:NM_005454.1


NM_022645
Homo sapiens
CSH1
−2.30
0.33
2.95
0.31
−2.78
0.24
2.45
0.34



chorionic



somatomammotropin



hormone 2 (CSH2),



transcript



variant 3, mRNA.



/PROD=chorionic



somatomammotropin



hormone 2,



isoform 3precursor



/FL=gb:NM_022645.1


AI821586
ESTs, Moderately
LOC440981
−3.22
0.97
0.66
3.19
−2.97
0.13
4.22
2.76



similar to JE0284



Mm-1 cell derived



transplantability-



associated



protein 1b



(H. sapiens)


AL121722
Human DNA sequence
—
−2.95
0.36
−0.01
2.69
−3.43
0.38
2.95
2.66



from clone



RP4-788L20 on



chromosome 20 Contains



the HNF3B (hepatocyte



nuclear factor 3,



beta) gene. a novel



gene based on ESTs,



ESTs, STSs, GSSs



and CpG Islands


NM_001311
Homo sapiens
CRIP1
1.66
0.17
1.90
0.07
−2.96
0.66
1.80
0.24



cysteine-rich



protein 1 (intestinal)



(CRIP1), mRNA.



/PROD=cysteine-rich



protein 1 (intestinal)



/FL=gb:U58630.1



gb:BC002738.1



gb:NM_001311.1



gb:U09770.1


AY177407
Homo sapiens
GSC
−4.59
0.18
−1.08
2.89
−4.64
0.06
1.89
2.79



homeobox protein



goosecoid mRNA,



complete cds.



/PROD=homeobox



protein goosecoid



/FL=gb:AY177407.1



gb:NM_173849.1


NM_005442
Homo sapiens
EOMES
−0.16
0.29
2.89
1.70
0.13
0.16
4.90
1.34



eomesodermin



(Xenopus laevis)



homolog (EOMES),



mRNA.



/PROD=eomesodermin



(Xenopus laevis)



homolog



/FL=gb:AB031038.1



gb:NM_005442.1


L01639
Human (clone
CXCR4
0.64
0.26
3.71
1.78
−0.16
0.50
5.48
1.77



HSY3RR) neuropeptide



Y receptor (NPYR)



mRNA, complete cds.



/PROD=neuropeptide Y



receptor



/FL=gb:L06797.1



gb:NM_003467.1



gb:AF025375.1



gb:AF147204.1



gb:M99293.1



gb:L01639.1


NM_022646
Homo sapiens
—
−1.57
0.60
2.67
0.26
−1.88
0.98
2.22
0.35



chorionic



somatomammotropin



hormone 2 (CSH2),



transcript variant 4,



mRNA.



/PROD=chorionic



somatomammotropin



hormone 2, isoform4



/FL=gb:NM_022646.1


AW007532
Human insulin-like
IGFBP5
0.31
0.25
4.59
1.53
0.72
0.09
6.19
1.58



growth factor binding



protein 5 (IGFBP5)



mRNA


NM_002160
Homo sapiens
TNC
−0.24
0.29
2.23
0.80
−0.81
0.81
2.85
0.82



hexabrachion (tenascin



C, cytotactin) (HXB),



mRNA.



/PROD=hexabrachion



(tenascin C,



cytotactin)



/FL=gb:M55618.1



gb:NM_002160.1


AA149250
ESTs, Weakly similar
LOC645638
1.27
0.61
4.23
1.26
−0.64
0.40
2.47
1.23



to WDNM RAT WDNM1



PROTEIN PRECURSOR



(R. norvegicus)


AW977527
ESTs
—
−0.91
0.99
1.18
0.68
−2.52
1.01
1.59
0.64


NM_022454
Homo sapiens
SOX17
−1.01
0.33
2.29
2.08
−0.14
0.15
4.60
1.73



hypothetical protein



FLJ22252 similar to



SRY-box containing



gene 17 (FLJ22252),



mRNA.



/PROD=hypothetical



protein FLJ22252



similar to



SRY-boxcontaining



gene 17



/FL=gb:NM_022454.1


AI640307
protocadherin 10
PCDH10
−1.89
1.37
1.53
1.32
−1.33
0.38
2.99
1.19


AJ224869
Homo sapiens
—
0.98
0.18
4.22
1.88
1.34
0.17
6.36
1.49



CXCR4 gene encoding



receptor CXCR4


AI824037
ESTs, Weakly
FREM1
−1.42
0.36
1.22
1.95
−1.37
0.61
3.48
1.49



similar to FCE2



MOUSE LOW AFFINITY



IMMUNOGLOBULIN



EPSILON FC



RECEPTOR



(M. musculus)


BE222344
splicing factor,
—
0.50
0.05
3.01
0.93
−0.94
1.18
4.27
0.94



arginineserine-rich 5


NM_001643
Homo sapiens
APOA2
−2.25
1.05
1.72
2.60
−1.20
0.44
4.47
2.42



apolipoprotein A-II



(APOA2), mRNA.



/PROD=apolipoprotein



A-II precursor



/FL=gb:M29882.1



gb:NM_001643.1



gb:BC005282.1


AI821669
ESTs
—
−0.94
0.79
1.71
2.19
−0.89
0.21
3.91
1.94


NM_002608
Homo sapiens
PDGFB
−0.23
0.82
1.87
0.15
−2.27
0.65
1.92
0.13



platelet-derived



growth factor beta



polypeptide



(simian sarcoma viral



(v-sis) oncogene



homolog) (PDGFB),



mRNA.



/PROD=platelet-derived



growth factor beta



polypeptide(simian



sarcoma viral (v-sis)



oncogene homolog)



/FL=gb:M12783.1



gb:NM_002


AW444761
ESTs
CDKN2B
−3.35
0.90
0.39
0.52
−3.11
0.88
0.80
0.25


BF223214
ESTs
—
−0.48
0.08
1.69
2.04
−1.51
0.25
4.09
1.98


AF154054
Homo sapiens
GREM1
1.68
0.26
4.61
0.74
0.62
0.04
3.58
0.87



DRM (DRM) mRNA,



complete cds.



/PROD=DRM



/FL=gb:NM_013372.1



gb:AF110137.2



gb:AF045800.1



gb:AF154054.1


NM_021223
Homo sapiens
MYL7
1.81
0.05
4.28
0.81
0.17
0.08
4.87
0.96



myosin light chain 2a



(LOC58498), mRNA.



/PROD=myosin light



chain 2a



/FL=gb:NM_021223.1


AI817041
G protein-coupled
CMKOR1
−0.19
0.27
2.67
1.97
0.06
0.18
5.05
1.64



receptor


NM_003670
Homo sapiens
BHLHB2
1.09
0.08
3.85
0.10
−0.11
0.17
3.46
0.03



basic helix-loop-helix



domain containing,



class B, 2 (BHLHB2),



mRNA.



/PROD=differentiated



embryo chondrocyte



expressed gene1



/FL=gb:AB004066.1



gb:NM_003670.1


NM_023915
Homo sapiens
GPR87
−1.70
0.61
1.64
0.18
−2.99
0.06
1.40
0.23



G protein-coupled



receptor 87 (GPR87),



mRNA.



/PROD=G protein-



coupled receptor 87



/FL=gb:NM_023915.1



gb:AF237763.1


NM_003867
Homo sapiens
FGF17
−3.05
0.39
0.03
2.07
−2.13
0.41
2.49
1.35



fibroblast growth



factor 17 (FGF17),



mRNA.



/PROD=fibroblast growth



factor 17



/FL=gb:NM_003867.1



gb:AB009249.1


NM_024426
Homo sapiens
WT1
−3.23
0.37
−1.11
0.62
−4.20
0.56
−2.44
1.26



Wilms tumor 1 (WT1),



transcript variant D,



mRNA.



/PROD=Wilms tumor 1



isoform D



/FL=gb:NM_024424.1



gb:NM_024426.1


NM_033136
Homo sapiens
FGF1
−3.10
1.42
0.09
0.83
−3.16
0.63
−0.78
1.21



fibroblast growth



factor 1 (acidic)



(FGF1), transcript



variant 2, mRNA.



/PROD=fibroblast growth



factor 1 (acidic)



isoform 2precursor



/FL=gb:NM_033137.1



gb:NM_033136.1


X99268
H. sapiens
TWIST1
0.10
0.33
3.94
0.24
0.34
0.22
3.45
0.34



mRNA for B-HLH DNA



binding protein.



/PROD=B-HLH DNA



binding protein



/FL=gb:NM_000474.1


AL524520
G protein-coupled
LGR5
−2.27
1.43
0.76
1.40
−1.58
0.40
2.51
1.35



receptor 49


NM_022557
Homo sapiens
CSH1
−0.91
0.19
1.40
0.22
−2.12
0.08
1.47
0.14



growth hormone 2



(GH2),



transcript variant 2,



mRNA.



/PROD=growth hormone



2, isoform 2 precursor



/FL=gb:J03756.1



gb:NM_022557.1


AL544576
ESTs
TMEM88
−1.96
0.68
1.75
0.58
−1.45
0.86
2.08
0.70


NM_022580
Homo sapiens
CSH1
−1.20
0.86
2.30
0.39
−1.78
0.64
1.55
0.63



chorionic



somatomammotropin



hormone-like



1 (CSHL1), transcript



variant 4,



mRNA.



/PROD=chorionic



somatomammotropin



hormone-like



1, isoform 4



/FL=gb:NM_022580.1


J03580
Human, parathyroid-
PTHLH
−2.72
0.33
−0.80
0.40
−4.05
0.41
−1.42
0.73



like protein



(associated with



humoral



hypercalcemia of



malignancy) mRNA,



complete cds.



/FL=gb:J03580.1


BC029835
Homo sapiens,
LOC646867
−2.66
1.12
1.01
1.85
−1.40
0.41
3.03
1.61



clone



IMAGE:5169759, mRNA.


AI452798
ESTs
MYOCD
0.98
0.13
3.31
0.66
−0.07
0.28
2.80
0.98


NM_022559
Homo sapiens
CSH1
−1.56
0.38
2.00
0.32
−2.07
0.42
1.47
0.28



growth hormone 1



(GH1), transcript



variant 2, mRNA.



/PROD=growth hormone



1, isoform 2



precursor



/FL=gb:NM_022559.1


NM_001318
Homo sapiens
CSH1
0.15
0.41
2.83
0.40
−1.30
0.34
2.37
0.50



chorionic



somatomammotropin



hormone-like



1 (CSHL1),



transcript



variant 1, mRNA.



/PROD=chorionic



somatomammotropin



hormone-like



1, isoform 1



/FL=gb:NM_001318.2


M65062
Human insulin-like
IGFBP5
−2.80
1.17
2.19
2.00
−0.99
0.23
4.11
1.81



growth factor binding



protein 5 (IGFBP-5)



mRNA, complete cds.



/PROD=insulin-like



growth factor binding



protein 5



/FL=gb:M65062.1



gb:M62782.1



gb:NM_000599.1



gb:AF055033.1


AF207990
Homo sapiens
FER1L3
0.49
0.40
3.00
0.81
−0.16
0.09
4.02
0.92



fer-1 like protein 3



(FER1L3) mRNA,



complete cds.



/PROD=fer-1 like



protein 3



/FL=gb:AF207990.1


AI079944
ESTs
—
−0.78
0.22
0.08
0.59
−3.56
0.24
−0.13
0.73


BC003070
Homo sapiens,
GATA3
1.07
0.04
3.23
0.96
0.31
0.26
4.45
0.80



GATA-binding



protein 3, clone



MGC:2346, mRNA,



complete cds.



/PROD=GATA-binding



protein 3



/FL=gb:NM_002051.1



gb:M69106.1



gb:BC003070.1


BE877796
collagen, type VIII,
COL8A1
−3.76
1.17
−1.28
0.73
−4.89
0.54
−1.33
1.64



alpha 1



/FL=gb:NM_001850.1


NM_022560
Homo sapiens
CSH1
−2.23
0.74
1.95
0.08
−2.01
0.51
1.47
0.31



growth hormone 1



(GH1), transcript



variant 3, mRNA.



/PROD=growth hormone



1, isoform 3



precursor



/FL=gb:NM_022560.1


BE328496
hypothetical
—
−0.59
0.25
1.76
0.13
−2.01
0.97
1.73
0.26



protein PRO2032



/FL=gb:AF116683.1



gb:NM_018615.1


NM_022469
Homo sapiens
GREM2
−1.15
0.25
0.09
0.88
−3.43
0.90
0.76
0.47



hypothetical protein



FLJ21195 similar to



protein related to



DAC and cerberus



(FLJ21195), mRNA.



/PROD=hypothetical



protein FLJ21195



similar to



proteinrelated to



DAC and cerberus



/FL=gb:NM_022469.1


NM_001362
Homo sapiens
DIO3
−1.73
0.70
1.99
1.92
−1.05
0.79
4.23
1.51



deiodinase,



iodothyronine,



type III (DIO3),



mRNA.



/PROD=thyroxine



deiodinase type III



/FL=gb:NM_001362.1



gb:S79854.1


NM_022581
Homo sapiens
CSH1
−1.56
0.67
2.08
0.42
−1.63
0.80
1.47
0.53



chorionic



somatomammotropin



hormone-like 1



(CSHL1), transcript



variant 5, mRNA.



/PROD=chorionic



somatomammotropin



hormone-like 1,



isoform 5 precursor



/FL=gb:NM_022581.1


NM_013372
Homo sapiens
GREM1
1.61
0.07
4.15
0.64
0.91
0.15
3.20
0.81



cysteine knot



superfamily 1, BMP



antagonist 1



(CKTSF1B1), mRNA.



/PROD=cysteine knot



superfamily 1, BMP



antagonist 1



/FL=gb:NM_013372.1



gb:AF110137.2



gb:AF045800.1



gb:AF154054.1


NM_022561
Homo sapiens
CSH1
−1.32
0.60
1.91
0.25
−1.66
0.46
1.57
0.22



growth hormone 1



(GH1), transcript



variant 4, mRNA.



/PROD=growth hormone



1, isoform 4



precursor



/FL=gb:NM_022561.1


NM_022659
Homo sapiens
—
−1.58
0.75
−1.24
1.79
−3.62
0.20
−1.81
1.25



hypothetical protein



FLJ11500 similar to



EBF2 (FLJ11500),



mRNA.



/PROD=hypothetical



protein FLJ11500



similar to EBF2



/FL=gb:NM_022659.1


AF006060
Homo sapiens
CSH1
−1.21
0.06
1.13
0.05
−3.08
0.63
0.50
0.17



placental growth



hormone 20 kDa



isoform (hGH-V)



mRNA, complete



cds.



/PROD=placental growth



hormone 20 kDa



isoform



/FL=gb:AF006060.1



gb:NM_022556.1


AI688418
plexin A2
PLXNA2
−0.18
0.14
0.84
1.58
−1.28
0.53
2.74
0.96


M86849
Homo sapiens
—
−5.08
0.26
0.54
0.47
−1.64
0.19
−0.09
0.44



connexin 26 (GJB2)



mRNA, complete cds.



/PROD=connexin 26



/FL=gb:NM_004004.1



gb:M86849.2


N71923
fibronectin
—
0.30
0.24
2.83
1.46
0.53
0.12
4.38
1.46



leucine rich



transmembrane



protein 3



/FL=gb:AF169677.1



gb:NM_013281.1


NM_013281
Homo sapiens
FLRT3
−0.20
0.33
2.14
1.61
0.00
0.15
3.96
1.39



fibronectin leucine



rich transmembrane



protein 3 (FLRT3),



mRNA.



/PROD=fibronectin



leucine rich



transmembrane protein3



/FL=gb:AF169677.1



gb:NM_013281.1


AI601101
Homo sapiens
FAM84A
0.46
0.38
3.07
0.46
−0.32
0.46
3.82
0.48



cDNA: FLJ21410 fis,



clone COL03938


NM_000325
Homo sapiens
PITX2
1.37
0.17
3.51
0.47
0.89
0.16
4.02
0.47



paired-like



homeodomain



transcription factor 2



(PITX2), mRNA.



/PROD=paired-like



homeodomain



transcription



factor 2



/FL=gb:NM_000325.1



gb:U69961.1



gb:AF048720.1


AI692659
heat shock
PRDM1
0.21
0.25
1.67
0.77
−0.67
0.17
2.61
0.80



90 kD protein 1, alpha


NM_000602
Homo sapiens
SERPINE1
2.97
0.16
4.75
1.89
1.55
0.22
2.75
1.87



serine (or cysteine)



proteinase inhibitor,



clade E (nexin,



plasminogen activator



inhibitor type 1),



member 1



(SERPINE1), mRNA.



/PROD=serine (or



cysteine) proteinase



inhibitor, cladeE



(nexin, plasminogen



activator



inhibitor type



1), membe


NM_001480
Homo sapiens
GALR1
−1.90
0.68
0.04
0.73
−2.42
0.72
−0.34
0.73



galanin receptor 1



(GALR1), mRNA.



/PROD=galanin



receptor 1



/FL=gb:NM_001480.2



gb:U23854.1



gb:L34339.1



gb:U53511.1


NM_000393
Homo sapiens
COL5A2
4.29
0.14
5.25
0.21
2.62
0.02
5.48
0.13



collagen, type V,



alpha 2 (COL5A2),



mRNA.



/PROD=collagen,



type V, alpha 2



/FL=gb:NM_000393.1


N63706
ESTs
—
0.06
0.18
1.73
1.89
−0.74
0.13
3.75
1.75


AF132818
Homo sapiens
KLF5
0.63
0.11
3.16
0.50
0.07
0.42
3.40
0.44



colon Kruppel-like



factor (CKLF) mRNA,



complete cds.



/PROD=colon Kruppel-



like factor



/FL=gb:AF132818.1



gb:AF287272.1



gb:AB030824.1



gb:NM_001730.1



gb:D14520.1


X59065
H. sapiens
—
−1.17
1.11
−0.39
1.06
−2.61
0.26
−0.96
1.01



FGF gene, exon 3



/FL=gb:NM_000800.1



gb:M13361.1


R73554
Human insulin-like
IGFBP5
−0.12
0.15
2.63
1.41
−0.69
0.16
4.00
1.65



growth factor binding



protein 5 (IGFBP5)



mRNA


NM_002149
Homo sapiens
HPCAL1
−0.18
0.28
1.81
0.44
−1.98
0.86
1.18
0.22



hippocalcin-like 1



(HPCAL1), mRNA.



/PROD=hippocalcin-



like 1



/FL=gb:NM_002149.1



gb:D16227.1


AI093327
ESTs
—
0.69
0.11
3.09
0.58
0.45
0.07
2.29
0.85


NM_003240
Homo sapiens
PYCR2
6.22
0.16
6.97
0.60
4.16
0.07
7.51
0.68



endometrial bleeding



associated factor



(left-right



determination,



factor A; transforming



growth factor beta



superfamily) (EBAF),



mRNA.



/PROD=transforming



growth factor, beta 4



/FL=gb:U81523.1



gb:NM_003240.1



gb:AF081513.1


AI263909
ras homolog gene
RHOB
3.89
0.16
5.88
0.13
3.01
0.15
5.64
0.10



family, member B



/FL=gb:NM_004040.1


NM_001792
Homo sapiens
CDH2
3.83
0.17
6.01
0.11
2.85
0.13
5.74
0.17



cadherin 2, type 1,



N-cadherin (neuronal)



(CDH2), mRNA.



/PROD=cadherin 2,



type 1, N-cadherin



(neuronal)



/FL=gb:NM_001792.1



gb:M34064.1


NM_003897
Homo sapiens
IER3
5.45
0.15
6.97
0.17
4.33
0.11
6.75
0.27



immediate early



response 3 (IER3),



mRNA.



/PROD=immediate early



response 3



/FL=gb:BC005080.1



gb:BC000844.1



gb:AF083421.1



gb:NM_003897.1


AF278532
Homo sapiens
NTN4
−0.16
0.30
1.70
0.91
−0.67
0.48
2.66
0.74



beta-netrin mRNA,



complete cds.



/PROD=beta-netrin



/FL=gb:AF119916.1



gb:AF297711.1



gb:NM_021229.1



gb:AF278532.1


AF348491
Homo sapiens
CXCR4
1.39
0.21
4.05
1.58
1.45
0.07
5.67
1.65



chemokine receptor



CXCR4 mRNA,



complete cds.



/PROD=chemokine



receptor CXCR4



/FL=gb:AF348491.1


NM_030781
Homo sapiens
COLEC12
1.93
0.13
3.64
1.66
1.96
0.18
5.68
1.51



scavenger receptor



with C-type lectin



(SRCL), mRNA.



/PROD=scavenger



receptor with



C-type lectin



/FL=gb:NM_030781.1


NM_000599
Homo sapiens
IGFBP5
−0.31
0.34
3.03
1.85
0.12
0.24
4.51
1.75



insulin-like growth



factor binding protein



5 (IGFBP5), mRNA.



/PROD=insulin-like



growth factor



binding protein 5



/FL=gb:M65062.1



gb:M62782.1



gb:NM_000599.1



gb:AF055033.1


AI348094
KIAA0882 protein
TBC1D9
0.13
0.26
3.21
1.13
0.96
0.13
4.67
1.08


BG287862
AHNAK nucleoprotein
AHNAK
1.47
0.20
3.72
0.51
1.37
0.17
4.34
0.58



(desmoyokin)


AI676059
ESTs
FOXQ1
−0.32
0.55
3.08
1.63
0.50
0.17
4.81
1.47


AI127440
ESTs
—
−0.85
0.36
0.60
1.22
−0.65
0.27
2.13
1.13


AL574210
serine (or cysteine)
SERPINE1
2.81
0.24
5.07
0.88
2.13
0.15
3.96
0.86



proteinase inhibitor,



clade E (nexin,



plasminogen activator



inhibitor type 1),



member 1



/FL=gb:NM_000602.1



gb:M16006.1


AB037810
Homo sapiens
SIPA1L2
3.88
0.04
5.66
0.08
3.18
0.11
5.85
0.11



mRNA for KIAA1389



protein, partial cds.



/PROD=KIAA1389



protein


NM_001394
Homo sapiens
DUSP4
0.22
0.37
2.88
1.09
0.50
0.14
4.12
0.99



dual specificity



phosphatase 4



(DUSP4), mRNA.



/PROD=dual



specificity



phosphatase 4



/FL=gb:NM_001394.2



gb:BC002671.1



gb:U48807.1



gb:U21108.1


BC029442
Homo sapiens,
—
2.04
0.20
3.80
0.74
1.18
0.05
4.50
0.65



Similar to immunity



associated protein 1,



clone MGC:32707



IMAGE:4618467, mRNA,



complete cds.



/PROD=Similar to



immunity associated



protein 1



/FL=gb:BC029442.1


NM_000700
Homo sapiens
ANXA1
5.00
0.18
6.27
1.28
3.67
0.05
4.96
1.25



annexin A1 (ANXA1),



mRNA.



/PROD=annexin I



/FL=gb:BC001275.1



gb:NM_000700.1


BC000740
Homo sapiens,
CCKBR
1.08
0.36
3.93
1.84
1.93
0.03
5.86
1.82



cholecystokinin B



receptor, clone



MGC:2199, mRNA,



complete cds.



/PROD=cholecystokinin



B receptor



/FL=gb:L07746.1



gb:L08112.1



gb:S70057.1



gb:BC000740.1



gb:L04473.1



gb:NM_000731.1


N36408
hypothetical protein
FOSL2
−0.09
0.40
2.07
0.04
−0.85
0.24
1.71
0.28



FLJ23306



/FL=gb:NM_024530.1


AF072242
Homo sapiens
MBD2
−3.98
0.38
−0.59
0.27
−3.07
0.22
−1.70
0.68



methyl-CpG binding



protein MBD2 (MBD2)



mRNA, complete cds.



/PROD=methyl-CpG



binding protein MBD2



/FL=gb:NM_003927.2



gb:AF072242.1


AF211891
Homo sapiens
MIXL1
−0.63
0.38
0.77
0.95
−2.29
0.44
0.92
1.71



Mix-like homeobox



protein 1 (MILD1)



mRNA, complete cds.



/PROD=Mix-like



homeobox protein 1



/FL=gb:AF211891.1


BF063186
ESTs
CALD1
1.16
0.19
2.03
0.42
−1.20
1.02
1.65
0.27


NM_000362
Homo sapiens
TIMP3
0.08
0.32
1.98
0.09
−0.34
0.33
1.80
0.26



tissue inhibitor of



metalloproteinase 3



(Sorsby fundus



dystrophy,



pseudoinflammatory)



(TIMP3), mRNA.



/PROD=tissue



inhibitor of



metalloproteinase



3precursor



/FL=gb:NM_000362.2



gb:U14394.1



gb:U67195.1



gb:U02571.1


AK022852
Homo sapiens
SIPA1L2
2.87
0.12
4.50
0.09
1.87
0.01
4.55
0.09



cDNA FLJ12790 fis,



clone



NT2RP2001985,



weakly similar to



Homo sapiens



high-risk human



papilloma viruses E6



oncoproteins targeted



protein E6TP1 alpha



mRNA.


BE500942
Homo sapiens
C6orf155
3.23
0.10
4.68
0.86
1.95
0.03
3.77
0.96



mRNA; cDNA



DKFZp761M0111 (from



clone



DKFZp761M0111)


AW665892
paternally
MFAP5
−1.82
0.54
0.37
0.60
−1.54
0.39
−0.01
0.28



expressed 3


AK025063
Homo sapiens
FAM84A
−1.34
0.84
0.75
0.45
−2.35
0.85
1.01
0.53



cDNA: FLJ21410



fis, clone COL03938.


NM_001828
Homo sapiens
CLC
2.17
0.21
3.53
1.07
0.45
0.08
2.11
1.27



Charot-Leyden crystal



protein (CLC), mRNA.



/PROD=Charot-Leyden



crystal protein



/FL=gb:NM_001828.3



gb:L01664.1


M15329
Human interleukin 1-
IL1A
0.85
0.32
2.72
0.30
−0.88
0.60
2.20
0.22



alpha (IL1A) mRNA,



complete cds.



/PROD=interleukin



1-alpha



/FL=gb:M15329.1


BC002671
Homo sapiens,
DUSP4
1.79
0.03
4.30
1.39
2.10
0.10
5.60
1.22



dual specificity



phosphatase 4, clone



MGC:3713, mRNA,



complete cds.



/PROD=dual



specificity



phosphatase 4



/FL=gb:NM_001394.2



gb:BC002671.1



gb:U48807.1



gb:U21108.1


AA524250
deleted in liver
DLC1
1.02
0.05
2.35
0.98
0.10
0.38
3.26
0.96



cancer 1


BC001211
Homo sapiens,
KIFC3
−1.53
0.72
0.81
0.38
−2.08
0.42
0.75
0.16



kinesin family



member C3, clone



MGC:3226, mRNA,



complete cds.



/PROD=kinesin family



member C3



/FL=gb:BC001211.1



gb:NM_005550.1



gb:AF004426.1


NM_004560
Homo sapiens
ROR2
0.76
0.08
2.20
0.81
0.08
0.14
3.06
0.95



receptor tyrosine



kinase-like orphan



receptor 2 (ROR2),



mRNA.



/PROD=receptor



tyrosine kinase-like



orphan receptor 2



/FL=gb:M97639.1



gb:NM_004560.1


BC000125
Homo sapiens,
TGFB1
1.16
0.18
3.43
0.08
0.72
0.14
3.30
0.16



Similar to



transforming growth



factor, beta 1, clone



MGC:3119, mRNA,



complete cds.



/PROD=Similar to



transforming growth



factor, beta 1



/FL=gb:M38449.1



gb:BC001180.1



gb:BC000125.1



gb:NM_000660.1


NM_016931
Homo sapiens
NOX4
1.83
0.06
3.31
1.29
1.20
0.14
2.28
1.27



NADPH oxidase 4



(NOX4), mRNA.



/PROD=NADPH oxidase 4



/FL=gb:AF261943.1



gb:NM_016931.1



gb:AF254621.1



gb:AB041035.1


BC001830
Homo sapiens,
TGFB1I1
0.95
0.17
3.59
0.73
1.52
0.09
2.74
0.69



Similar to



transforming growth



factor beta 1 induced



transcript 1, clone



MGC:4078, mRNA,



complete cds.



/PROD=Similar to



transforming



growth factor beta



1induced transcript 1



/FL=gb:NM_015927.1



gb:BC001830.1



gb:AF116343.1


NM_024576
Homo sapiens
OGFRL1
3.15
0.13
4.49
0.82
1.63
0.06
3.30
0.98



hypothetical protein



FLJ21079 (FLJ21079),



mRNA.



/PROD=hypothetical



protein FLJ21079



/FL=gb:NM_024576.1


NM_001963
Homo sapiens
EGF
0.12
0.22
1.82
1.15
−0.62
0.36
2.68
1.23



epidermal growth



factor (beta-



urogastrone)



(EGF), mRNA.



/PROD=epidermal



growth factor (beta-



urogastrone)



/FL=gb:NM_001963.2


BE620374
ESTs
C6orf155
1.97
0.05
3.35
1.01
0.59
0.17
2.10
1.05


AL359062
Homo sapiens
COL8A1
−1.31
0.25
2.32
0.94
0.03
0.12
3.51
1.02



mRNA full length



insert cDNA clone



EUROIMAGE 1913076.


AL117653
Homo sapiens
MITF
−0.12
0.61
2.32
0.10
0.21
0.13
2.56
0.06



mRNA; cDNA



DKFZp586C0224 (from



clone



DKFZp586C0224).


AL021977
Cluster Incl.
—
1.92
0.14
4.53
0.43
2.15
0.12
4.66
0.22



AL021977:b



K447C4.1



(novel MAFF (v-maf



musculoaponeurotic



fibrosarcoma (avian)



oncogene family,



protein F)



LIKE protein)



/cds=(0.494)



/gb=AL021977



/gi=4914526



/ug=Hs.51305



/len=2128


NM_003564
Homo sapiens
TAGLN2
5.43
0.12
6.76
0.06
3.48
0.27
6.69
0.04



transgelin 2



(TAGLN2), mRNA.



/PROD=transgelin 2



/FL=gb:D21261.1



gb:NM_003564.1


BC005107
Homo sapiens,
—
7.08
0.07
6.42
0.26
2.60
0.07
6.93
0.22



clone



IMAGE:3840937, mRNA,



partial cds.



/PROD=Unknown



(protein for



IMAGE:3840937)


NM_001124
Homo sapiens
ADM
4.70
0.22
7.61
0.19
5.21
0.06
7.58
0.21



adrenomedullin (ADM),



mRNA.



/PROD=adrenomedullin



/FL=gb:NM_001124.1



gb:D14874.1


AF280545
Homo sapiens
NRP2
−0.29
0.33
1.40
0.30
−1.67
0.61
1.22
0.33



neuropilin-2b(5)



(NRP2) mRNA,



complete cds,



alternatively



spliced.



/PROD=neuropilin-2b(5)



/FL=gb:AF280544.1



gb:AF280545.1


NM_014624
Homo sapiens
S100A6
3.08
0.38
3.10
0.18
−0.57
0.37
3.24
0.19



S100 calcium-binding



protein A6



(calcyclin) (S100A6),



mRNA.



/PROD=S100 calcium-



binding protein A6



/FL=gb:NM_014624.2



gb:BC001431.1


AB030824
Homo sapiens
KLF5
0.57
0.24
2.16
0.49
−0.41
0.05
2.53
0.17



mRNA for



transcription factor



BTEB2, complete cds.



/PROD=transcription



factor BTEB2



/FL=gb:AF132818.1



gb:AF287272.1



gb:AB030824.1



gb:NM_001730.1



gb:D14520.1


NM_015675
Homo sapiens
GADD45B
2.47
0.22
4.44
0.46
2.02
0.09
4.64
0.33



growth arrest and DNA-



damage-inducible,



beta (GADD45B),



mRNA.



/PROD=DKFZP566B133



protein



/FL=gb:NM_015675.1



gb:AF090950.1


BF347089
tissue inhibitor of
TIMP3
0.57
0.13
2.01
0.29
−0.19
0.20
2.09
0.44



metalloproteinase 3



(Sorsby fundus



dystrophy,



pseudoinflammatory)



/FL=gb:NM_000362.2



gb:U14394.1



gb:U67195.1



gb:U02571.1


BF056473
ESTs
—
−0.64
0.87
1.79
0.10
−0.73
0.34
1.57
0.23


AA809487
Homo sapiens
—
0.52
0.22
3.68
0.43
1.39
0.22
3.89
0.48



cDNA: FLJ21715



fis, clone COL10287,



highly similar to



AF071569



Homo sapiens



multifunctional



calciumcalmodulin-



dependent protein



kinase II delta2



isoform mRNA


AL575735
collagen,
—
5.54
0.11
6.65
0.14
4.57
0.08
6.68
0.07



type V, alpha 2



/FL=gb:NM_000393.1


AF003934
Homo sapiens
GDF15
2.74
0.20
4.01
0.41
0.72
0.17
4.34
0.50



prostate



differentiation



factor mRNA,



complete cds.



/PROD=prostate



differentiation



factor



/FL=gb:U88323.1



gb:BC000529.1



gb:AF003934.1



gb:NM_004864.1



gb:AF019770.1



gb:AB000584.1


NM_000313
Homo sapiens
PROS1
−1.54
0.10
0.98
0.83
−0.62
0.09
1.79
0.86



protein S (alpha)



(PROS1), mRNA.



/PROD=protein S



(alpha)



/FL=gb:M15036.1



gb:NM_000313.1


NM_016651
Homo sapiens
DACT1
2.37
0.26
4.79
0.67
2.46
0.14
3.92
0.78



heptacellular



carcinoma novel



gene-3 protein



(LOC51339), mRNA.



/PROD=heptacellular



carcinoma novel



gene-3 protein



/FL=gb:NM_016651.2



gb:AF251079.2


NM_020129
Homo sapiens
LGALS14
1.38
0.30
3.15
0.70
0.79
0.04
2.47
0.66



placental protein



13-like protein



(LOC56891), mRNA.



/PROD=placental



protein 13-like



protein



/FL=gb:NM_020129.1



gb:AF267852.1


NM_013451
Homo sapiens
FER1L3
1.75
0.15
4.02
0.97
1.59
0.13
5.14
0.82



fer-1



(C. elegans)-like 3



(myoferlin) (FER1L3),



mRNA.



/PROD=fer-1



(C. elegans)-like 3



(myoferlin)



/FL=gb:NM_013451.1



gb:AF182316.1


R72286
microfibrillar-
MFAP4
−1.15
0.60
−1.76
0.61
−2.37
0.22
−0.60
0.72



associated



protein 4


AI417362
ESTs, Moderately
FUT1
2.42
0.14
2.32
0.72
−0.46
0.53
1.70
0.59



similar to



ALU1_HUMAN ALU



SUBFAMILY J



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


NM_001553
Homo sapiens
IGFBP7
3.41
0.19
4.63
1.07
1.65
0.10
3.34
1.16



insulin-like growth



factor binding



protein 7 (IGFBP7),



mRNA.



/PROD=insulin-like



growth factor



binding protein 7



/FL=gb:NM_001553.1



gb:L19182.1


BG285011
Homo sapiens
ARID5B
0.05
0.22
1.79
0.21
−0.95
0.42
1.16
0.55



mRNA; cDNA



DKFZp586N012 (from



clone



DKFZp586N012)


BE967311
Homo sapiens
—
1.99
0.06
4.08
0.92
2.28
0.17
5.01
0.87



mRNA; cDNA



DKFZp762O1615 (from



clone



DKFZp762O1615)


BC005047
Homo sapiens,
DUSP6
2.50
0.32
4.04
0.67
1.56
0.16
2.94
0.84



clone MGC:12852,



mRNA, complete cds.



/PROD=Unknown



(protein for



MGC:12852)



/FL=gb:NM_001946.1



gb:AB013382.1



gb:BC003562.1



gb:BC003143.1



gb:BC005047.1


AW005572
putative 47 kDa
ANKS1B
−0.59
0.12
0.19
0.70
−1.59
0.07
0.86
0.92



protein


AW294092
ESTs
RERG
0.54
0.16
−0.68
1.55
−2.78
0.92
−0.58
0.97


NM_001899
Homo sapiens
CST4
0.14
0.52
2.46
1.59
0.53
0.14
3.87
1.40



cystatin S (CST4),



mRNA.



/PROD=cystatin S



/FL=gb:NM_001899.1


AI917371
ESTs
—
−1.47
0.95
0.18
1.43
−1.24
0.33
2.21
1.17


NM_000515
Homo sapiens
CSH1
−2.36
0.29
1.92
0.10
−0.73
0.57
1.37
0.21



growth hormone 1



(GH1), transcript



variant 1, mRNA.



/PROD=growth hormone



1, isoform 1



precursor



/FL=gb:NM_000515.2


NM_004414
Homo sapiens
DSCR1
2.14
0.03
3.85
0.26
1.74
0.16
3.34
0.36



Down syndrome



critical region



gene 1 (DSCR1),



mRNA.



/PROD=Down syndrome



critical region



protein 1



/FL=gb:U28833.2



gb:NM_004414.2


AI355441
sprouty (Drosophila)
—
0.24
0.48
1.72
0.50
−1.12
0.40
2.03
0.61



homolog 4


AB032953
Homo sapiens
ODZ2
0.00
0.14
0.65
0.35
−1.74
0.74
0.88
0.03



mRNA for KIAA1127



protein, partial cds.



/PROD=KIAA1127



protein


BE048571
ESTs
MGC16121
−1.66
0.78
1.44
1.00
−1.31
0.21
0.10
1.22


AW471145
ESTs
PRSS23
0.87
0.10
3.46
0.35
1.13
0.11
3.37
0.53


BF196943
ESTs
USP53
1.43
0.03
3.24
0.92
0.85
0.26
2.32
0.88


AF498927
Homo sapiens
ARHGDIB
−0.03
0.19
0.04
0.96
−3.00
0.54
−0.85
0.93



Rho GDP dissociation



inhibitor beta



(ARHGDIB) mRNA,



complete cds.



/PROD=Rho GDP



dissociation



inhibitor beta



/FL=gb:AF498927.1


AF329092
Homo sapiens
DOC1
−2.38
0.45
1.00
0.19
−1.62
0.74
0.75
0.11



GPBP-interacting



protein 90 mRNA,



complete cds.



/PROD=GPBP-interacting



protein 90



/FL=gb:AF329092.1


BG250721
Homo sapiens
—
2.48
0.04
4.54
0.95
2.63
0.14
5.31
0.83



mRNA; cDNA



DKFZp564C2063 (from



clone



DKFZp564C2063)


N69091
ESTs
PCDH17
0.59
0.10
1.49
0.88
−1.11
0.69
2.49
0.78


BF589359
ESTs
PAG1
−1.11
0.52
−0.09
0.43
−1.54
0.12
0.50
0.13


BF968270
ESTs
SLC35F3
0.37
0.02
1.84
0.30
0.10
0.17
2.46
0.36


NM_006183
Homo sapiens
NTS
4.77
0.15
4.73
1.45
3.01
0.19
3.21
1.47



neurotensin (NTS),



mRNA.



/PROD=neurotensin



precursor



/FL=gb:NM_006183.2



gb:U91618.1


D28124
Cluster Incl.
NBL1
2.90
0.03
4.24
0.37
2.09
0.08
4.44
0.38



D28124:Human mRNA for



unknown product,



complete cds



/cds=(61,603)



/gb=D28124



/gi=641821



/ug=Hs.76307



/len=1929


AW129593
tudor repeat
TDRD7
1.19
0.19
2.40
0.87
0.92
0.04
3.27
0.89



associator with



PCTAIRE 2


BE675435
core promoter element
KLF6
0.29
0.30
2.91
1.02
0.54
0.24
3.68
0.94



binding protein



/FL=gb:AF001461.1



gb:BC000311.1



gb:NM_001300.2



gb:AB017493.1



gb:BC004301.1


AI202327
ESTs
CPEB2
1.88
0.09
3.42
0.07
0.99
0.10
3.36
0.03


NM_002228
Homo sapiens
JUN
2.12
0.16
4.09
0.56
1.62
0.13
4.53
0.41



v-jun avian sarcoma



virus 17 oncogene



homolog (JUN), mRNA.



/PROD=v-jun avian



sarcoma virus 17



oncogene homolog



/FL=gb:NM_002228.2



gb:BC002646.1


AF005775
Homo sapiens
CFLAR
2.88
0.18
3.70
1.56
0.35
0.17
5.22
1.69



caspase-like apoptosis



regulatory protein 2



(clarp) mRNA,



alternatively spliced,



complete cds.



/PROD=caspase-like



apoptosis regulatory



protein 2



/FL=gb:AF005775.1


NM_007173
Homo sapiens
PRSS23
3.02
0.10
5.11
0.21
2.90
0.05
5.22
0.23



protease, serine, 23



(SPUVE), mRNA.



/PROD=protease,



serine, 23



/FL=gb:BC001278.1



gb:AF193611.1



gb:AF015287.1



gb:AL136914.1



gb:NM_007173.1


NM_012413
Homo sapiens
QPCT
1.44
0.09
0.97
0.85
−2.46
0.65
0.19
1.10



glutaminyl-peptide



cyclotransferase



(glutaminyl cyclase)



(QPCT), mRNA.



/PROD=glutaminyl-



peptide



cyclotransferase



precursor



/FL=gb:NM_012413.2


BU683415
Homo sapiens,
KLF6
3.40
0.06
5.28
0.85
3.46
0.09
6.01
0.85



clone



IMAGE:4096273, mRNA


AV729634
DnaJ (Hsp40)
DNAJC6
0.62
0.20
2.59
0.79
0.99
0.19
3.35
0.65



homolog, subfamily B,



member 6



/FL=gb:AB007942.1



gb:NM_014787.1


BC038556
Homo sapiens,
—
−0.79
0.40
0.12
0.99
−2.17
0.62
−0.08
0.38



clone



IMAGE:3446976, mRNA.


NM_014942
Homo sapiens
ANKRD6
0.62
0.04
2.17
1.11
0.59
0.23
3.30
1.07



KIAA0957 protein



(KIAA0957), mRNA.



/PROD=KIAA0957



protein



/FL=gb:AB023174.1



gb:NM_014942.1


AF260333
Homo sapiens
C4orf18
−1.58
0.10
0.21
1.21
−3.19
1.07
1.60
1.12



AD036 mRNA,



complete cds.



/PROD=AD036



/FL=gb:AF260333.1


AA448956
Homo sapiens
CAMK2D
1.47
0.18
2.75
0.42
0.21
0.21
2.97
0.39



cDNA: FLJ21715 fis,



clone COL10287,



highly similar to



AF071569



Homo sapiens



multifunctional



calciumcalmodulin-



dependent protein



kinase II delta2



isoform mRNA



/FL=gb:AF071569.1


BE349115
ESTs
COL22A1
−0.02
0.52
2.04
0.25
0.24
0.06
2.38
0.22


BF209337
Homo sapiens
LOC541471
4.83
0.17
5.80
0.73
3.13
0.02
4.84
0.81



cDNA FLJ10934 fis,



clone



OVARC1000640


AB019695
Homo sapiens
—
2.43
0.01
4.15
0.40
1.42
0.05
3.50
0.29



mRNA for thioredoxin



reductase II beta,



complete cds.



/PROD=thioredoxin



reductase II beta



/FL=gb:AB019695.1


AK090497
Homo sapiens
LOC284576
−3.78
0.56
−2.87
1.42
−4.94
0.07
−4.74
0.53



cDNA FLJ33178 fis,



clone



ADRGL2002753.


NM_006763
Homo sapiens
BTG2
2.30
0.10
3.28
0.62
0.85
0.25
3.84
0.74



BTG family, member 2



(BTG2), mRNA.



/PROD=BTG family,



member 2



/FL=gb:U72649.1



gb:NM_006763.1


BC002616
Homo sapiens,
TAGLN2
5.61
0.20
5.56
0.17
2.91
0.18
5.40
0.12



transgelin 2, clone



MGC:2989, mRNA,



complete cds.



/PROD=transgelin 2



/FL=gb:BC002616.1


AF078077
Homo sapiens
GADD45B
1.30
0.17
3.41
0.31
0.61
0.12
3.05
0.29



growth arrest and



DNA-damage-inducible



protein GADD45beta



mRNA, complete cds.



/PROD=growth arrest



and DNA-damage-



inducible



proteinGADD45beta



/FL=gb:AF087853.1



gb:AF078077.1


NM_001854
Homo sapiens
COL11A1
2.90
0.02
4.06
1.44
1.87
0.06
2.68
1.43



collagen, type XI,



alpha 1 (COL11A1),



mRNA.



/PROD=collagen,



type XI, alpha 1



/FL=gb:J04177.1



gb:NM_001854.1


AI830201
ESTs
KIAA0773
−0.75
0.82
1.24
0.22
−1.87
0.51
0.40
0.22


N95437
ESTs
LMCD1
0.74
0.12
2.72
0.57
0.39
0.14
3.05
0.42


BC002511
Homo sapiens,
CBR1
3.72
0.02
1.03
2.43
−4.10
0.16
−1.64
2.36



carbonyl reductase 1,



clone MGC:1920,



mRNA, complete cds.



/PROD=carbonyl



reductase 1



/FL=gb:BC002511.1



gb:NM_001757.1



gb:J04056.1


AV682252
HIV-1 rev binding
—
−0.33
0.15
1.78
1.36
−0.24
0.17
0.41
1.29



protein 2


AW263497
ESTs
SYTL5
−1.05
0.25
1.44
0.38
−0.55
0.41
2.11
0.47


AF130095
Homo sapiens
—
5.80
0.14
6.80
0.49
4.54
0.10
7.35
0.36



clone FLC0562



PRO2841 mRNA,



complete cds.



/PROD=PRO2841



/FL=gb:AF130095.1


H92988
tyrosine
C9orf19
2.00
0.06
3.54
0.91
1.93
0.14
4.61
0.81



3-monooxygenase-



tryptophan



5-monooxygenase



activation protein,



eta polypeptide


X02761
Human mRNA for
FN1
5.67
0.17
6.65
0.54
4.52
0.21
7.21
0.42



fibronectin (FN



precursor).



/PROD=fibronectin



precursor


AI016316
ESTs
—
0.24
0.18
1.19
1.17
−0.33
0.18
0.25
1.18


NM_006622
Homo sapiens
PLK2
4.64
0.14
5.88
0.45
3.50
0.06
5.19
0.47



serum-inducible kinase



(SNK), mRNA.



/PROD=serum-inducible



kinase



/FL=gb:AF059617.1



gb:NM_006622.1



gb:AF223574.1


NM_013238
Homo sapiens
DNAJC15
−2.27
0.60
3.79
2.03
4.07
0.09
5.58
2.31



DNAJ domain-



containing (MCJ),



mRNA.



/PROD=DNAJ domain-



containing



/FL=gb:NM_013238.1



gb:AF126743.1


AK026737
Homo sapiens
FN1
5.86
0.14
6.85
0.49
4.66
0.08
7.39
0.37



cDNA: FLJ23084 fis,



clone LNG06602,



highly similar to



HSFIB1 Human mRNA for



fibronectin (FN



precursor).


NM_001458
Homo sapiens
FLNC
3.28
0.17
4.17
0.70
2.11
0.17
3.39
0.64



filamin C, gamma



(actin-binding



protein-280) (FLNC),



mRNA.



/PROD=gamma filamin



/FL=gb:AF089841.1



gb:NM_001458.1


AK025843
Homo sapiens
PALLD
1.58
0.31
3.38
0.15
1.03
0.20
3.29
0.19



cDNA: FLJ22190 fis,



clone HRC01053.



/FL=gb:AF151909.1



gb:AF077041.1



gb:NM_016081.1


BC005858
Homo sapiens,
FN1
5.86
0.10
6.86
0.49
4.70
0.19
7.37
0.36



clone MGC:3255, mRNA,



complete cds.



/PROD=Unknown



(protein for MGC:3255)



/FL=gb:BC005858.1


BG491844
v-jun avian sarcoma
JUN
3.53
0.08
5.45
0.47
3.42
0.11
5.91
0.34



virus 17 oncogene



homolog



/FL=gb:NM_002228.2



gb:BC002646.1


AA284532
tyrosine
C9orf19
2.19
0.11
4.12
0.84
2.43
0.08
4.93
0.90



3-monooxygenase-



tryptophan



5-monooxygenase



activation protein,



eta polypeptide


AA192306
triadin
TRDN
−2.54
0.69
0.02
0.61
−1.99
0.31
0.57
0.63



/FL=gb:U18985.1



gb:NM_006073.1


AF116676
Homo sapiens
—
1.50
0.18
3.31
1.11
1.37
0.15
4.23
1.05



PRO1957 mRNA,



complete cds.



/PROD=PRO1957



/FL=gb:AF116676.1


NM_003033
Homo sapiens
ST3GAL1
−0.08
0.18
1.68
1.04
0.20
0.16
2.74
0.74



sialyltransferase 4A



(beta-galactosidase



alpha-2,3-



sialytransferase)



(SIAT4A), mRNA.



/PROD=sialyl-



transferase 4A



(beta-galactosidase-



alpha-2,3-



sialytransferase)



/FL=gb:L13972.1



gb:L29555.1



gb:NM_003033.1


AI222435
ESTs
—
−4.14
1.52
−0.73
0.32
−2.33
0.62
−0.33
0.12


NM_001924
Homo sapiens
GADD45A
3.59
0.09
5.04
0.44
3.06
0.17
5.38
0.48



growth arrest and



DNA-damage-inducible,



alpha (GADD45A), mRNA.



/PROD=growth arrest



and DNA-damage-



inducible, alpha



/FL=gb:M60974.1



gb:NM_001924.2


NM_001425
Homo sapiens
EMP3
2.76
0.18
3.90
0.64
1.51
0.06
3.13
0.57



epithelial membrane



protein 3 (EMP3),



mRNA.



/PROD=epithelial



membrane protein 3



/FL=gb:U52101.1



gb:NM_001425.1



gb:U87947.1


AB017493
Homo sapiens
KLF6
1.69
0.20
4.02
1.01
1.80
0.08
4.87
0.87



mRNA for DNA-binding



zinc finger(GBF),



complete cds.



/PROD=DNA-binding



zinc finger(GBF)



/FL=gb:AF001461.1



gb:BC000311.1



gb:NM_001300.2



gb:AB017493.1



gb:BC004301.1


X58851
Human MLC1emb
—
0.99
0.16
2.75
1.28
1.12
0.15
4.04
1.14



gene for embryonic



myosin alkaline light



chain, promoter and



exon 1


BE327172
v-jun avian sarcoma
—
0.86
0.15
2.94
0.51
1.14
0.08
3.57
0.45



virus 17 oncogene



homolog


U37283
Human microfibril-
MFAP5
0.48
0.19
1.87
0.26
0.15
0.35
1.36
0.47



associated



glycoprotein-2



MAGP-2 mRNA,



complete cds.



/PROD=microfibril-



associated



glycoprotein-2



MAGP-2



/FL=gb:NM_003480.1



gb:U37283.1


AI819043
ESTs
CREB5
0.92
0.26
2.63
0.73
0.65
0.15
1.80
0.79


NM_001511
Homo sapiens
CXCL1
1.01
0.13
2.95
0.35
0.31
0.08
2.39
0.27



GRO1 oncogene



(melanoma growth



stimulating activity,



alpha) (GRO1), mRNA.



/PROD=GRO1 oncogene



(melanoma growth



stimulatingactivity,



alpha)



/FL=gb:NM_001511.1


NM_006736
Homo sapiens
DNAJB2
2.23
0.04
3.46
0.44
1.10
0.02
3.75
0.41



heat shock protein,



neuronal DNAJ-like 1



(HSJ1), mRNA.



/PROD=heat shock



protein, neuronal



DNAJ-like 1



/FL=gb:NM_006736.1


AA534817
ESTs, Weakly similar
EDG3
2.32
0.06
3.28
1.04
2.08
0.04
4.58
1.04



to ALU8_HUMAN ALU



SUBFAMILY SX



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


U82164
Human transmembrane
CD99
4.73
0.15
5.69
0.73
3.19
0.07
6.36
0.93



protein CD99 type II



mRNA, complete cds.



/PROD=CD99 typeII



/FL=gb:BC002584.1



gb:NM_002414.1



gb:M16279.1



gb:BC003147.1



gb:U82164.1


NM_000389
Homo sapiens
CDKN1A
4.03
0.02
4.20
0.12
1.81
0.07
4.16
0.05



cyclin-dependent



kinase inhibitor 1A



(p21, Cip1)



(CDKN1A), mRNA.



/PROD=cyclin-dependent



kinase inhibitor 1A



(p21, Cip1)



/FL=gb:U03106.1



gb:BC000275.1



gb:BC001935.1



gb:L25610.1



gb:U09579.1



gb:NM_000389.1



gb:L26165.1


NM_001299
Homo sapiens
CNN1
3.75
0.15
5.75
0.50
3.47
0.11
5.14
0.43



calponin 1, basic,



smooth muscle (CNN1),



mRNA.



/PROD=calponin 1,



basic, smooth muscle



/FL=gb:U37019.1



gb:NM_001299.1



gb:D17408.1


M36172
Human embryonic myosin
MYL4
1.55
0.21
3.30
1.05
1.21
0.13
4.11
1.06



alkali light chain



(MLC1) mRNA, complete



cds.



/FL=gb:M36172.1



gb:M24121.1



gb:NM_002476.1


AB033831
Homo sapiens
PDGFC
0.48
0.21
0.63
0.15
−1.87
0.96
0.21
0.16



hSCDGF mRNA for



spinal cord-derived



growth factor,



complete cds.



/PROD=spinal cord-



derived growth factor



/FL=gb:NM_016205.1



gb:AB033831.1



gb:AF091434.1



gb:AF244813.1


NM_014333
Homo sapiens
IGSF4
3.11
0.09
4.12
0.27
1.85
0.06
3.79
0.24



immunoglobulin



superfamily, member 4



(IGSF4), mRNA.



/PROD=immunoglobulin



superfamily,



member 4



/FL=gb:NM_014333.1



gb:AF132811.1


AF345910
Homo sapiens
TTC29
0.35
0.26
1.53
0.93
−0.51
0.52
0.91
0.72



NYD-SP14 mRNA,



complete cds.



/PROD=NYD-SP14



/FL=gb:AF345910.1


NM_004297
Homo sapiens
GNA14
4.59
0.06
4.52
1.59
1.95
0.11
2.95
1.50



guanine nucleotide



binding protein (G



protein), alpha 14



(GNA14), mRNA.



/PROD=guanine



nucleotide binding



protein (G protein),



alpha 14



/FL=gb:AF105201.1



gb:NM_004297.1


AK057525
Homo sapiens
—
3.60
0.08
4.45
0.44
2.00
0.16
4.80
0.46



cDNA FLJ32963 fis,



clone



TESTI2008405.


BC000893
Homo sapiens,
HIST1H2BK
2.80
0.06
3.99
0.90
2.48
0.09
4.81
0.82



H2B histone family,



member A, clone



MGC:5132, mRNA,



complete cds.



/PROD=H2B histone



family, member A



/FL=gb:BC000893.1


NM_007038
Homo sapiens
ADAMTS5
0.23
0.17
1.80
0.48
0.45
0.28
1.11
0.73



a disintegrin-like



and metalloprotease



(reprolysin type)



with thrombospondin



type 1 motif, 5



(aggrecanase-2)



(ADAMTS5), mRNA.



/PROD=a disintegrin



and metalloprotease



withthrombospondin



motifs-5



preproprotein



/FL=gb:NM_007038.1



gb:AF14209


AW241910
ESTs, Weakly similar
COL22A1
−0.21
0.12
1.15
0.47
−0.57
0.18
1.90
0.51



to JX0369 collagen



alpha 1(XIX) chain



precursor



(H. sapiens)


AI860150
ESTs, Weakly similar
FOSL2
−0.43
0.55
1.59
0.37
−0.34
0.10
1.08
0.20



to A49134 Ig kappa



chain V-I region



(H. sapiens)


NM_005902
Homo sapiens
SMAD3
1.09
0.41
1.49
0.25
−1.10
0.89
1.73
0.10



MAD (mothers against



decapentaplegic,



Drosophila) homolog 3



(MADH3), mRNA.



/PROD=MAD (mothers



against



decapentaplegic.



Drosophila) homolog 3



/FL=gb:U68019.1



gb:U76622.1



gb:NM_005902.1


AA777512
Homo sapiens
CAMK2D
2.27
0.15
3.67
0.47
1.67
0.08
3.99
0.51



cDNA: FLJ21715 fis,



clone COL10287,



highly similar to



AF071569



Homo sapiens



multifunctional



calciumcalmodulin-



dependent protein



kinase II delta2



isoform mRNA


AI130705
ESTs, Weakly similar
FAM89A
0.80
0.00
1.96
0.99
0.43
0.18
2.70
0.97



to A46302 PTB-



associated splicing



factor, long form



(H. sapiens)


NM_007061
Homo sapiens
CDC42EP1
1.86
0.11
2.51
0.29
0.16
0.27
2.00
0.17



serum constituent



protein (MSE55),



mRNA.



/PROD=serum



constituent protein



/FL=gb:M88338.1



gb:NM_007061.1


NM_003407
Homo sapiens
ZFP36
2.55
0.14
3.53
0.55
1.49
0.28
3.98
0.47



zinc finger protein



homologous to



Zfp-36 in mouse



(ZFP36), mRNA.



/PROD=zinc finger



protein homologous



to Zfp-36 inmouse



/FL=gb:NM_003407.1



gb:M92843.1



gb:M63625.1


BC033088
Homo sapiens,
LMNA
2.03
0.22
2.89
0.45
0.69
0.24
1.79
0.50



Similar to lamin AC,



clone MGC:45654



IMAGE:3623265, mRNA,



complete cds.



/PROD=Similar to



lamin AC



/FL=gb:BC033088.1


U97075
Homo sapiens
CFLAR
2.50
0.09
3.39
1.45
0.73
0.08
4.93
1.54



FLICE-like inhibitory



protein short form



mRNA, complete cds.



/PROD=FLICE-like



inhibitory protein



short form



/FL=gb:U97075.1


AF133207
Homo sapiens
HSPB8
2.54
0.07
4.47
0.62
2.22
0.04
4.88
0.58



protein kinase (H11)



mRNA, complete cds.



/PROD=protein kinase



/FL=gb:AF133207.1


NM_005979
Homo sapiens
S100A13
4.10
0.11
5.67
1.17
3.92
0.02
6.81
1.20



S100 calcium-binding



protein A13



(S100A13), mRNA.



/PROD=S100 calcium-



binding protein A13



/FL=gb:BC000632.1



gb:NM_005979.1


AL040178
ESTs
PEAR1
−0.87
0.08
0.51
0.36
−1.16
0.30
0.48
0.26


AL117523
Homo sapiens
SAMD4A
0.47
0.06
1.59
0.61
−0.24
0.30
1.28
0.53



mRNA; cDNA



DKFZp434H0350 (from



clone



DKFZp434H0350);



partial cds.



/PROD=hypothetical



protein


AB051826
Homo sapiens
RHOU
0.55
0.23
2.18
0.61
0.17
0.29
2.73
0.65



hG28K mRNA for



GTP-binding protein



like 1, complete cds.



/PROD=GTP-binding



protein like 1



/FL=gb:AF282258.1



gb:NM_021205.1



gb:AB051826.1


BC005961
Homo sapiens,
PTHLH
−3.33
0.69
−0.88
0.81
−2.86
0.77
−1.97
1.36



parathyroid



hormone-like hormone,



clone



MGC:14611, mRNA,



complete cds.



/PROD=parathyroid



hormone-like hormone



/FL=gb:BC005961.1


AI670948
ESTs
NODAL
2.13
0.04
2.37
0.22
0.42
0.13
2.78
0.38


AI685060
caldesmon 1
CALD1
4.34
0.33
6.42
0.73
4.45
0.09
5.63
0.82



/FL=gb:M64110.1



gb:NM_004342.2


BF797381
Homo sapiens
CAMK2D
3.16
0.13
4.91
0.65
3.10
0.10
5.49
0.61



cDNA:



FLJ21715 fis, clone



COL10287, highly



similar to AF071569



Homo sapiens



multifunctional



calciumcalmodulin-



dependent protein



kinase II delta2



isoform mRNA


AF026219
Homo sapiens
DLC1
1.16
0.07
2.28
1.13
0.61
0.09
3.52
0.84



HP protein (HP) mRNA,



complete cds.



/PROD=HP protein



/FL=gb:AF026219.1



gb:AF035119.1



gb:NM_006094.2


AK024480
Homo sapiens
LOC126917
1.64
0.11
2.86
0.24
1.19
0.06
2.58
0.25



mRNA for FLJ00074



protein, partial cds.



/PROD=FLJ00074



protein


N29837
ESTs
LIX1
−1.43
0.15
−0.11
0.33
−1.50
0.45
−0.22
0.22


AK001022
Homo sapiens
ISL2
0.41
0.34
2.14
0.39
0.32
0.08
1.57
0.49



cDNA FLJ10160 fis,



clone



HEMBA1003545, highly



similar to INSULIN



GENE ENHANCER



PROTEIN ISL-2.


NM_000047
Homo sapiens
ARSE
1.37
0.11
2.45
1.03
0.59
0.05
3.18
1.23



arylsulfatase E



(chondrodysplasia



punctata 1) (ARSE),



mRNA.



/PROD=arylsulfatase E



precursor



/FL=gb:X83573.1



gb:NM_000047.1


NM_006379
Homo sapiens
SEMA3C
0.24
0.25
1.08
0.00
−0.34
0.05
0.86
0.18



sema domain,



immunoglobulin domain



(Ig), short basic



domain, secreted,



(semaphorin) 3C



(SEMA3C), mRNA.



/PROD=sema domain,



immunoglobulin



domain(Ig), shortbasic



domain, secreted,



(semaphorin) 3C



/FL=gb:NM_006379.1



gb:AB000220.1


NM_007127
Homo sapiens
VIL1
0.31
0.30
1.92
0.49
0.02
0.22
2.54
0.68



villin 1 (VIL1),



mRNA.



/PROD=villin 1



/FL=gb:NM_007127.1


U76549
Human cytokeratin 8
KRT8
5.41
0.16
5.87
0.56
3.79
0.05
6.34
0.57



mRNA, complete cds.



/PROD=cytokeratin 8



/FL=gb:BC000654.1



gb:U76549.1



gb:NM_002273.1



gb:M26324.1



gb:M34225.1


NM_004904
Homo sapiens
CREB5
0.92
0.23
1.84
0.37
0.00
0.06
1.21
0.80



cAMP response



element-binding



protein CRE-BPa



(H_GS165L15.1), mRNA.



/PROD=cAMP response



element-binding



protein CRE-BPa



/FL=gb:NM_004904.1



gb:L05911.1


AW082836
ESTs, Weakly
WNK4
−1.17
0.26
0.98
0.38
−0.97
0.20
0.05
0.86



similar to B34087



hypothetical



protein



(H. sapiens)


BE568134
death receptor 6
TNFRSF21
4.76
0.06
5.90
0.54
3.94
0.08
6.28
0.58



/FL=gb:NM_014452.1



gb:AF068868.1


NM_002845
Homo sapiens
PTPRM
2.50
0.10
3.43
0.46
1.34
0.19
3.76
0.50



protein tyrosine



phosphatase, receptor



type, M (PTPRM),



mRNA.



/PROD=protein tyrosine



phosphatase, receptor



type, mupolypeptide



/FL=gb:NM_002845.1


AI949419
ESTs
—
−0.11
0.21
1.72
0.64
−0.47
0.01
2.39
0.77


AK024680
Homo sapiens
NRP2
0.42
0.02
2.65
0.17
0.98
0.09
2.86
0.17



cDNA: FLJ21027



fis, clone CAE07110.



/FL=gb:NM_018534.1


BE542563
ESTs
LOC643277
2.20
0.10
0.54
1.49
−3.73
0.40
−1.74
1.96


AW005572
putative 47 kDa
ANKS1B
−1.21
0.52
−0.24
0.82
−1.19
0.55
1.05
0.64



protein


AW665892
paternally expressed 3
MFAP5
−3.87
0.82
−1.56
1.24
−2.40
0.24
−3.49
2.10


NM_006206
Homo sapiens
PDGFRA
0.92
0.18
2.34
0.74
0.72
0.09
3.18
0.62



platelet-derived



growth factor



receptor, alpha



polypeptide



(PDGFRA), mRNA.



/PROD=platelet-



derived growth factor



receptor,



alphapolypeptide



/FL=gb:NM_006206.1



gb:M21574.1


NM_002425
Homo sapiens
MMP10
−1.21
0.70
1.21
0.88
−0.37
0.28
0.52
0.45



matrix



metalloproteinase 10



(stromelysin 2)



(MMP10), mRNA.



/PROD=matrix



metalloproteinase 10



preproprotein



/FL=gb:BC002591.1



gb:NM_002425.1


NM_004338
Homo sapiens
C18orf1
−0.79
0.22
−0.09
0.39
−1.57
0.43
0.50
0.02



chromosome 18 open



reading frame 1



(C18ORF1), mRNA.



/PROD=chromosome 18



open reading frame 1



/FL=gb:NM_004338.1



gb:AF009426.1


AF052094
Homo sapiens
EPAS1
0.75
0.12
2.28
0.19
0.42
0.21
1.74
0.33



clone 23698 mRNA



sequence.



/FL=gb:U51626.1



gb:U81984.1



gb:NM_001430.1


BF126155
ESTs
S100A10
−0.32
0.25
1.24
0.28
−0.65
0.33
1.02
0.44


AI860212
phosphoprotein
PAG1
−0.17
0.22
1.37
0.28
−0.40
0.23
1.25
0.13



associated with GEMs



/FL=gb:AF240634.1



gb:NM_018440.1


AL110298
Homo sapiens
SLC2A14
5.13
0.06
6.03
0.58
3.97
0.24
6.33
0.73



mRNA; cDNA



DKFZp564K1672 (from



clone



DKFZp564K1672);



partial cds.



/PROD=hypothetical



protein


AY048775
Homo sapiens
MANEA
−0.47
0.40
0.71
0.90
−0.69
0.35
1.23
0.81



mandaselin long form



mRNA, complete cds.



/PROD=mandaselin long



form



/FL=gb:AY048775.1


M99436
Cluster Incl.
TLE2
2.65
0.07
4.05
0.66
1.95
0.13
4.76
0.61



M99436:Human



transducin-like



enhancer protein



(TLE2) mRNA,



complete cds



/cds=(25,2256)



/gb=M99436



/gi=307511



/ug=Hs.173063



/len=2271


NM_014061
Homo sapiens
MAGEH1
2.04
0.23
3.84
1.17
1.08
0.24
4.86
1.12



APR-1 protein



(APR-1), mRNA.



/PROD=APR-1 protein



/FL=gb:AF320912.1



gb:AF143235.3



gb:NM_014061.1


AL577531
caldesmon 1
CALD1
5.79
0.06
6.06
0.75
4.07
0.06
5.13
0.77



/FL=gb:M64110.1



gb:NM_004342.2


AI082237
proprotein
TAGLN
1.38
0.21
3.20
0.33
1.55
0.15
2.74
0.44



convertase



subtilisinkexin



type 7


BF055171
acyl-Coenzyme A
ACOX3
0.88
0.22
2.52
0.83
1.27
0.06
3.73
0.81



oxidase 3,



pristanoyl



/FL=gb:NM_003501.1


AF231124
Homo sapiens
SPOCK1
2.84
0.09
4.17
0.95
2.71
0.07
5.38
1.00



testican-1 mRNA,



complete cds.



/PROD=testican-1



/FL=gb:NM_004598.1



gb:AF231124.1


AA588092
ESTs
SLC40A1
−1.53
0.23
−0.94
0.51
−2.42
0.33
0.27
0.27


AK094809
Homo sapiens
RASGRF2
3.20
0.05
3.64
0.77
1.95
0.24
2.95
0.74



cDNA FLJ37490 fis,



clone



BRAWH2014934, highly



similar to



GUANINE NUCLEOTIDE



RELEASING PROTEIN.


NM_013959
Homo sapiens
NRG1
1.50
0.20
3.22
0.68
1.34
0.13
3.78
0.75



neuregulin 1



(NRG1), transcript



variant SMDF, mRNA.



/PROD=neuregulin 1



isoform SMDF



/FL=gb:L41827.1



gb:NM_013959.1


NM_004887
Homo sapiens
CXCL14
0.70
0.07
1.70
0.37
−0.15
0.11
2.36
0.63



small inducible



cytokine subfamily B



(Cys-X-Cys), member



14 (BRAK) (SCYB14),



mRNA.



/PROD=small inducible



cytokine subfamily



B(Cys-X-Cys),



member 14 (BRAK)



/FL=gb:AF144103.1



gb:AF106911.1



gb:AF073957.1



gb:BC003513.1



gb:NM_004887.1


T77995
Homo sapiens
—
−0.20
0.16
0.82
0.50
−1.70
0.24
1.17
0.06



cDNA FLJ13392 fis,



clone PLACE1001280


NM_030971
Homo sapiens
SFXN3
−0.43
0.59
−0.01
0.78
−1.92
0.17
−1.25
0.43



similar to rat



tricarboxylate



carrier-like protein



(BA108L7.2), mRNA.



/PROD=similar to rat



tricarboxylate



carrier-likeprotein



/FL=gb:NM_030971.1


H25097
KIAA1350 protein
USP53
3.25
0.05
4.57
0.20
2.96
0.04
4.47
0.13


NM_004932
Homo sapiens
CDH6
−3.58
1.16
−1.12
0.51
−2.14
0.63
−0.88
0.39



cadherin 6, type 2,



K-cadherin (fetal



kidney) (CDH6), mRNA.



/PROD=cadherin 6, type



2, K-cadherin



(fetal kidney)



/FL=gb:D31784.1



gb:NM_004932.1


N21426
hypothetical protein
SYTL2
2.41
0.09
3.44
1.22
1.70
0.06
2.02
1.21



FLJ20163


AA234096
KIAA0963 protein
MGC16121
0.38
0.16
1.74
0.63
−0.13
0.04
1.10
0.65


AV734843
hypothetical protein
OBFC2A
0.62
0.06
1.62
0.79
0.09
0.02
1.25
0.73



FLJ22833


AL519710
immunoglobulin
IGSF4
3.86
0.02
4.95
0.26
3.22
0.12
4.66
0.27



superfamily, member 4



/FL=gb:NM_014333.1



gb:AF132811.1


N32834
HIV-1 rev binding
—
−0.28
0.36
1.58
0.97
−0.23
0.18
0.33
1.17



protein 2


AF132811
Homo sapiens
IGSF4
2.23
0.07
3.85
0.28
2.17
0.10
3.58
0.34



nectin-like protein 2



(NECL2) mRNA,



complete cds.



/PROD=nectin-like



protein 2



/FL=gb:NM_014333.1



gb:AF132811.1


J04177
Cluster Incl.
COL11A1
3.28
0.22
4.44
1.22
2.32
0.03
3.03
1.31



J04177:Human alpha-1



type XI collagen



(COL11A1) mRNA,



complete cds



/cds=(161,5581)



/gb=J04177



/gi=179729



/ug=Hs.82772



/len=6158


AI982754
clusterin (complement
CLU
0.17
0.14
1.86
0.94
0.39
0.13
0.97
0.83



lysis inhibitor,



SP-40,40, sulfated



glycoprotein 2,



testosterone-



repressed prostate



message 2,



apolipoprotein J)


NM_003501
Homo sapiens
ACOX3
0.43
0.32
2.10
0.91
0.79
0.08
3.19
0.95



acyl-Coenzyme A



oxidase 3, pristanoyl



(ACOX3), mRNA.



/PROD=acyl-Coenzyme A



oxidase 3, pristanoyl



/FL=gb:NM_003501.1


AF144103
Homo sapiens
CXCL14
1.69
0.13
2.16
0.55
0.40
0.21
2.73
0.69



NJAC protein (NJAC)



mRNA, complete cds.



/PROD=NJAC protein



/FL=gb:AF144103.1



gb:AF106911.1



gb:AF073957.1



gb:BC003513.1



gb:NM_004887.1


NM_005451
Homo sapiens
PDLIM7
2.58
0.12
3.12
0.50
1.30
0.16
2.65
0.37



enigma (LIM domain



protein) (ENIGMA),



mRNA.



/PROD=enigma protein



/FL=gb:BC001093.1



gb:NM_005451.2



gb:AF265209.1


NM_004472
Homo sapiens
FOXD1
−0.85
0.80
2.04
0.55
0.71
0.23
1.70
0.74



forkhead box D1



(FOXD1), mRNA.



/PROD=forkhead box D1



/FL=gb:U59832.1



gb:NM_004472.1


AF332197
Homo sapiens
SIX2
0.36
0.24
0.72
0.12
−0.44
0.35
1.05
0.15



adult SIX2 (SIX2)



mRNA, complete cds.



/PROD=SIX2



/FL=gb:AF332197.1



gb:NM_016932.1



gb:AF136940.1


AB046817
Homo sapiens
SYTL2
2.88
0.17
4.09
1.09
2.50
0.05
3.12
1.03



mRNA for KIAA1597



protein, partial cds.



/PROD=KIAA1597



protein


AK093435
Homo sapiens
FLJ36116
4.75
0.02
4.59
1.53
2.46
0.18
6.15
1.46



cDNA FLJ36116 fis,



clone



TESTI2022338.


NM_004815
Homo sapiens
ARHGAP29
2.18
0.02
3.18
0.82
1.69
0.03
4.15
0.69



PTPL1-associated



RhoGAP 1 (PARG1),



mRNA.



/PROD=PTPL1-



associated RhoGAP 1



/FL=gb:U90920.1



gb:NM_004815.1


BG028597
ESTs
COL11A1
0.19
0.02
1.61
1.02
−0.11
0.38
0.48
1.09


AB019562
Homo sapiens
SPP1
0.58
0.14
2.79
1.07
1.10
0.02
1.21
1.06



mRNA expressed only



in placental villi,



clone SMAP41.


NM_002346
Homo sapiens
LY6E
3.54
0.06
3.92
1.16
1.81
0.23
4.93
1.39



lymphocyte antigen 6



complex, locus E



(LY6E), mRNA.



/PROD=lymphocyte



antigen 6 complex,



locus E



/FL=gb:U42376.1



gb:NM_002346.1



gb:U56145.1


BF589515
ESTs
TMEM16D
0.63
0.24
1.94
0.85
0.90
0.20
1.47
0.75


AL037401
nuclear receptor
NR2F2
−2.00
0.30
0.58
0.67
−2.19
0.13
0.82
0.66



subfamily 2, group F,



member 2



/FL=gb:M64497.1


NM_000783
Homo sapiens
CYP26A1
3.92
0.27
6.28
1.06
6.29
0.09
7.31
0.98



cytochrome P450,



subfamily XXVIA,



polypeptide 1



(CYP26A1), mRNA.



/PROD=cytochrome



P450, subfamily



XXVIA,



polypeptide 1



/FL=gb:NM_000783.1



gb:AF005418.1


U16307
Human glioma
GLIPR1
−0.11
0.03
1.16
0.95
−0.46
0.77
0.35
0.77



pathogenesis-related



protein (GliPR) mRNA,



complete cds.



/PROD=glioma



pathogenesis-related



protein



/FL=gb:NM_006851.1



gb:U16307.1


NM_001233
Homo sapiens
CAV2
4.07
0.07
4.42
0.90
2.82
0.08
3.44
0.85



caveolin 2 (CAV2),



mRNA.



/PROD=caveolin 2



/FL=gb:AF035752.1



gb:BC005256.1



gb:NM_001233.1


AA211909
ESTs
C20orf100
0.62
0.17
1.93
1.00
−0.21
0.11
0.72
0.99


AK057525
Homo sapiens
—
2.17
0.13
2.97
0.77
0.97
0.08
3.68
0.60



cDNA FLJ32963 fis,



clone



TESTI2008405.


BF344237
Homo sapiens
—
−2.82
0.20
−0.92
0.64
−0.73
0.67
−1.25
1.03



mRNA; cDNA



DKFZp564N1116 (from



clone



DKFZp564N1116)


NM_014481
Homo sapiens
APEX2
2.76
0.15
3.18
0.80
1.57
0.11
3.91
0.93



apurinicapyrimidinic



endonuclease(APEX



nuclease)-like 2



protein (APEXL2),



mRNA.



/PROD=apurini-



capyrimidinic endo-



nuclease(APEXnuclease)-



like 2 protein



/FL=gb:AB049211.1



gb:NM_014481.1



gb:BC002959.1



gb:AB021260.1



gb:AF119046.1


AI912583
HIV-1 rev binding
KRR1
1.10
0.23
2.68
1.04
1.31
0.06
1.53
1.01



protein 2


BI254089
Homo sapiens
ADAMTS5
−1.40
0.26
−0.01
0.31
−1.71
0.64
−0.79
0.52



full length insert



cDNA clone ZD50E03


BF197655
caveolin 2
CAV2
3.51
0.10
3.54
0.94
2.32
0.09
2.70
0.85



/FL=gb:AF035752.1



gb:BC005256.1



gb:NM_001233.1


NM_001955
Homo sapiens
EDN1
2.84
0.15
2.97
0.95
1.01
0.14
1.75
1.03



endothelin 1 (EDN1),



mRNA.



/PROD=endothelin 1



/FL=gb:NM_001955.1


NM_003319
Homo sapiens
TTN
1.28
0.19
0.62
0.86
−0.92
0.42
2.00
0.55



titin (TTN), mRNA.



/PROD=titin



/FL=gb:NM_003319.1


BE965029
Homo sapiens
MICAL2
0.60
0.21
1.95
0.64
0.21
0.19
0.83
0.78



cDNA: FLJ22463 fis,



clone



HRC10126


AI452457
ESTs
C1orf168
−1.48
0.53
0.02
0.93
−1.87
0.07
1.05
0.94


AI733465
collagen, type IX,
COL9A2
0.96
0.13
1.99
1.00
1.17
0.14
2.94
0.86



alpha 2



/FL=gb:NM_001852.1


NM_006103
Homo sapiens
WFDC2
4.02
0.11
5.11
1.05
3.39
0.04
6.19
1.10



epididymis-specific,



whey-acidic protein



type, four-disulfide



core; putative



ovarian carcinoma



marker (HE4), mRNA.



/PROD=epididymis-



specific, whey-acidic



protein type, four-



disulfide core;



putative ovarian



carcinoma marker



/FL=gb:NM_00610


NM_017540
Homo sapiens
GALNT10
1.46
0.18
2.68
0.78
0.97
0.23
3.47
0.82



hypothetical protein



DKFZp586H0623



(DKFZp586H0623),



mRNA.



/PROD=hypothetical



protein



DKFZp586H0623



/FL=gb:NM_017540.1


W72527
phosphoserine
SLC1A4
−0.65
0.09
0.38
0.80
−0.63
0.62
−0.72
0.09



aminotransferase


NM_003468
Homo sapiens
FZD5
1.35
0.26
3.43
1.21
1.45
0.10
4.43
1.16



frizzled (Drosophila)



homolog 5 (FZD5),



mRNA.



/PROD=frizzled 5



/FL=gb:NM_003468.1



gb:U43318.1


H15920
ESTs, Weakly similar
MRGPRF
2.34
0.18
3.62
1.02
2.11
0.11
2.87
0.84



to RTA RAT PROBABLE



G PROTEIN-COUPLED



RECEPTOR RTA



(R. norvegicus)


U83508
Human angiopoietin-1
ANGPT1
1.06
0.11
1.49
0.44
−0.32
0.27
0.32
0.79



mRNA, complete cds.



/PROD=angiopoietin-1



/FL=gb:NM_001146.1



gb:D13628.1



gb:U83508.1


AF043179
Homo sapiens
PRSS1
2.42
0.12
3.07
0.81
1.71
0.10
2.30
0.60



T cell receptor beta



chain (TCRBV13S1-



TCRBJ2S1) mRNA,



complete cds.



/PROD=T cell receptor



beta chain



/FL=gb:AF043179.1


AU157541
hypothetical protein
—
1.39
0.08
2.12
0.42
0.55
0.18
1.41
0.77



FLJ22833



/FL=gb:NM_022837.1


AF114264
Homo sapiens
NEXN
1.37
0.21
2.86
0.51
1.72
0.16
1.96
0.76



clone HH409 unknown



mRNA.



/PROD=unknown


BE965029
Homo sapiens
MICAL2
1.58
0.19
2.58
0.51
1.32
0.13
1.40
0.74



cDNA: FLJ22463 fis,



clone HRC10126


AB028976
Homo sapiens
SAMD4A
2.89
0.08
3.65
0.66
2.04
0.17
2.78
0.81



mRNA for KIAA1053



protein, partial cds.



/PROD=KIAA1053



protein


AI670862
ESTs, Weakly similar
FOSL2
0.17
0.21
2.05
0.57
0.45
0.16
1.18
0.65



to A49134 Ig kappa



chain V-I region



(H. sapiens)


L03203
Human peripheral
PMP22
0.37
0.24
1.95
0.84
0.31
0.26
2.77
0.91



myelin protein 22



(GAS3) mRNA,



complete cds.



/PROD=peripheral



myelin protein 22



/FL=gb:L03203.1


AI571798
Rho GDP dissociation
ARHGDIA
0.89
0.06
−0.54
0.26
−3.00
0.89
−2.58
0.87



inhibitor (GDI) alpha


X57348
Cluster Incl.
—
2.94
0.04
4.07
0.41
2.70
0.08
3.69
0.41



X57348: H. sapiens mRNA



(clone 9112)



/cds=(165,911)



/gb=X57348



/gi=23939



/ug=Hs.184510



/len=1407


AF051851
Homo sapiens
SVIL
2.03
0.18
2.28
0.62
1.13
0.20
1.71
0.69



supervillin mRNA,



complete cds.



/PROD=supervillin



/FL=gb:AF051851.1



gb:NM_003174.2



gb:AF051850.1


M95929
Human homeobox protein
—
0.07
0.43
1.27
0.58
−0.02
0.19
0.64
0.61



(PHOX1) mRNA, 3 end.



/PROD=homeobox



protein


BG251266
FOS-like antigen-1
FOSL1
1.94
0.06
2.41
0.46
1.25
0.05
1.93
0.36



/FL=gb:NM_005438.1


AW298375
ESTs
—
−0.40
0.30
0.46
0.63
−0.09
0.20
−0.58
1.07


NM_004362
Homo sapiens
CLGN
0.90
0.30
1.95
0.96
0.69
0.11
3.11
1.01



calmegin (CLGN),



mRNA.



/PROD=calmegin



/FL=gb:NM_004362.1



gb:D86322.1


AF001540
calcineurin-binding
—
0.10
0.15
2.84
0.70
0.42
0.44
1.64
0.39



protein calsarcin-1


NM_001191
Homo sapiens
BCL2L1
1.21
0.22
0.58
0.35
−1.89
0.35
−1.09
1.03



BCL2-like 1 (BCL2L1),



mRNA.



/PROD=BCL 2-like 1



/FL=gb:NM_001191.1


NM_003316
Homo sapiens
TTC3
3.90
0.15
5.15
0.98
3.68
0.12
6.19
0.97



tetratricopeptide



repeat domain 3



(TTC3), mRNA.



/PROD=tetra-



tricopeptide repeat



domain 3



/FL=gb:D84295.1



gb:NM_003316.1


L16895
Human lysyl
—
−0.66
0.24
0.53
0.14
−0.63
0.15
0.14
0.19



oxidase (LOX) gene,



exon 7


AI912976
ESTs
RASGRF2
3.13
0.08
4.05
0.56
2.66
0.16
3.54
0.64


NM_012242
Homo sapiens
DKK1
0.47
0.29
0.73
0.04
0.49
0.51
1.09
0.23



dickkopf



(Xenopus laevis)



homolog 1 (DKK1),



mRNA.



/PROD=dickkopf



(Xenopus laevis)



homolog 1



/FL=gb:AF177394.1



gb:NM_012242.1



gb:AF127563.1


AL096776
Human DNA sequence
—
1.94
0.07
3.41
0.16
2.40
0.07
3.40
0.14



from clone



RP4-646B12 on



chromosome



1q42.11-42.3.



Contains an FTH1



(ferritin, heavy



polypeptide 1)



(FTHL6) pseudogene,



the gene for a



novel Ras family



protein, ESTs, STSs,



GSSs and a putative



CpG island



/FL=gb:AF282258.1



gb:NM_0212


BC005997
Homo sapiens,
—
1.12
0.25
0.95
0.44
2.04
0.07
1.51
0.22



clone MGC:14801,



mRNA, complete cds.



/PROD=Unknown



(protein for



MGC:14801)



/FL=gb:BC005997.1


AF074979
Homo sapiens
RGS20
−0.52
0.18
−0.04
0.38
−1.30
0.09
−0.34
0.13



regulator of G protein



signaling-Z (RGSZ1)



mRNA, complete cds.



/PROD=regulator of G



protein signaling



/FL=gb:AF060877.2



gb:AF074979.1



gb:NM_003702.2


BF060767
ESTs
ADAMTS5
−0.36
0.15
0.80
0.33
0.22
0.14
0.40
0.56


AU151151
Homo sapiens
LEPR
1.72
0.18
2.38
0.80
1.85
0.10
1.71
0.69



cDNA FLJ13536 fis,



clone



PLACE1006521


L27624
Homo sapiens
TFPI2
3.77
0.06
3.57
0.77
2.59
0.11
2.44
0.89



tissue factor pathway



inhibitor-2 mRNA,



complete cds.



/PROD=tissue factor



pathway inhibitor-2



/FL=gb:D29992.1



gb:L27624.1



gb:NM_006528.1



gb:BC005330.1


NM_003174
Homo sapiens
SVIL
3.15
0.09
3.68
0.44
2.91
0.01
3.27
0.46



supervillin (SVIL),



transcript variant 1,



mRNA.



/PROD=supervillin,



isoform 1



/FL=gb:AF051851.1



gb:NM_003174.2



gb:AF051850.1


AF052127
Homo sapiens
RELN
−3.13
1.04
−1.36
0.26
−0.88
0.17
−0.40
0.11



clone 23850 mRNA



sequence.


AL031290
Human DNA sequence
—
0.38
0.06
1.39
0.46
0.40
0.05
0.95
0.44



from clone 774I24 on



chromosome



1q24.1-24.3



Contains protein



similar to pregnancy-



associated plasma



protein A precursor



neuronal migration



protein astrotactin,



ESTs, STS and GSS


AI129628
ESTs
SAMD3
−0.30
0.18
−0.01
0.25
0.72
0.06
0.17
0.21


NM_016206
Homo sapiens
VGLL3
−0.39
0.36
0.05
1.03
−0.31
0.29
−1.05
1.01



colon carcinoma



related protein



(LOC51159), mRNA.



/PROD=colon carcinoma



related protein



/FL=gb:NM_016206.1



gb:AF099505.1


BE348291
ESTs
—
1.62
0.06
−0.19
0.98
−3.65
0.10
−2.75
1.84


AW242720
Homo sapiens
LOC143381
−1.62
0.34
−1.20
0.94
−0.08
0.24
−0.08
0.77



cDNA FLJ10561 fis,



clone



NT2RP2002672


NM_001146
Homo sapiens
ANGPT1
1.86
0.06
2.09
0.81
1.10
0.16
0.87
1.05



angiopoietin 1



(ANGPT1), mRNA.



/PROD=angiopoietin 1



/FL=gb:NM_001146.1



gb:D13628.1



gb:U83508.1


AU152579
Homo sapiens
PCSK5
2.41
0.14
2.47
0.86
2.04
0.15
1.11
1.20



cDNA FLJ13034 fis,



clone



NT2RP3001232


NM_006200
Homo sapiens
PCSK5
3.01
0.21
2.88
0.92
2.28
0.11
1.52
1.25



proprotein convertase



subtilisinkexin type



5 (PCSK5), mRNA.



/PROD=proprotein



convertase



subtilisinkexin



type 5



/FL=gb:U56387.2



gb:NM_006200.1


BF342661
KIAA0036 gene product
MAP2
−0.77
0.30
−0.19
0.44
0.12
0.17
−2.21
1.64


AF063824
Homo sapiens
TRPC4
−3.03
0.39
0.05
0.49
1.28
0.17
0.21
0.92



trp-related protein 4



truncated variant



delta mRNA,



complete cds.



/PROD=trp-related



protein 4 truncated



variant delta



/FL=gb:AF063824.1


AA723810
cDNA for
LY6K
0.07
0.20
−0.13
1.23
0.15
0.14
−0.38
1.27



differentially



expressed CO16 gene



/FL=gb:BC001291.1


N29877
interleukin 14
—
1.94
0.13
0.51
0.48
0.78
0.64
−1.57
0.65


NM_007287
Homo sapiens
MME
2.59
0.03
1.81
0.40
1.89
0.21
2.14
0.30



membrane metallo-



endopeptidase (neutral



endopeptidase,



enkephalinase, CALLA,



CD10) (MME),



transcript variant



1bis, mRNA.



/PROD=membrane



metallo-endopeptidase



/FL=gb:NM_007288.1



gb:NM_007287.1



gb:J03779.1


AB050856
Homo sapiens
B3GALNT1
1.39
0.05
0.20
0.22
−0.13
0.21
−0.10
0.17



beta3GalNAcT-1 mRNA



for globoside



synthase, complete



cds, clone:type 2.



/PROD=globoside



synthase



/FL=gb:AB050856.1


AI827455
Homo sapiens
BCL6B
0.65
0.31
0.62
0.21
1.07
0.24
0.90
0.29



cDNA: FLJ21042 fis,



clone



CAE11204


AF017987
Homo sapiens
SFRP1
4.15
0.22
4.37
1.24
6.02
0.06
5.74
1.01



secreted apoptosis



related protein 2



(SARP2) mRNA,



complete cds.



/PROD=secreted



apoptosis related



protein 2



/FL=gb:AF056087.1



gb:NM_003012.2



gb:AF017987.1



gb:AF001900.1


AL117451
Homo sapiens
C1orf108
−0.73
0.20
0.68
0.24
1.06
0.05
0.82
0.21



mRNA; cDNA



DKFZp586E2317 (from



clone



DKFZp586E2317).


AW268357
ESTs, Highly similar
USP32
0.73
0.20
0.36
0.19
0.70
0.03
0.09
0.18



to AF155116



1 NY-REN-60



antigen



(H. sapiens)


BE465243
ESTs
ARFGEF1
0.50
0.23
0.16
0.32
1.03
0.08
0.47
0.06


AA993400
ESTs
ADAL
0.89
0.22
0.48
0.28
0.83
0.10
0.29
0.43


AI970898
Cluster Incl.
ACACB
0.69
0.15
0.51
0.53
1.46
0.11
−0.87
0.83



AI970898:wr



21c03.x1



Homo sapiens



cDNA, 3 end/



clone=IMAGE-2488324



/clone_end=3



/gb=AI970898



/gi=5767724



/ug=Hs.234898



/len=382


BG026457
ESTs, Weakly
KIAA1909
0.60
0.07
0.23
0.59
0.98
0.03
−2.11
1.28



similar to



ALU5_HUMAN ALU



SUBFAMILY SC



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


BC041933
Homo sapiens,
UBE3C
0.83
0.23
0.11
0.51
0.55
0.05
0.41
0.92



clone



IMAGE:5300703, mRNA.


AI638611
KIAA1373 protein
STAMBPL1
2.20
0.17
1.37
0.58
2.32
0.08
−0.39
1.24


AI341686
ESTs, Highly similar to
MTRF1
1.36
0.14
0.45
0.48
1.43
0.09
−1.09
0.99



RF1M_HUMAN



MITOCHONDRIAL



PEPTIDE CHAIN



RELEASE FACTOR 1



PRECURSOR



(H. sapiens)


NM_003182
Homo sapiens
TAC1
1.89
0.20
1.65
0.13
2.46
0.09
1.48
0.26



tachykinin, precursor



1 (substance K,



substance P,



neurokinin 1,



neurokinin 2,



neuromedin L,



neurokinin alpha,



neuropeptide K,



neuropeptide gamma)



(TAC1), transcript



variant beta, mRNA.



/PROD=tachykinin 2



precursor, isoform



beta



/FL=gb:U3


M80634
Human keratinocyte
FGFR2
3.63
0.06
2.25
0.39
2.79
0.19
2.07
0.84



growth factor



receptor mRNA,



complete cds.



/PROD=keratinocyte



growth factor



receptor



/FL=gb:M80634.1



gb:NM_022969.1



gb:M97193.1


AK021452
Homo sapiens
ZNF521
−0.20
0.16
−1.15
1.12
1.31
0.05
0.36
0.18



cDNA FLJ11390 fis,



clone



HEMBA1000561, weakly



similar to ZINC



FINGER PROTEIN 91.


AA541622
ESTs
SYNPO2
−0.49
0.30
−0.90
0.06
0.35
0.09
−2.58
0.93


N50714
ESTs
—
−1.13
0.13
−1.34
0.52
0.10
0.12
−0.21
0.28


NM_002674
Homo sapiens
PMCH
0.62
0.24
1.10
0.48
1.64
0.03
0.09
0.80



pro-melanin-



concentrating hormone



(PMCH), mRNA.



/PROD=pro-melanin-



concentrating hormone



/FL=gb:NM_002674.1



gb:M57703.1


BM666010
Homo sapiens
LOC200169
1.36
0.16
0.45
0.74
1.49
0.12
−1.65
1.41



cDNA FLJ23803 fis,



clone



HEP22811.


AI343467
Homo sapiens
—
−0.44
0.08
−1.50
0.82
1.11
0.19
−0.64
0.20



cDNA FLJ11041 fis,



clone



PLACE1004405


AA046424
ESTs, Weakly
ACOT4
0.58
0.27
−0.94
0.66
−1.08
0.38
−1.09
0.25



similar to



YZ28_HUMAN



HYPOTHETICAL



PROTEIN ZAP128



(H. sapiens)


R38389
olfactomedin related
OLFM1
4.27
0.11
3.02
0.76
4.53
0.07
2.17
0.96



ER localized protein


BF724270
ESTs
—
2.03
0.22
0.72
0.42
2.36
0.06
0.01
0.97


NM_001819
Homo sapiens
CHGB
1.40
0.18
0.50
0.75
1.66
0.05
−0.74
1.03



chromogranin B



(secretogranin 1)



(CHGB), mRNA.



/PROD=chromogranin B



precursor



/FL=gb:BC000375.1



gb:NM_001819.1


AK026387
Homo sapiens
—
0.03
0.28
−0.05
0.52
1.23
0.26
0.52
0.67



cDNA: FLJ22734 fis,



clone



HUV00109.


BF062139
polymerase (RNA) III
—
5.23
0.06
4.31
0.74
5.80
0.07
3.37
0.86



(DNA directed) (32 kD)



/FL=gb:NM_006467.1



gb:U93868.1


BG540454
ESTs
SCGB3A2
4.64
0.02
2.91
0.84
4.13
0.07
1.72
0.83


AI659533
ArgAbl-interacting
SORBS2
0.02
0.12
1.12
0.71
2.17
0.05
−1.08
1.48



protein ArgBP2


AA531287
ESTs
—
2.94
0.27
1.32
0.72
2.57
0.04
0.22
0.90


NM_013243
Homo sapiens
SCG3
3.30
0.20
2.13
0.95
3.67
0.19
0.87
1.06



secretogranin III



(SCG3), mRNA.



/PROD=secretogranin



III



/FL=gb:AF078851.1



gb:NM_013243.1


AI307586
Homo sapiens
C10orf95
0.34
0.22
−1.15
0.24
−0.49
0.16
−3.61
0.99



mRNA; cDNA



DKFZp566H0124 (from



clone



DKFZp566H0124)


BC032004
Homo sapiens,
GRIA3
−0.02
0.23
−0.15
0.33
0.47
0.04
0.17
0.07



Similar to glutamate



receptor, ionotrophic,



AMPA3, clone



IMAGE:4753474, mRNA.


AW205739
ESTs, Weakly
TYW3
0.47
0.01
0.81
1.35
3.72
0.18
2.46
1.15



similar to ORF



YGL050w



(S. cerevisiae)


NM_153262
Homo sapiens
SYT14
−0.01
0.17
−0.84
0.51
0.00
0.18
−3.88
1.64



hypothetical protein



FLJ34198 (FLJ34198),



mRNA.



/FL=gb:NM_153262.1


AA156873
albumin
PELO
0.77
0.12
−0.23
0.93
0.65
0.14
−2.33
1.01


BC012375
Homo sapiens,
ARSG
−0.46
0.48
−0.78
0.42
0.57
0.22
−0.41
0.55



Similar to KIAA1001



protein, clone



MGC:8996



IMAGE:3882163, mRNA,



complete cds.



/PROD=Similar to



KIAA1001 protein



/FL=gb:AB023218.1



gb:NM_014960.1



gb:BC012375.1


AA843242
ESTs
BNC2
2.12
0.37
1.03
0.47
2.89
0.06
0.43
0.80


BF792954
ESTs
HDLBP
−1.53
1.04
−2.75
0.91
0.30
0.16
−0.72
0.26


AA780067
heparan sulfate
HS3ST3B1
0.41
0.19
0.30
0.61
2.61
0.18
0.78
0.60



(glucosamine) 3-O-



sulfotransferase 3B1


AA909330
ESTs
RP1-32F7.2
0.98
0.17
−1.67
0.51
0.79
0.02
−0.38
0.12


AF141339
Homo sapiens
ZNF521
−0.41
0.12
−0.97
0.93
1.67
0.11
−0.31
0.80



LYST-interacting



protein LIP3 mRNA,



partial cds.



/PROD=LYST-



interacting



protein LIP3


BF435123
bromodomain and
—
2.28
0.17
1.31
0.51
2.70
0.12
1.30
1.00



PHD finger



containing, 3


AK056212
Homo sapiens
—
1.03
0.14
0.14
0.27
1.10
0.07
−1.41
0.51



cDNA FLJ31650 fis,



clone



NT2RI2004079.


NM_001446
Homo sapiens
FABP7
2.07
0.15
0.74
0.29
2.20
0.04
1.08
0.16



fatty acid binding



protein 7, brain



(FABP7), mRNA.



/PROD=fatty acid



binding protein 7,



brain



/FL=gb:U81235.1



gb:D88648.1



gb:U51338.1



gb:NM_001446.1



gb:D50373.1


AW051591
ESTs, Moderately
RNF175
1.41
0.32
0.22
0.10
2.60
0.06
0.96
0.20



similar to unnamed



protein product



(H. sapiens)


BC041970
Homo sapiens,
C9orf122
0.74
0.03
−0.35
0.49
0.76
0.06
−2.31
1.25



clone



IMAGE:5302687, mRNA.


BC013077
Homo sapiens,
—
2.51
0.11
0.80
1.15
2.85
0.06
0.08
0.89



clone



IMAGE:3459334, mRNA.


AW572379
ESTs
—
1.80
0.03
0.60
0.84
1.45
0.07
1.88
0.53


BE644917
nuclear receptor
XIST
−3.88
1.26
−0.54
1.28
1.94
0.13
−0.20
1.94



subfamily 1,



group I, member 3


NM_171999
Homo sapiens
SALL3
2.97
0.18
2.04
0.71
3.79
0.08
1.02
0.74



sal-like 3



(Drosophila) (SALL3),



mRNA.



/PROD=sal-like 3



/FL=gb:NM_171999.1


AI654224
ESTs
—
0.46
0.36
0.14
0.44
1.03
0.07
−0.81
0.85


AA167449
nuclear receptor
XIST
−3.08
0.10
−0.47
2.59
4.59
0.07
2.80
1.86



subfamily 1, group I,



member 3


BF977837
KIAA0527 protein
SUSD5
1.64
0.02
0.50
0.68
1.96
0.16
−1.52
1.66


BC029425
Homo sapiens,
FILIP1
−0.83
0.45
−0.03
0.62
0.76
0.20
−0.29
0.44



Similar to KIAA1275



protein, clone



IMAGE:4616553, mRNA.


AI978754
ESTs
—
2.76
0.14
1.54
0.57
3.57
0.06
2.31
0.69


AA628440
nuclear receptor
XIST
0.18
0.13
1.42
1.52
4.80
0.02
3.01
1.50



subfamily 1, group I,



member 3


L36861
L36861
—
2.75
0.17
2.01
0.82
3.63
0.09
3.00
0.97



/FEATURE= expanded_cds



/DEFINITION=HUMGCAPB



Homo sapiens



guanylate cyclase



activating protein



(GCAP) gene exons



1-4, complete cds


AW023227
ESTs
MKX
−0.91
0.60
−1.31
0.06
0.27
0.13
−2.90
0.52


NM_021614
Homo sapiens
KCNN2
3.45
0.13
2.52
0.83
4.40
0.14
1.54
0.80



potassium



intermediate small



conductance calcium-



activated channel,



subfamily N, member 2



(KCNN2), mRNA.



/PROD=potassium



intermediate small



conductance calcium-



activated channel,



subfamily N, member 2



/FL=gb:NM_021614.1



gb:AF239613.1


NM_000956
Homo sapiens
PTGER2
−0.71
0.71
−0.73
0.57
0.63
0.33
−2.38
0.61



prostaglandin E



receptor 2 (subtype



EP2), 53 kD (PTGER2),



mRNA.



/PROD=prostaglandin E



receptor 2 (subtype



EP2), 53 kD



/FL=gb:U19487.1



gb:NM_000956.1


NM_013381
Homo sapiens
TRHDE
1.96
0.16
0.25
0.36
1.81
0.03
0.82
0.23



thyrotropin-releasing



hormone degrading



ectoenzyme (TRHDE),



mRNA.



/PROD=thyrotropin-



releasing hormone



degradingectoenzyme



/FL=gb:AF126372.1



gb:NM_013381.1


NM_016354
Homo sapiens
SLCO4A1
2.18
0.06
1.01
0.37
2.35
0.05
−0.91
1.23



solute carrier family



21 (organic anion



transporter),



member 12



(SLC21A12), mRNA.



/PROD=organic anion



transporter OATP-E



/FL=gb:AB031051.1



gb:NM_016354.1



gb:AF205072.1



gb:AF187817.1


BC028359
Homo sapiens,
ZNF141
−1.05
0.39
−0.59
0.65
1.25
0.17
−1.48
0.72



clone



IMAGE:4828836, mRNA.


AI193252
ESTs, Weakly
LRRN6A
4.38
0.11
2.47
0.43
4.23
0.11
1.90
0.37



similar to



AF133270 1 SLIT2



(H. sapiens)


H09780
Human
—
2.04
0.19
0.56
0.07
2.50
0.11
0.12
0.40



(clone CTG-A4) mRNA



sequence


BC040605
Homo sapiens,
—
2.64
0.16
1.61
1.08
3.59
0.09
0.45
1.14



clone



IMAGE:5271039, mRNA.


AW057589
ESTs
—
−0.96
0.26
−2.77
0.81
0.25
0.24
−1.21
0.35


M31213
Human papillary
RET
0.42
0.10
−1.86
0.83
1.77
0.12
−0.64
0.15



thyroid carcinoma-



encoded protein



mRNA, complete cds.



/FL=gb:M31213.1


Z92546
Human DNA sequence
—
0.58
0.13
−1.16
0.54
1.07
0.12
−1.27
0.14



from clone CTA-65A6



on chromosome



22q11-12



Contains the 3 part



of the gene for the



ortholog of rat CAIN



(KIAA0330), the gene



for a novel Sushi



domain (SCR repeat)



containing protein



similar to Mucins,



ESTs, an STS, GSSs



and two . . .


AA974416
protein phosphatase
PPP2R2B
4.02
0.16
3.52
0.23
5.22
0.07
3.12
0.15



2 (formerly 2A),



regulatory subunit B



(PR 52), beta



isoform


AW138143
ESTs
SORBS2
3.44
0.16
2.89
0.88
4.47
0.10
1.88
1.15


NM_014862
Homo sapiens
ARNT2
1.29
0.08
−0.68
0.97
2.00
0.08
0.02
0.24



KIAA0307 gene product



(KIAA0307), mRNA.



/PROD=KIAA0307 gene



product



/FL=gb:AB002305.1



gb:NM_014862.1


AI765540
ESTs
—
1.28
0.22
0.07
0.39
1.50
0.07
−0.11
0.26


NM_018013
Homo sapiens
FLJ10159
1.30
0.21
0.38
0.39
2.89
0.02
0.62
0.21



hypothetical protein



FLJ10159 (FLJ10159),



mRNA.



/PROD=hypothetical



protein FLJ10159



/FL=gb:NM_018013.1


BF382322
ESTs, Weakly
—
−0.40
0.09
−2.09
0.67
−0.12
0.08
−1.73
0.50



similar to unnamed



protein product



(H. sapiens)


AV699347
nuclear receptor
XIST
−1.37
0.38
0.45
1.69
4.28
0.02
2.25
1.53



subfamily 1,



group I, member 3


BC011549
Homo sapiens,
ATP5S
0.69
0.19
0.96
0.68
2.57
0.29
−0.02
0.52



clone MGC:19945



IMAGE:4554461, mRNA,



complete cds.



/PROD=Unknown



(protein for



MGC:19945)



/FL=gb:BC011549.1


NM_001889
Homo sapiens
CRYZ
−1.68
0.26
0.23
1.38
3.75
0.07
1.78
1.11



crystallin, zeta



(quinone reductase)



(CRYZ), mRNA.



/PROD=crystallin,



zeta (quinone



reductase)



/FL=gb:NM_001889.1



gb:L13278.1



gb:S58039.1


BC002665
Homo sapiens,
PLP1
4.57
0.15
2.95
0.64
4.70
0.06
2.32
0.52



proteolipid protein



(Pelizaeus-Merzbacher



disease, spastic



paraplegia 2,



uncomplicated), clone



MGC:3940, mRNA,



complete cds.



/PROD=proteolipid



protein (Pelizaeus-



Merzbacherdisease,



spastic paraplegia 2,



uncomplicated)



/FL=gb:BC002665.1


NM_001243
Homo sapiens
TNFRSF8
3.75
0.03
1.45
0.35
3.24
0.11
0.89
0.46



tumor necrosis factor



receptor superfamily,



member 8 (TNFRSF8),



mRNA.



/PROD=CD30 antigen



(Ki-1 antigen)



/FL=gb:NM_001243.1



gb:D86042.1



gb:M83554.1


NM_001195
Homo sapiens
BFSP1
0.57
0.33
−1.65
1.30
0.60
0.14
−0.86
0.73



beaded filament



structural protein 1,



filensin (BFSP1),



mRNA.



/PROD=filensin



/FL=gb:AF039655.1



gb:NM_001195.2



gb:Y16717.2


NM_024582
Homo sapiens
FAT4
2.16
0.27
−0.49
0.30
1.54
0.28
−1.35
1.01



hypothetical protein



FLJ23056 (FLJ23056),



mRNA.



/PROD=hypothetical



protein FLJ23056



/FL=gb:NM_024582.1


NM_000767
Homo sapiens
CYP2B6
2.60
0.18
0.87
0.96
1.47
0.12
−0.69
1.03



cytochrome P450,



subfamily IIB



(phenobarbital-



inducible),



polypeptide 6



(CYP2B6), mRNA.



/PROD=cytochrome



P450, subfamily IIB



(phenobarbital-



inducible),



polypeptide 6



/FL=gb:NM_000767.2



gb:AF182277.1



gb:M29874.1


AW665509
ESTs
MGC42174
−0.10
0.33
−0.87
0.72
1.20
0.08
−2.60
1.40


NM_001104
Homo sapiens
ACTN3
2.12
0.13
1.64
0.28
3.35
0.03
1.67
0.36



actinin, alpha 3



(ACTN3), mRNA.



/PROD=skeletal muscle



specific actinin,



alpha 3



/FL=gb:M86407.1



gb:NM_001104.1


NM_021069
Homo sapiens
SORBS2
−0.49
0.43
0.30
1.36
2.03
0.07
−1.13
1.15



ArgAbl-interacting



protein ArgBP2



(ARGBP2), transcript



variant 2, mRNA.



/PROD=ArgAbl-



interacting



protein 2, isoform 2



/FL=gb:AB018320.1



gb:NM_021069.1


T65020
ESTs
—
0.51
0.16
−0.84
0.26
1.10
0.15
−1.50
0.38


NM_152647
Homo sapiens
GALK2
0.08
0.56
−1.42
0.52
0.03
0.17
−2.34
0.91



hypothetical protein



FLJ32800 (FLJ32800),



mRNA.



/FL=gb:NM_152647.1


BC036917
Homo sapiens,
C6orf141
−0.82
0.30
−1.18
0.40
0.88
0.11
−2.18
0.62



clone MGC:46457



IMAGE:5201433, mRNA,



complete cds.



/PROD=Unknown



(protein for



MGC:46457)



/FL=gb:BC036917.1


AI969112
Homo sapiens,
PHIP
−1.07
0.13
−2.88
0.91
−0.37
0.02
−2.23
0.33



clone



IMAGE:5260603, mRNA,



partial cds


AW449813
KIAA0918 protein
SLITRK5
0.32
0.26
−1.89
0.50
0.46
0.10
−0.57
0.13


AW044658
ESTs
—
0.16
0.16
−1.47
0.25
0.90
0.14
−0.89
0.16


AI694300
ESTs
—
−0.52
0.02
−1.34
0.52
0.27
0.08
−1.00
0.08


NM_017631
Homo sapiens
FLJ20035
1.35
0.13
0.05
0.19
1.51
0.17
0.32
0.15



hypothetical protein



FLJ20035 (FLJ20035),



mRNA.



/PROD=hypothetical



protein FLJ20035



/FL=gb:NM_017631.1


AF298547
Homo sapiens
NALP2
1.54
0.08
1.22
1.61
5.04
0.05
3.23
1.36



nucleotide-binding



site protein 1



mRNA, complete cds.



/PROD=nucleotide-



binding site



protein 1



/FL=gb:AF298547.1


NM_030631
Homo sapiens
SLC25A21
1.96
0.25
0.33
0.81
2.47
0.11
−0.28
0.63



oxodicarboxylate



carrier (ODC1),



mRNA.



/PROD=oxodi-



carboxylate carrier



/FL=gb:NM_030631.1


BF196255
ESTs
—
2.94
0.05
1.18
0.43
3.15
0.08
0.54
0.57


NM_003247
Homo sapiens
THBS2
2.62
0.20
1.04
0.28
2.70
0.04
0.27
0.45



thrombospondin 2



(THBS2), mRNA.



/PROD=thrombospondin 2



/FL=gb:NM_003247.1



gb:L12350.1


NM_001446
Homo sapiens
FABP7
0.95
0.09
−0.75
0.37
0.20
0.22
−1.73
0.99



fatty acid binding



protein 7, brain



(FABP7), mRNA.



/PROD=fatty acid



binding protein 7,



brain



/FL=gb:U81235.1



gb:D88648.1



gb:U51338.1



gb:NM_001446.1



gb:D50373.1


AW004016
ESTs
ST6GAL2
1.57
0.14
0.89
0.32
3.30
0.07
1.48
0.43


AW072790
contactin 1
CNTN1
0.50
0.20
0.18
0.98
1.84
0.10
1.01
1.39


AL512686
Homo sapiens
GNAO1
2.16
0.50
−0.28
1.03
1.67
0.18
−1.12
0.37



mRNA; cDNA



DKFZp761I177 (from



clone



DKFZp761I177).


AI638063
ESTs
CBX5
0.49
0.16
−0.34
0.34
0.98
0.11
−2.71
1.22


AU157049
Homo sapiens
LOC153346
2.24
0.20
0.34
0.47
2.24
0.09
−0.44
0.96



cDNA FLJ14284 fis,



clone



PLACE1005898


NM_002738
Homo sapiens
PRKCB1
0.99
0.28
−0.15
0.45
1.42
0.08
−1.02
1.04



protein kinase C,



beta 1 (PRKCB1),



mRNA.



/PROD=protein kinase C,



beta 1



/FL=gb:NM_002738.1


AI185136
ESTs
DYDC2
0.12
0.31
−0.80
0.08
0.72
0.08
−0.60
0.09


AI928037
ESTs
RPIB9
−0.55
0.55
−2.71
0.74
0.49
0.12
−0.89
0.27


NM_016179
Homo sapiens
TRPC4
−1.51
0.85
−1.87
1.42
1.71
0.11
−0.13
1.23



transient receptor



potential channel 4



(TRPC4), mRNA.



/PROD=transient



receptor potential 4



/FL=gb:NM_016179.1



gb:AF175406.1


AW138143
ESTs
SORBS2
3.29
0.13
2.35
1.05
4.28
0.10
1.32
1.07


NM_001876
Homo sapiens
CPT1A
1.63
0.18
−0.15
0.21
1.32
0.07
−0.20
0.33



carnitine



palmitoyltransferase I,



liver (CPT1A),



nuclear gene



encoding mitochondrial



protein, mRNA.



/PROD=liver carnitine



palmitoyltransferase I



/FL=gb:L39211.1



gb:NM_001876.1


AA903862
ESTs
C20orf54
1.61
0.15
−1.17
0.30
0.68
0.21
−1.49
0.23


AI670947
phosphatidyl-
—
3.91
0.05
0.55
1.54
3.33
0.10
1.17
0.68



inositol-4-phosphate



5-kinase, type I, beta


AV648405
polymerase (RNA) III
—
−0.01
0.11
0.23
0.52
1.73
0.14
−1.23
1.15



(DNA directed) (32 kD)


BF435123
bromodomain and PHD
—
1.80
0.13
−1.35
1.01
1.14
0.03
0.37
0.24



finger containing, 3


NM_016582
Homo sapiens
SLC15A3
1.77
0.16
−0.88
1.35
1.30
0.23
−2.04
1.23



peptide transporter 3



(LOC51296), mRNA.



/PROD=peptide



transporter 3



/FL=gb:NM_016582.1



gb:AB020598.1


AI830490
glycerol kinase
GK
0.61
0.01
−0.65
0.79
1.25
0.20
−0.38
0.33


AW 134979
HSPC156 protein
STXBP6
2.28
0.03
0.55
0.28
2.05
0.03
0.30
0.44


AI354636
ESTs
—
2.81
0.11
0.68
1.17
3.02
0.20
0.19
0.76


BE672659
ESTs
—
0.20
0.64
−0.43
0.61
1.98
0.12
−0.69
0.45


AF284095
Homo sapiens
ADRA2A
0.63
0.21
−0.74
0.98
1.85
0.03
0.55
0.31



alpha-2A adrenergic



receptor mRNA,



complete cds.



/PROD=alpha-2A



adrenergic receptor



/FL=gb:AF284095.1



gb:NM_000681.1


NM_021136
Homo sapiens
RTN1
−0.30
0.03
−1.39
0.35
0.80
0.07
−1.23
0.39



reticulon 1 (RTN1),



mRNA.



/PROD=reticulon 1



/FL=gb:L10333.1



gb:L10334.1



gb:NM_021136.1


NM_014729
Homo sapiens
TOX
0.34
0.03
−0.46
0.12
0.90
0.18
−1.86
0.40



KIAA0808 gene product



(KIAA0808), mRNA.



/PROD=KIAA0808 gene



product



/FL=gb:AB018351.1



gb:NM_014729.1


BE674118
ESTs
—
0.78
0.07
−1.32
0.73
0.28
0.19
−0.96
0.74


BC026969
Homo sapiens,
WDR67
2.95
0.11
0.75
0.92
3.08
0.08
0.45
0.61



clone



IMAGE:5116073, mRNA,



partial cds.


AF131783
Homo sapiens
PAP2D
−0.42
0.26
−2.00
1.05
0.31
0.22
−1.56
0.70



clone 25181 mRNA



sequence.


U11058
Homo sapiens
KCNMA1
1.56
0.09
−0.16
1.08
2.29
0.18
−0.79
0.86



large conductance



calcium- and voltage-



dependent potassium



channel alpha



subunit (MaxiK)



mRNA, complete cds.



/PROD=large



conductance



calcium- and voltage-



dependentpotassium



channel alpha



subunit



/FL=gb:U23767.1



gb:NM_002247.1



gb:AF025999


BF510715
fibroblast growth
—
2.67
0.27
0.84
0.17
3.29
0.01
0.64
0.51



factor 4 (heparin



secretory transforming



protein 1, Kaposi



sarcoma oncogene)



/FL=gb:M17446.1



gb:NM_002007.1


BE897866
ESTs
ACADSB
1.41
0.23
0.08
0.34
3.10
0.10
−0.27
0.88


AI735586
ESTs
LOC152573
0.60
0.13
0.00
1.14
2.84
0.07
1.12
1.30


AL573058
complement component
C1R
0.92
0.14
−1.67
0.38
0.73
0.02
−0.96
0.77



1, r subcomponent


AF429305
Homo sapiens
RMST
−0.48
0.03
−2.49
0.51
−0.25
0.20
−1.89
0.27



C23up NCRMS mRNA,



partial sequence;



alternatively



spliced.


BF195118
ESTs, Weakly
ATP5J
0.09
0.13
−0.92
0.42
1.57
0.16
−0.53
0.08



similar to



ALU7_HUMAN ALU



SUBFAMILY SQ



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


NM_005460
Homo sapiens
SNCAIP
1.76
0.16
−0.84
0.52
1.61
0.19
−0.92
0.88



synuclein, alpha



interacting protein



(synphilin)



(SNCAIP), mRNA.



/PROD=synuclein alpha



interacting protein



/FL=gb:AF076929.1



gb:NM_005460.1


NM_024893
Homo sapiens
C20orf39
1.16
0.29
−0.56
0.42
2.36
0.15
−0.06
0.17



hypothetical



protein FLJ14220



(FLJ14220), mRNA.



/PROD=hypothetical



protein FLJ14220



/FL=gb:NM_024893.1


NM_022034
Homo sapiens
CUZD1
1.31
0.08
0.80
0.28
2.78
0.07
0.24
0.58



estrogen regulated



gene 1 (ERG-1),



mRNA.



/PROD=estrogen



regulated gene 1



/FL=gb:AF305835.1



gb:NM_022034.1


NM_014788
Homo sapiens
TRIM14
3.12
0.09
1.22
0.59
3.54
0.08
1.02
0.45



KIAA0129 gene product



(KIAA0129), mRNA.



/PROD=KIAA0129 gene



product



/FL=gb:D50919.1



gb:NM_014788.1


AV646597
ESTs, Weakly
XIST
−0.77
0.18
0.06
1.55
3.78
0.25
2.28
0.98



similar to



ALU7_HUMAN ALU



SUBFAMILY SQ



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


BF062629
DKFZP586E1621 protein
TMEM158
4.00
0.19
2.41
0.27
4.87
0.00
1.93
0.19


AW440492
ATPase, Na+K+
ATP1A2
1.33
0.07
−2.12
1.07
1.27
0.06
−0.53
0.11



transporting,



alpha 2 (+)



polypeptide



/FL=gb:NM_000702.1


AF283777
Homo sapiens
CD72
−0.08
0.45
−1.88
1.19
0.41
0.24
−1.42
1.13



clone



TCBAP0702



mRNA sequence.


NM_005375
Homo sapiens
MYB
2.11
0.17
−0.52
0.18
2.24
0.03
0.10
0.08



v-myb avian



myeloblastosis viral



oncogene homolog



(MYB), mRNA.



/PROD=v-myb avian



myeloblastosis viral



oncogenehomolog



/FL=gb:NM_005375.1



gb:AF104863.1



gb:M15024.1


NM_017671
Homo sapiens
C20orf42
3.19
0.12
0.97
0.70
3.26
0.07
0.16
0.94



hypothetical protein



FLJ20116 (FLJ20116),



mRNA.



/PROD=hypothetical



protein FLJ20116



/FL=gb:NM_017671.1


BG283790
ESTs
MATR3
2.27
0.11
0.95
0.57
3.53
0.04
0.89
0.51


NM_000702
Homo sapiens
ATP1A2
2.12
0.17
0.76
0.38
2.93
0.16
0.51
0.12



ATPase, Na+K+



transporting, alpha



2 (+) polypeptide



(ATP1A2), mRNA.



/PROD=ATPase, Na+K+



transporting,



alpha 2



(+)polypeptide



/FL=gb:NM_000702.1


NM_025135
Homo sapiens
FHOD3
1.02
0.18
0.80
0.40
2.34
0.10
−0.77
0.72



hypothetical protein



FLJ22297 (KIAA1695),



mRNA.



/PROD=hypothetical



protein KIAA1695



/FL=gb:NM_025135.1


NM_012281
Homo sapiens
KCND2
1.67
0.30
0.05
0.15
2.70
0.09
−0.26
0.38



potassium voltage-



gated channel,



ShaI-related



subfamily, member 2



(KCND2), mRNA.



/PROD=potassium



voltage-gated channel,



ShaI-relatedsubfamily,



member 2



/FL=gb:NM_012281.1



gb:AB028967.1



gb:AF121104.1


BF449063
collagen, type XIV,
COL14A1
1.61
0.15
0.30
0.38
2.68
0.17
−0.33
0.68



alpha 1 (undulin)


AA280904
ESTs
C9orf39
0.23
0.21
−1.58
1.26
1.13
0.14
−0.78
0.42


NM_022467
Homo sapiens
CHST8
2.11
0.20
−0.51
0.30
2.17
0.10
0.48
0.02



N-acetylgalactos-



amine-4-O-sulfo-



transferase



(GALNAC-4-ST1),



mRNA.



/PROD=N-acetyl-



galactosamine-4-O-



sulfotransferase



/FL=gb:NM_022467.1



gb:AF300612.1


BE468066
ESTs
RMST
3.26
0.09
1.49
0.10
3.80
0.09
1.63
0.08


AL120674
ESTs
—
1.00
0.11
−1.35
1.73
1.55
0.15
−2.20
0.86


NM_133329
Homo sapiens
KCNG3
2.18
0.27
0.74
0.29
3.67
0.01
0.26
0.28



potassium voltage-



gated channel,



subfamily G, member 3



(KCNG3), transcript



variant 1, mRNA.



/PROD=potassium



voltage-gated



channel, subfamily



G, member 3 isoform 1



/FL=gb:AF454548.1



gb:AF348982.1



gb:AB070604.1



gb:NM_133329.4


AI742043
ESTs
—
0.94
0.34
−0.76
0.37
1.77
0.10
−1.45
0.52


NM_005103
Homo sapiens
FEZ1
2.80
0.11
2.08
0.25
4.98
0.06
2.20
0.26



fasciculation and



elongation protein



zeta 1 (zygin I)



(FEZ1), transcript



variant 1, mRNA.



/PROD=zygin 1,



isoform 1



/FL=gb:U60060.1



gb:U69139.1



gb:NM_005103.2


NM_000277
Homo sapiens
PAH
0.65
0.11
−1.08
0.08
1.46
0.18
−0.42
0.12



phenylalanine



hydroxylase (PAH),



mRNA.



/PROD=phenylalanine



hydroxylase



/FL=gb:U49897.1



gb:NM_000277.1


BF698797
ESTs
—
0.93
0.10
−0.74
0.11
2.07
0.10
−0.40
0.44


BF437747
ESTs, Weakly
C20orf118
4.26
0.07
1.99
0.45
4.57
0.09
1.75
0.39



similar to



ALU7_HUMAN ALU



SUBFAMILY SQ



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


NM_003020
Homo sapiens
SCG5
4.03
0.14
2.53
0.32
5.01
0.12
3.11
0.24



secretory granule,



neuroendocrine



protein 1 (7B2



protein) (SGNE1),



mRNA.



/PROD=secretory



granule,



neuroendocrine



protein 1



(7B2protein)



/FL=gb:BC005349.1



gb:NM_003020.1


NM_002800
Homo sapiens
PSMB9
1.38
0.10
0.27
0.34
2.05
0.07
−1.43
1.14



proteasome (prosome,



macropain) subunit,



beta type, 9 (large



multifunctional



protease 2) (PSMB9),



mRNA.



/PROD=proteasome



(prosome, macropain)



subunit, betatype, 9



(large multifunctional



protease 2)



/FL=gb:U01025.1



gb:NM_002800.1


BE972639
ESTs
LOC646326
0.00
0.15
−2.71
0.55
0.33
0.22
−2.24
0.61


BC044830
Homo sapiens,
C10orf96
1.38
0.12
−0.32
0.82
2.28
0.06
−1.39
0.90



Similar to RIKEN



cDNA 1700011F14



gene, clone



MGC:35062



IMAGE:5166167, mRNA,



complete cds.



/PROD=Similar to RIKEN



cDNA 1700011F14 gene



/FL=gb:BC044830.1


AI961231
KIAA0808
TOX
3.28
0.13
1.23
0.06
4.00
0.01
1.13
0.37



gene product



/FL=gb:AB018351.1



gb:NM_014729.1


U17496
Human proteasome
PSMB8
3.43
0.21
0.46
1.17
3.30
0.06
0.09
0.64



subunit LMP7 (allele



LMP7B) mRNA, complete



cds.



/PROD=proteasome



subunit LMP7



/FL=gb:U17497.1



gb:U17496.1


N23651
ESTs
SDK2
−1.16
0.31
−2.65
0.42
1.27
0.09
−2.07
0.75


NM_007015
Homo sapiens
LECT1
4.83
0.12
2.27
0.84
5.36
0.09
1.83
0.69



chondromodulin I



precursor (CHM-I),



mRNA.



/PROD=chondromodulin



I precursor



/FL=gb:NM_007015.1



gb:AB006000.1


NM_015474
Homo sapiens
SAMHD1
2.63
0.17
0.57
0.33
2.89
0.09
0.29
0.22



DKFZP564A032 protein



(DKFZP564A032), mRNA.



/PROD=DKFZP564A032



protein



/FL=gb:AF228421.1



gb:AL050267.1



gb:AB013847.1



gb:NM_015474.1


AF147427
Homo sapiens
SAMHD1
1.33
0.03
−0.99
0.88
1.35
0.08
−2.30
0.97



full length insert



cDNA clone YP80A10.


NM_004688
Homo sapiens
NMI
3.54
0.04
0.96
0.07
3.80
0.07
1.13
0.34



N-myc (and STAT)



interactor (NMI),



mRNA.



/PROD=N-myc and



STAT interactor



/FL=gb:BC001268.1



gb:NM_004688.1



gb:U32849.1


AB040812
Homo sapiens
PAK7
−1.37
0.60
−3.89
1.62
−0.49
0.07
−4.18
0.12



mRNA for protein



kinase PAK5,



complete cds.



/PROD=protein kinase



PAK5



/FL=gb:AB040812.1


AI985987
ESTs, Moderately
SCNN1G
−0.28
0.31
−0.99
0.35
0.12
0.19
−2.70
0.59



similar to



ALU1_HUMAN ALU



SUBFAMILY J



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


NM_001877
Homo sapiens
CR2
1.50
0.13
−0.43
1.05
2.35
0.14
−1.44
1.51



complement component



(3dEpstein Barr virus)



receptor 2 (CR2),



mRNA.



/PROD=complement component



(3dEpstein Barr



virus)receptor 2



/FL=gb:NM_001877.1



gb:M26004.1


NM_001351
Homo sapiens
DAZL
1.69
0.24
−0.81
0.76
1.93
0.12
−0.73
0.06



deleted in



azoospermia-like



(DAZL), mRNA.



/PROD=deleted in



azoospermia-like



/FL=gb:U66726.2



gb:NM_001351.1



gb:U65918.1



gb:U66078.1


NM_022168
Homo sapiens
IFIH1
−2.03
1.08
−2.76
0.83
−0.10
0.15
−2.15
0.40



melanoma



differentiation



associated protein-5



(MDA5), mRNA.



/PROD=melanoma



differentiation



associated protein-5



/FL=gb:AY017378.1



gb:NM_022168.1



gb:AF095844.1


AF052108
Homo sapiens
LOC157627
1.49
0.33
0.22
1.02
2.26
0.40
−1.33
1.19



clone 23687 mRNA



sequence.


AI056877
Human DNA sequence
LOC200230
0.11
0.30
−2.37
0.94
1.15
0.18
−1.35
0.75



from clone



RP4-530I15 on



chromosome 20.



Contains the 3 end of



the PTPN1 gene for



protein tyrosine



phosphatase, non-



receptor type 1 (EC



3.1.3.48), the gene



for a novel protein



similar to placental



protein DIFF40, an



RPL36 (60S Ribos


NM_002522
Homo sapiens
NPTX1
2.35
0.25
−0.48
0.19
2.17
0.09
−1.19
0.83



neuronal pentraxin I



(NPTX1), mRNA.



/PROD=neuronal



pentraxin I precursor



/FL=gb:NM_002522.1



gb:U61849.1


N21096
ESTs
STXBP6
2.42
0.16
0.99
0.08
3.69
0.09
1.14
0.04


AI693516
ESTs
COL14A1
0.39
0.23
−1.63
0.96
0.96
0.17
−1.81
0.67


NM_018043
Homo sapiens
TMEM16A
−0.90
0.37
−1.93
0.70
0.25
0.43
−3.93
0.49



hypothetical protein



FLJ10261 (FLJ10261),



mRNA.



/PROD=hypothetical



protein FLJ10261



/FL=gb:NM_018043.1


AF110400
Homo sapiens
FGF19
2.12
0.14
−0.08
0.34
2.18
0.04
−0.91
0.18



fibroblast growth



factor 19 (FGF19)



mRNA, complete cds.



/PROD=fibroblast



growth factor 19



/FL=gb:AF110400.1



gb:NM_005117.1



gb:AB018122.1


AK098525
Homo sapiens
HHIP
1.76
0.14
−0.57
0.63
3.32
0.06
−0.11
0.15



cDNA FLJ25659 fis,



clone TST00427,



highly similar to



Mus musculus



hedgehog-interacting



protein (Hip) mRNA.


AV715309
ESTs, Weakly
C20orf118
4.28
0.13
1.89
0.39
4.76
0.10
1.48
0.42



similar to



ALU7_HUMAN ALU



SUBFAMILY SQ



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


U96136
Homo sapiens
CTNND2
0.78
0.11
−1.33
0.87
1.18
0.12
−1.17
0.67



delta-catenin mRNA,



complete cds.



/PROD=delta-catenin



/FL=gb:NM_001332.1



gb:U72665.1



gb:AB013805.1



gb:U96136.1



gb:AF035302.1


NM_002590
Homo sapiens
PCDH8
−0.80
0.34
−0.83
0.82
2.18
0.02
0.12
0.33



protocadherin 8



(PCDH8), mRNA.



/PROD=protocadherin 8



/FL=gb:NM_002590.2



gb:AF061573.2


AF107846
Homo sapiens
—
0.44
0.20
−0.25
0.12
2.12
0.14
−2.56
0.81



neuroendocrine-



specific Golgi



protein p55



(XLalphas) gene, exon



XL2 and complete cds


BG169832
adenylate kinase 5
AK5
0.62
0.36
−2.79
0.50
1.03
0.36
−1.94
0.32



/FL=gb:NM_012093.1



gb:AF062595.1


BE968806
ESTs, Weakly
ATP5S
−1.15
0.14
−3.03
0.71
−0.34
0.15
−4.73
0.74



similar to



ALU4_HUMAN ALU



SUBFAMILY SB2



SEQUENCE



CONTAMINATION



WARNING ENTRY



(H. sapiens)


BU729850
hypothetical protein
JAKMIP2
2.30
0.07
0.43
1.78
3.86
0.04
−1.56
1.92



LOC153469


AL832535
Homo sapiens
LOC157627
2.25
0.18
−0.04
1.00
3.14
0.25
−1.50
1.19



mRNA; cDNA



DKFZp547J1816 (from



clone



DKFZp547J1816).


NM_000439
Homo sapiens
PCSK1
−0.63
0.18
−2.68
0.53
0.69
0.30
−3.19
1.03



proprotein convertase



subtilisinkexin



type 1 (PCSK1), mRNA.



/PROD=proprotein



convertase



subtilisinkexin



type 1



/FL=gb:NM_000439.2



gb:M90753.1


M34455
Human interferon-
INDO
4.39
0.23
1.58
0.09
5.44
0.08
1.69
0.13



gamma-inducible



indoleamine 2,3-



dioxygenase (IDO)



mRNA, complete cds.



/FL=gb:NM_002164.1



gb:M34455.1


AB014737
Homo sapiens
SMOC2
1.25
0.12
−2.04
0.48
1.94
0.06
−2.27
1.12



mRNA for SMAP-2b,



complete cds.



/PROD=SMAP-2b



/FL=gb:AB014737.1


BM682352
Homo sapiens
HCN1
−0.41
0.14
−4.38
1.19
0.35
0.31
−3.88
0.32



cDNA FLJ37204 fis,



clone



BRALZ2006976.
















TABLE V







RELATIVE EXPRESSION OF DEFINITE ENDODERM MARKERS


FOR CONDITIONS OUTLINED IN EXAMPLE 10. ALL VALUES


WERE NORMALIZED TO GROUP 1 (CONTROL).














Sox17
CXCR4
Goosecoid
HNF-3B
SOX-2
Oct-4

















Group 1-
1
1
1
1
1
1


control


Group 2
45
19.5
2.8
0.64
0.91
1.1


Group 3
45
30
2.9
0.74
0.70
0.76


Group 4
8
14
2.7
1.11
0.18
0.36


Group 5
23
16
3.1
1.76
0.16
0.41


Group 6
41
5.8
3.0
1.87
0.61
0.57


Group 7
25
19.5
2.7
0.62
0.34
0.48


Group 8
6
15.9
2.9
2.0
0.13
0.43


Group 9
1
1.4
0.9
0.89
1.2
0.85


Group 10
22
1.5
1.4
1.20
1.36
0.68


Group 11
54
23
2.5
0.71
0.66
0.65


Group 12
68
0.7
0.9
1.51
0.02
0.30


Group 13
13.9
12.7
3.0
2.1
0.11
0.30


Group 14
52.6
20.6
2.9
0.82
0.69
0.70


Group 15
68
27.7
2.9
0.68
0.68
0.85


Group 16
13.9
21
2.4
0.79
0.46
0.72


Group 17
52
14.9
3.5
2.12
0.22
0.44
















TABLE VI







MEAN NORMALIZED INTENSITY VALUES (IN LOG


FORMAT) OF GENES FOR H9 EMBRYONIC STEM CELLS


DERIVED DEFINITIVE ENDODERM STAGE CELLS CULTURED


ON EITHER MATRIGEL TM OR MEFS +/− WNT-3A.













DE
DE





treatment in
treatment



DE treatment
low serum +
in low
Benjamini



in low serum +
AA + Wnt-
serum +
and



AA on
3A on
AA on
Hochberg-


Gene Title
MATRIGEL
MATRIGEL
MEFS
P-value














ESTs
−4.82708
2.63968
−4.26995
8.62E−05


microfibrillar-associated protein 4
0.063791
5.16358
−0.60091
3.48E−03


Homo sapiens, alpha-1 (VI) collagen
−3.66187
2.36459
−2.26934
1.45E−04


ESTs
−3.43712
2.14508
−2.6475
0.00E+00


Homo sapiens cystatin SN (CST1),
0.072931
7.53908
4.63955
4.46E−03


mRNA. /PROD = cystatin SN


/FL = gb: J03870.1 gb: NM_001898.1


solute carrier family 16 (monocarboxylic
−0.066405
5.23572
0.279005
4.27E−04


acid transporters), member 3


/FL = gb: U81800.1 gb: NM_004207.1


Homo sapiens fibroblast growth factor
−0.894644
5.75417
2.4872
4.93E−03


17 (FGF17), mRNA. /PROD = fibroblast


growth factor 17 /FL = gb: NM_003867.1


gb: AB009249.1


Human link protein mRNA, complete
−1.93991
3.31348
−1.26346
1.14E−02


cds. /PROD = link protein


/FL = gb: NM_001884.1 gb: U43328.1


Homo sapiens solute carrier family 16
0.710321
6.12971
1.72403
0.00E+00


(monocarboxylic acid transporters),


member 3 (SLC16A3), mRNA.


/PROD = solute carrier family 16


(monocarboxylic acidtransporters),


member 3 /FL = gb: U81800.1


gb: NM_004207.1


Homo sapiens apolipoprotein A-I
−1.47073
5.37558
2.46891
5.07E−03


(APOA1), mRNA. /PROD = apolipoprotein


A-I precursor /FL = gb: M27875.1


gb: M11791.1 gb: NM_000039.1


gb: BC005380.1


Homo sapiens cytidine deaminase
−3.89129
2.05822
−1.67035
2.23E−03


(CDA), mRNA. /PROD = cytidine


deaminase /FL = gb: L27943.1


gb: NM_001785.1


ESTs, Moderately similar to JE0284
−2.37712
5.75671
4.22227
2.56E−02


Mm-1 cell derived transplantability-


associated protein 1b (H. sapiens)


ESTs
−0.04716
5.28231
0.966974
0.00E+00


glycophorin B (includes Ss blood group)
−2.85201
3.32812
−0.12969
1.45E−04


Homo sapiens homeobox protein
−4.42042
3.55326
1.89424
2.50E−02


goosecoid mRNA, complete cds.


/PROD = homeobox protein goosecoid


/FL = gb: AY177407.1 gb: NM_173849.1


MCP-1 = monocyte chemotactic protein
−2.27571
5.13499
2.95543
2.92E−02


(human, aortic endothelial cells, mRNA,


661 nt). /PROD = MCP-1


Homo sapiens Mix-like homeobox
−1.54648
4.47601
0.921971
2.01E−02


protein 1 (MILD1) mRNA, complete cds.


/PROD = Mix-like homeobox protein 1


/FL = gb: AF211891.1


ESTs
−4.93603
2.17917
−0.23735
1.12E−04


Homo sapiens lumican (LUM), mRNA.
−4.05726
3.21064
0.948822
3.39E−02


/PROD = lumican /FL = gb: NM_002345.1


gb: U18728.1 gb: U21128.1


Homo sapiens HNF-3beta mRNA for
−2.71785
4.68666
2.82506
3.71E−02


hepatocyte nuclear factor-3 beta,


complete cds. /PROD = hepatocyte


nuclear factor-3 beta


/FL = gb: AB028021.1 gb: NM_021784.1


Homo sapiens reserved (KCNK12),
−0.468745
6.28184
3.77969
1.97E−02


mRNA. /PROD = tandem pore domain


potassium channel THIK-2


/FL = gb: NM_022055.1 gb: AF287302.1


Homo sapiens atrophin-1 interacting
−4.30828
1.80825
−1.32021
9.63E−03


protein 1; activin receptor interacting


protein 1 (KIAA0705), mRNA.


/PROD = atrophin-1 interacting protein 1;


activinreceptor interacting protein 1


/FL = gb: NM_012301.1 gb: AF038563.1


ESTs
−2.33636
2.25176
−2.32124
5.26E−04


Homo sapiens glutamate decarboxylase
−2.424
2.31908
−1.87965
4.07E−04


1 (brain, 67 kD) (GAD1), transcript


variant GAD25, mRNA.


/PROD = glutamate decarboxylase 1,


isoform GAD25 /FL = gb: NM_013445.1


gb: AF178853.1 gb: BC002815.1


Homo sapiens cardiac ventricular
−0.549728
4.89072
1.4377
2.99E−03


troponin C mRNA, complete cds.


/PROD = cardiac ventricular troponin C


/FL = gb: NM_003280.1 gb: AF020769.1


ESTs
−2.89554
3.42817
0.926036
2.62E−02


Homo sapiens fibroblast growth factor 8
−4.32791
2.19561
0.015827
5.91E−03


(androgen-induced) (FGF8), mRNA.


/PROD = fibroblast growth factor 8


(androgen-induced) /FL = gb: U36223.1


gb: U46212.1 gb: NM_006119.1


ESTs
−3.09818
1.66254
−2.20564
8.62E−05


Homo sapiens haptoglobin-related
−2.6068
2.38009
−1.19632
6.95E−03


protein (HPR), mRNA.


/PROD = haptoglobin-related protein


/FL = gb: NM_020995.1


collagen, type VI, alpha 1
−1.418
3.85952
0.604245
1.21E−02


Human (clone 8B1) Br-cadherin mRNA,
−2.17941
2.59894
−1.02624
1.24E−02


complete cds. /PROD = Br-cadherin


/FL = gb: L34057.1 gb: L33477.1


gb: NM_004061.1


ESTs
−1.40092
2.57297
−1.82509
1.45E−04


Homo sapiens cystatin SA (CST2),
−0.102178
5.21645
2.1671
2.40E−02


mRNA. /PROD = cystatin SA


/FL = gb: NM_001322.1


Human mRNA for apolipoprotein AI (apo
0.215086
5.51109
2.49684
9.57E−03


AI) =. /PROD = preproapolipoprotein AI


Homo sapiens MLL septin-like fusion
−3.29221
1.70902
−1.49951
2.25E−03


(MSF), mRNA. /PROD = MLL septin-like


fusion /FL = gb: AF123052.1


gb: NM_006640.1


Homo sapiens cystatin S (CST4),
0.92448
6.48842
3.87036
7.20E−03


mRNA. /PROD = cystatin S


/FL = gb: NM_001899.1


Homo sapiens phorbolin-like protein
−1.11883
4.73391
2.40782
7.86E−03


MDS019 (MDS019), mRNA.


/PROD = phorbolin-like protein MDS019


/FL = gb: AF182420.1 gb: NM_021822.1


Homo sapiens apolipoprotein A-II
−1.03333
5.80468
4.46856
3.23E−02


(APOA2), mRNA. /PROD = apolipoprotein


A-II precursor /FL = gb: M29882.1


gb: NM_001643.1 gb: BC005282.1


ESTs
−1.55475
3.48278
0.420447
2.50E−02


Homo sapiens glutamate decarboxylase
−3.86752
1.56384
−1.08675
2.52E−02


1 (brain, 67 kD) (GAD1), transcript


variant GAD67, mRNA.


/PROD = glutamate decarboxylase 1,


isoform GAD67 /FL = gb: NM_000817.1


gb: M81883.1 gb: L16888.1


ESTs
−0.731491
5.43249
3.52168
1.60E−02


ESTs
−2.03591
3.38924
0.760984
2.34E−03


Homo sapiens retinoid X receptor,
−2.37496
2.62934
−0.32035
8.83E−04


gamma (RXRG), mRNA.


/PROD = retinoid X receptor, gamma


/FL = gb: NM_006917.1 gb: U38480.1


ESTs
−0.648552
4.30576
1.43266
2.19E−03


Homo sapiens cDNA FLJ11550 fis,
−1.22228
5.37746
4.21644
1.68E−02


clone HEMBA1002970


ESTs
−1.782
3.50391
1.0501
1.85E−02


Homo sapiens haptoglobin (HP), mRNA.
−1.10114
3.5449
0.477027
8.13E−03


/PROD = haptoglobin /FL = gb: K00422.1


gb: L29394.1 gb: NM_005143.1


Homo sapiens hypothetical protein
−0.431989
5.14497
3.02045
2.62E−02


FLJ10970 (FLJ10970), mRNA.


/PROD = hypothetical protein FLJ10970


/FL = gb: NM_018286.1


Homo sapiens beta-site APP cleaving
−2.0354
3.70648
1.75385
8.00E−03


enzyme (BACE) mRNA, complete cds.


/PROD = beta-site APP cleaving enzyme


/FL = gb: AF200343.1 gb: AF204943.1


gb: AF190725.1 gb: AF201468.1


gb: NM_012104.1


Homo sapiens hypothetical protein
1.36784
6.82571
4.5979
2.10E−02


FLJ22252 similar to SRY-box containing


gene 17 (FLJ22252), mRNA.


/PROD = hypothetical protein FLJ22252


similar to SRY-boxcontaining gene 17


/FL = gb: NM_022454.1


Cluster Incl. AB028021: Homo sapiens
−1.5339
5.12418
4.11704
4.47E−02


HNF-3beta mRNA for hepatocyte


nuclear factor-3 beta, complete cds


/cds = (196, 1569) /gb = AB028021


/gi = 4958949 /ug = Hs.155651 /len = 1944


Homo sapiens gastrin-releasing peptide
−2.74071
2.70077
0.509757
2.49E−04


(GRP), mRNA. /PROD = gastrin-releasing


peptide /FL = gb: NM_002091.1


gb: K02054.1 gb: BC004488.1


Homo sapiens sema domain, seven
−1.53335
3.78503
1.48732
4.71E−02


thrombospondin repeats (type 1 and


type 1-like), transmembrane domain


(TM) and short cytoplasmic domain,


(semaphorin) 5A (SEMA5A), mRNA.


/PROD = sema domain, seven


thrombospondin repeats (type1 and type


1-like), transmem


Homo sapiens mRNA; cDNA
−0.835182
5.22406
3.69882
2.08E−02


DKFZp586J0624 (from clone


DKFZp586J0624); complete cds.


/PROD = hypothetical protein


/FL = gb: AF215636.1 gb: NM_014585.1


gb: AF231121.1 gb: AF226614.1


gb: AL136944.1


Human mRNA for alpha-1 type II
−2.8736
2.155
−0.38021
5.70E−03


collagen.


Rho GDP dissociation inhibitor (GDI)
−1.54385
1.7147
−2.58241
1.71E−02


alpha


neuropilin 1 /FL = gb: AF016050.1
−1.62253
1.95432
−1.9667
8.83E−04


gb: NM_003873.1 gb: AF018956.1


Human DNA sequence from clone RP1-
−3.72313
1.68755
−0.37308
1.38E−03


181C24 on chromosome 6p11.1-12.2.


Contains the 3 end of the BMP5 gene for


bone morphogenetic protein 5, ESTs,


STSs and GSSs /FL = gb: M60314.1


gb: NM_021073.1


myristoylated alanine-rich protein kinase
−0.71724
3.51728
0.335725
4.29E−03


C substrate (MARCKS, 80K-L)


/FL = gb: M68956.1 gb: D10522.1


gb: NM_002356.4


hypothetical protein FLJ23403
−1.45618
1.81423
−2.31327
1.20E−02


/FL = gb: NM_022068.1


hepatocyte nuclear factor 4, alpha
−4.26574
1.7879
0.445241
3.25E−02


Homo sapiens cell adhesion molecule
−0.541188
2.1751
−2.5002
1.16E−03


with homology to L1CAM (close


homologue of L1) (CHL1), mRNA.


/PROD = cell adhesion molecule with


homology to L1CAM (close homologue of


L1) /FL = gb: AF002246.1


gb: NM_006614.1


matrix metalloproteinase 14 (membrane-
−2.05734
2.36236
−0.5185
1.66E−02


inserted) /FL = gb: U41078.1


gb: NM_004995.2


Homo sapiens glycophorin B (includes
−0.947308
3.26089
0.180293
4.83E−04


Ss blood group) (GYPB), mRNA.


/PROD = glycophorin B precursor


/FL = gb: J02982.1 gb: NM_002100.2


WAS protein family, member 2
−2.18746
1.99129
−1.05968
4.00E−03


/FL = gb: NM_006990.1 gb: AB026542.1


Homo sapiens frizzled-related protein
0.56502
5.7261
3.67629
1.75E−02


(FRZB), mRNA. /PROD = frizzled-related


protein /FL = gb: U24163.1 gb: U68057.1


gb: NM_001463.1 gb: U91903.1


Homo sapiens glutamate decarboxylase
−1.68495
2.27067
−0.96944
8.02E−03


1 (brain, 67 kD) (GAD1), transcript


variant GAD25, mRNA.


/PROD = glutamate decarboxylase 1,


isoform GAD25 /FL = gb: NM_013445.1


gb: AF178853.1 gb: BC002815.1


ESTs
0.812766
5.93144
3.91314
4.15E−02


Homo sapiens clone 23736 mRNA
−0.047182
5.79006
4.50744
1.74E−02


sequence


Homo sapiens glycophorin E (GYPE),
−2.01601
1.79002
−1.50134
1.97E−02


mRNA. /PROD = glycophorin E


/FL = gb: NM_002102.1 gb: M29610.1


ESTs
1.06767
5.63319
3.12487
2.00E−02


ESTs
−1.41162
2.5396
−0.57029
1.29E−02


Human Fritz mRNA, complete cds.
0.436589
5.69814
3.91514
1.99E−02


/PROD = Fritz /FL = gb: U24163.1


gb: U68057.1 gb: NM_001463.1


gb: U91903.1


Homo sapiens, clone MGC: 4655,
2.3772
5.9184
2.47596
1.20E−02


mRNA, complete cds. /PROD = Unknown


(protein for MGC: 4655)


/FL = gb: BC004908.1


KIAA0878 protein /FL = gb: NM_014899.1
1.1189
6.41747
4.78882
2.01E−02


gb: AB020685.1


Homo sapiens sema domain,
−0.785987
3.69668
1.27624
2.10E−02


immunoglobulin domain (Ig), short basic


domain, secreted, (semaphorin) 3E


(SEMA3E), mRNA. /PROD = sema


domain, immunoglobulin domain (Ig),


shortbasic domain, secreted,


(semaphorin) 3E /FL = gb: NM_012431.1


gb: AB002329.1


ESTs
1.48084
6.59709
4.81395
1.36E−02


noggin /FL = gb: NM_005450.1
−1.63627
3.28161
1.32958
2.53E−02


Homo sapiens hypothetical protein
−0.904749
3.35854
0.755319
1.12E−04


FLJ11316 (FLJ11316), mRNA.


/PROD = hypothetical protein FLJ11316


/FL = gb: NM_018388.1


Homo sapiens angiopoietin 2 (ANGPT2),
−2.93044
2.23779
0.59685
3.43E−02


mRNA. /PROD = angiopoietin 2


/FL = gb: AB009865.1 gb: AF004327.1


gb: NM_001147.1


Homo sapiens matrix metalloproteinase
−0.723489
2.97262
−0.09689
5.44E−03


14 (membrane-inserted) (MMP14),


mRNA. /PROD = matrix metalloproteinase


14 preproprotein /FL = gb: U41078.1


gb: NM_004995.2


G protein-coupled receptor
1.50709
6.65228
5.05327
2.00E−02


collagen, type IX, alpha 2
1.27026
5.4659
2.93507
3.19E−03


/FL = gb: NM_001852.1


ESTs
0.521638
3.93176
0.620223
0.00E+00


KIAA1462 protein
−3.84563
1.65452
0.437064
3.27E−02


Homo sapiens cartilage linking protein 1
−1.31515
2.27271
−0.80521
2.22E−02


(CRTL1), mRNA. /PROD = cartilage


linking protein 1 /FL = gb: NM_001884.1


gb: U43328.1


Homo sapiens solute carrier family 21
0.711428
4.89808
2.43304
6.84E−03


(prostaglandin transporter), member 2


(SLC21A2), mRNA. /PROD = solute


carrier family 21


(prostaglandintransporter), member 2


/FL = gb: U70867.1 gb: NM_005630.1


ESTs
0.307173
4.78515
2.65653
1.72E−02


6-phosphofructo-2-kinasefructose-2,6-
−0.242865
3.97929
1.59985
5.28E−03


biphosphatase 4


Homo sapiens dual specificity
0.953857
5.82811
4.12159
4.00E−03


phosphatase 4 (DUSP4), mRNA.


/PROD = dual specificity phosphatase 4


/FL = gb: NM_001394.2 gb: BC002671.1


gb: U48807.1 gb: U21108.1


ESTs, Weakly similar to T00331
−1.57372
2.70797
0.443622
6.74E−03


hypothetical protein KIAA0555


(H. sapiens)


ESTs
−3.57414
3.15167
3.37198
8.82E−03


ESTs, Highly similar to IHH_HUMAN
−0.653989
3.22059
0.590533
5.18E−03


INDIAN HEDGEHOG PROTEIN


PRECURSOR (H. sapiens)


ESTs, Weakly similar to FCE2 MOUSE
0.494192
5.22522
3.48031
1.97E−02


LOW AFFINITY IMMUNOGLOBULIN


EPSILON FC RECEPTOR (M. musculus)


homeo box HB9 /FL = gb: NM_005515.1
−1.65563
3.2238
1.63092
5.58E−03


Homo sapiens arylsulfatase E
0.283004
4.95903
3.18424
9.22E−03


(chondrodysplasia punctata 1) (ARSE),


mRNA. /PROD = arylsulfatase E


precursor /FL = gb: X83573.1


gb: NM_000047.1


ESTs
−0.05909
3.0455
−0.29817
1.47E−02


Homo sapiens hypothetical protein
−1.48452
1.97473
−1.00431
5.72E−03


FLJ23403 (FLJ23403), mRNA.


/PROD = hypothetical protein FLJ23403


/FL = gb: NM_022068.1


ESTs
−0.182403
3.01548
−0.18954
3.72E−03


hypothetical protein FLJ23091
0.323388
5.25192
3.77987
3.57E−02


Human dipeptidyl peptidase IV (CD26)
−3.61145
0.760585
−1.25595
3.07E−02


mRNA, complete cds. /PROD = dipeptidyl


peptidase IV /FL = gb: M74777.1


hypothetical protein FLJ21032
0.355672
4.67756
2.61753
4.59E−02


Homo sapiens Kell blood group (KEL),
−2.20519
1.89439
−0.38393
1.91E−02


mRNA. /PROD = Kell blood group antigen


/FL = gb: BC003135.1 gb: NM_000420.1


splicing factor, arginineserine-rich 5
0.7481
5.68934
4.27169
2.97E−03


Human prostatic secretory protein 57
−3.01313
1.46338
−0.40767
1.97E−02


mRNA, complete cds. /PROD = PSP57


/FL = gb: U22178.1


Homo sapiens KIAA0878 protein
2.0265
7.00937
5.65368
2.85E−02


(KIAA0878), mRNA. /PROD = KIAA0878


protein /FL = gb: NM_014899.1


gb: AB020685.1


Homo sapiens cryptic mRNA, complete
0.104874
2.87319
−0.67353
1.61E−03


cds. /PROD = cryptic /FL = gb: AF312769.1


Homo sapiens cDNA FLJ13221 fis,
0.355743
3.98782
1.30963
1.38E−03


clone NT2RP4002075


phorbolin-like protein MDS019
−1.11756
2.83853
0.503523
1.59E−02


Homo sapiens mRNA for KIAA1409
0.368334
2.8009
−1.03191
1.78E−02


protein, partial cds. /PROD = KIAA1409


protein


Homo sapiens mRNA; cDNA
−2.63427
1.64513
−0.3056
2.52E−02


DKFZp434D0818 (from clone


DKFZp434D0818)


ESTs
0.35393
5.14775
3.74875
3.16E−02


Homo sapiens TBX3-iso protein (TBX3-
−2.34566
2.45238
1.07038
1.31E−02


iso), mRNA. /PROD = TBX3-iso protein


/FL = gb: NM_016569.1 gb: AF216750.1


Homo sapiens chromosome 19, cosmid
−0.258871
3.0636
0.223926
1.58E−03


R31181


Homo sapiens mRNA for GATA-6,
2.21862
6.8609
5.34263
4.29E−02


complete cds. /PROD = GATA-6


/FL = gb: U66075.1 gb: NM_005257.1


gb: D87811.1


Homo sapiens ankyrin-like with
−1.10879
3.93484
2.81939
1.29E−02


transmembrane domains 1 (ANKTM1),


mRNA


G protein-coupled receptor 49
0.265509
4.46257
2.50537
2.19E−02


Homo sapiens growth differentiation
1.67253
5.34944
2.87486
8.62E−05


factor 3 (GDF3), mRNA. /PROD = growth


differentiation factor 3 precursor


/FL = gb: NM_020634.1 gb: AF263538.1


Human (clone HSY3RR) neuropeptide Y
1.77461
6.70301
5.47995
3.00E−02


receptor (NPYR) mRNA, complete cds.


/PROD = neuropeptide Y receptor


/FL = gb: L06797.1 gb: NM_003467.1


gb: AF025375.1 gb: AF147204.1


gb: M99293.1 gb: L01639.1


Homo sapiens type VI collagen alpha 2
1.7011
5.33126
2.84458
5.91E−03


chain precursor (COL6A2) mRNA,


complete cds, alternatively spliced.


/PROD = type VI collagen alpha 2 chain


precursor /FL = gb: AY029208.1


ESTs
−0.349726
3.29119
0.824929
4.11E−03


ESTs
−0.903317
1.89777
−1.36429
6.84E−03


Homo sapiens hypothetical protein
2.60483
7.10633
5.55242
1.41E−02


FLJ10718 (FLJ10718), mRNA.


/PROD = hypothetical protein FLJ10718


/FL = gb: NM_018192.1


Homo sapiens Rho GTPase activating
−1.10389
1.83053
−1.28521
1.28E−02


protein 6 (ARHGAP6), transcript variant


2, mRNA. /PROD = Rho GTPase


activating protein 6 isoform 2


/FL = gb: AF022212.2 gb: NM_001174.2


stanniocalcin 1 /FL = gb: U25997.1
2.41135
7.29563
6.14284
2.51E−02


gb: NM_003155.1 gb: U46768.1


Human glycophorin HeP2 mRNA, partial
−0.843493
2.71108
0.233547
2.98E−04


cds. /PROD = glycophorin HeP2


Homo sapiens cDNA FLJ12993 fis,
0.147259
4.12241
2.06949
6.84E−03


clone NT2RP3000197


Homo sapiens presenilin stabilization
−0.86173
2.85614
0.56745
6.09E−03


factor b (PSF) mRNA, complete cds;


alternatively spliced. /PROD = presenilin


stabilization factor b /FL = gb: AY113699.1


Homo sapiens glycophorin Erik STA
−1.19362
2.17108
−0.45439
1.45E−04


(GPErik) gene complete cds.


/PROD = glycophorin Erik (STA)


/FL = gb: U00178.1


bromodomain and PHD finger
−2.8472
1.75573
0.369397
8.62E−05


containing, 3


ESTs
0.784344
4.74104
2.71543
7.55E−03


ESTs
−1.26251
3.4693
2.27614
3.80E−03


ESTs
−1.71713
1.23763
−1.71122
2.99E−02


Homo sapiens microsomal glutathione
2.06233
6.81536
5.6918
3.79E−02


S-transferase 2 (MGST2), mRNA.


/PROD = microsomal glutathione S-


transferase 2 /FL = gb: NM_002413.1


gb: U77604.1


Homo sapiens eomesodermin (Xenopus
2.65926
6.71627
4.89839
2.76E−02


laevis) homolog (EOMES), mRNA.


/PROD = eomesodermin (Xenopus laevis)


homolog /FL = gb: AB031038.1


gb: NM_005442.1


Homo sapiens mRNA for MSX-2,
0.407211
4.11145
1.94529
1.35E−02


complete cds. /PROD = MSX-2


/FL = gb: D89377.1


Homo sapiens apolipoprotein A-II
−1.10237
5.10066
4.75899
1.34E−02


(APOA2), mRNA. /PROD = apolipoprotein


A-II precursor /FL = gb: M29882.1


gb: NM_001643.1 gb: BC005282.1


Homo sapiens adenylate cyclase 8
−1.3408
1.12773
−2.26149
1.07E−02


(brain) (ADCY8), mRNA.


/PROD = adenylate cyclase 8


/FL = gb: NM_001115.1


Homo sapiens glucose-6-phosphate
−0.193516
3.03064
0.407738
1.38E−03


transporter (G6PT) gene, G6PT-Dt


allele, complete cds


Homo sapiens glutathione S-transferase
−3.10645
1.11704
−0.4221
5.37E−04


A2 (GSTA2), mRNA. /PROD = glutathione


S-transferase A2 /FL = gb: BC002895.1


gb: M25627.1 gb: M16594.1 gb: M14777.1


gb: M15872.1 gb: M21758.1


gb: NM_000846.1


Homo sapiens sodium dependent
−0.397133
2.78341
0.231961
1.11E−02


phosphate transporter isoform NaPi-IIb


mRNA, complete cds. /PROD = sodium


dependent phosphate transporter


isoformNaPi-IIb /FL = gb: AF111856.1


gb: NM_006424.1 gb: AF146796.1


ESTs
2.77413
7.19192
5.906
3.17E−02


Homo sapiens partial LHX9 gene for
−2.2486
1.64952
−0.13424
7.89E−03


LIM-homeobox 9, 3UTR.


Homo sapiens LYST-interacting protein
−0.682149
2.34271
−0.31253
9.87E−03


LIP3 mRNA, partial cds. /PROD = LYST-


interacting protein LIP3


putative 47 kDa protein
−0.123937
3.2914
1.05247
3.72E−03


Homo sapiens protein S (alpha)
0.577604
4.00035
1.7883
7.89E−03


(PROS1), mRNA. /PROD = protein S


(alpha) /FL = gb: M15036.1


gb: NM_000313.1


ESTs
−1.63711
0.915477
−2.15328
1.83E−02


protocadherin 10
1.76718
5.1503
2.98569
2.52E−02


KIAA1511 protein
0.620278
3.86886
1.57543
1.86E−03


Homo sapiens cDNA FLJ13221 fis,
0.282785
3.44178
1.08178
1.31E−03


clone NT2RP4002075


Homo sapiens fibroblast growth factor
−1.72385
2.26674
0.747239
6.86E−03


receptor 4, soluble-form splice variant


(FGFR4) mRNA, complete cds.


/PROD = fibroblast growth factor receptor


4, soluble-form splice variant


/FL = gb: NM_022963.1 gb: AF202063.1


X75208 /FEATURE = cds
−1.36729
2.60456
1.08204
1.58E−02


/DEFINITION = HSPTKR H. sapiens


HEK2 mRNA for protein tyrosine kinase


receptor


Homo sapiens chemokine receptor
2.30557
6.72969
5.66933
3.31E−02


CXCR4 mRNA, complete cds.


/PROD = chemokine receptor CXCR4


/FL = gb: AF348491.1


KIAA1415 protein
0.185854
3.70346
1.74255
7.78E−04


ESTs
−0.048335
3.11786
0.836448
1.29E−02


Homo sapiens stanniocalcin 1 (STC1),
2.39435
6.55737
5.28007
2.19E−02


mRNA. /PROD = stanniocalcin 1


/FL = gb: U25997.1 gb: NM_003155.1


gb: U46768.1


Homo sapiens high mobility group
1.04695
3.2786
0.095286
2.11E−02


protein-R mRNA, complete cds.


/PROD = high mobility group protein-R


/FL = gb: AF176039.1


ESTs, Moderately similar to
−1.10358
1.66331
−0.98371
3.37E−02


ALU4_HUMAN ALU SUBFAMILY SB2


SEQUENCE CONTAMINATION


WARNING ENTRY (H. sapiens)


Homo sapiens arrestin, beta 1 (ARRB1),
−0.158844
2.41791
−0.40915
1.12E−02


transcript variant 1, mRNA.


/PROD = arrestin beta 1, isoform A


/FL = gb: BC003636.1 gb: AF084040.1


gb: NM_004041.2


ESTs
−1.34399
1.80279
−0.44529
1.60E−03


Human mRNA for pro-alpha 1 (II)
0.85982
4.1935
2.15657
1.09E−02


collagen 3end C-term. triple helical and


C-terminal non-helical domain.


/PROD = pro-alpha 1 (II) collagen (313


AA; AA 975-271c) /FL = gb: NM_001844.2


Homo sapiens hypothetical protein
1.47773
4.42479
2.00326
1.16E−03


DKFZp564B052 (DKFZp564B052),


mRNA. /PROD = hypothetical protein


DKFZp564B052 /FL = gb: NM_030820.1


testis enhanced gene transcript (BAX
−0.695228
2.99328
1.31679
1.17E−03


inhibitor 1)


Homo sapiens fasciculation and
1.86433
4.71354
2.20315
8.62E−05


elongation protein zeta 1 (zygin I)


(FEZ1), transcript variant 1, mRNA.


/PROD = zygin 1, isoform 1


/FL = gb: U60060.1 gb: U69139.1


gb: NM_005103.2


Homo sapiens matrix metalloproteinase
−0.298504
3.44582
1.8336
4.80E−03


15 (membrane-inserted) (MMP15),


mRNA. /PROD = matrix metalloproteinase


15 preproprotein /FL = gb: D86331.1


gb: NM_002428.1


heparan sulfate proteoglycan 2
−2.53327
0.924726
−0.97212
4.31E−02


(perlecan) /FL = gb: M85289.1


gb: NM_005529.2


Homo sapiens dickkopf (Xenopus laevis)
0.981469
3.70716
1.09014
1.76E−03


homolog 1 (DKK1), mRNA.


/PROD = dickkopf (Xenopus laevis)


homolog 1 /FL = gb: AF177394.1


gb: NM_012242.1 gb: AF127563.1


ESTs
−0.801487
3.37342
2.21393
1.22E−02


ESTs
−2.57626
1.40231
0.050493
6.60E−03


ESTs
0.499579
4.10541
2.39934
4.29E−03


Homo sapiens bone morphogenetic
2.04506
6.19114
5.02927
4.98E−02


protein 2 (BMP2), mRNA. /PROD = bone


morphogenetic protein 2 precursor


/FL = gb: NM_001200.1


Human extracellular matrix protein 1
0.634778
3.65829
1.37564
6.11E−04


(ECM1) mRNA, complete cds.


/PROD = extracellular matrix protein 1


/FL = gb: NM_004425.2 gb: U65932.1


gb: U68186.1


Homo sapiens nuclear receptor
−1.92783
2.32456
1.27907
2.99E−02


subfamily 0, group B, member 1


(NR0B1), mRNA. /PROD = adrenal


hypoplasia protein /FL = gb: NM_000475.2


Homo sapiens cDNA: FLJ23067 fis,
−2.25626
1.95987
0.882498
6.62E−03


clone LNG04993.


Homo sapiens ADP-ribosylation factor 4-
1.63466
5.70351
4.48037
1.90E−02


like (ARF4L), mRNA. /PROD = ADP-


ribosylation factor 4-like


/FL = gb: U25771.1 gb: L38490.1


gb: NM_001661.1 gb: BC000043.1


Homo sapiens HT016 mRNA, complete
0.218762
3.97514
2.45222
3.37E−02


cds. /PROD = HT016 /FL = gb: AF225426.1


Homo sapiens, tropomodulin, clone
0.348796
3.54199
1.46427
1.29E−03


MGC: 3643, mRNA, complete cds.


/PROD = tropomodulin


/FL = gb: NM_003275.1 gb: M77016.1


gb: BC002660.1


Homo sapiens hypothetical protein
0.903825
4.65366
3.1349
2.48E−02


FLJ22471 (FLJ22471), mRNA.


/PROD = hypothetical protein FLJ22471


/FL = gb: NM_025140.1


ESTs
−0.120583
2.94229
0.738304
7.20E−03


ESTs, Moderately similar to JC4969 pig-
−2.39713
0.906454
−1.04495
1.69E−02


c protein (H. sapiens)


Homo sapiens LIM homeobox protein 1
−0.77166
2.27746
0.085559
4.26E−02


(LHX1), mRNA. /PROD = LIM homeobox


protein 1 /FL = gb: NM_005568.1


gb: U14755.1


Homo sapiens, hypothetical protein
−2.43105
0.646883
−1.50617
8.27E−03


MGC2865, clone MGC: 20246


IMAGE: 4635389, mRNA, complete cds.


/FL = gb: BC016043.1


ESTs
−2.01523
2.16773
1.12403
1.99E−02


Homo sapiens oxoglutarate
−0.032595
2.3997
−0.38533
1.45E−02


dehydrogenase (lipoamide) (OGDH),


mRNA. /PROD = oxoglutarate


dehydrogenase (lipoamide)


/FL = gb: D10523.1 gb: BC004964.1


gb: NM_002541.1


ESTs, Weakly similar to T32252
2.20966
5.35974
3.33255
1.12E−04


hypothetical protein T15B7.2-


Caenorhabditis elegans (C. elegans)


Homo sapiens, cleft lip and palate
1.31578
3.52357
0.556735
2.92E−03


associated transmembrane protein 1,


clone MGC: 10593, mRNA, complete


cds. /PROD = cleft lip and palate


associated transmembraneprotein 1


/FL = gb: BC004865.1


Homo sapiens clone TUA8 Cri-du-chat
0.09218
3.37126
1.49746
6.46E−03


region mRNA


Homo sapiens transcriptional activator of
0.666391
4.50481
3.22532
5.80E−03


the c-fos promoter (CROC4), mRNA.


/PROD = transcriptional activator of the c-


fos promoter /FL = gb: NM_006365.1


gb: U49857.1


Homo sapiens hypothetical protein
−1.38519
2.23626
0.763963
7.36E−03


FLJ21195 similar to protein related to


DAC and cerberus (FLJ21195), mRNA.


/PROD = hypothetical protein FLJ21195


similar to proteinrelated to DAC and


cerberus /FL = gb: NM_022469.1


ras homolog gene family, member B
−0.123101
2.52093
0.071885
1.86E−02


/FL = gb: AF498971.1 gb: NM_004040.1


cartilage linking protein 1
0.879449
3.47789
0.987243
1.03E−03


Homo sapiens BCL2adenovirus E1B
3.30506
6.91803
5.44746
1.79E−03


19 kD-interacting protein 3 (BNIP3)


mRNA, complete cds.


/PROD = BCL2adenovirus E1B 19 kD-


interacting protein 3 /FL = gb: AF002697.1


gb: NM_004052.2 gb: U15174.1


Homo sapiens polycythemia rubra vera
−1.19629
2.40987
0.93741
9.40E−03


1; cell surface receptor (PRV1), mRNA.


/PROD = polycythemia rubra vera 1; cell


surfacereceptor /FL = gb: NM_020406.1


gb: AF146747.1


Homo sapiens hypothetical protein
−0.044112
3.42017
1.80688
3.77E−03


FLJ11560 (FLJ11560), mRNA.


/PROD = hypothetical protein FLJ11560


/FL = gb: NM_025182.1


plexin A2
0.930527
4.36203
2.74167
1.52E−02


Homo sapiens mRNA; cDNA
1.37193
4.24502
2.08129
5.37E−04


DKFZp547H236 (from clone


DKFZp547H236). /PROD = hypothetical


protein


Homo sapiens cystatin C (amyloid
2.08601
5.30525
3.51965
2.20E−03


angiopathy and cerebral hemorrhage)


(CST3), mRNA. /PROD = cystatin C


(amyloid angiopathy and


cerebralhemorrhage)


/FL = gb: NM_000099.1


ESTs, Moderately similar to NFY-C
−0.749407
2.00148
−0.24268
2.34E−03


(H. sapiens)


ESTs
2.54243
5.75201
3.97307
2.60E−02


Homo sapiens sialyltransferase (STHM),
−0.68709
2.89586
1.50348
3.70E−02


mRNA. /PROD = sialyltransferase


/FL = gb: U14550.1 gb: NM_006456.1


Homo sapiens SG2NA beta isoform
−1.15566
1.13989
−1.53439
1.55E−02


mRNA, partial cds. /PROD = SG2NA beta


isoform


Homo sapiens hypothetical protein
1.49091
4.80138
3.15205
2.63E−02


FLJ12838 (FLJ12838), mRNA.


/PROD = hypothetical protein FLJ12838


/FL = gb: NM_024641.1


Homo sapiens solute carrier
0.403706
2.12385
−1.10043
1.78E−02


(SLC25A18) mRNA, complete cds;


nuclear gene for mitochondrial product.


/PROD = solute carrier


/FL = gb: AY008285.1


KIAA0346 protein
−1.34736
2.49605
1.41497
3.90E−03


Human clone 23826 mRNA sequence
1.80782
4.07112
1.44315
2.28E−04


Homo sapiens receptor tyrosine kinase-
1.53333
4.73839
3.05612
1.63E−02


like orphan receptor 2 (ROR2), mRNA.


/PROD = receptor tyrosine kinase-like


orphan receptor 2 /FL = gb: M97639.1


gb: NM_004560.1


Homo sapiens MYC-associated zinc
−0.055569
2.67075
0.511292
2.63E−02


finger protein (purine-binding


transcription factor) (MAZ), mRNA.


/PROD = MYC-associated zinc finger


protein (purine-binding transcription


factor) /FL = gb: D85131.1


gb: NM_002383.1


Homo sapiens cDNA FLJ30081 fis,
0.677905
3.33728
1.12499
1.12E−04


clone BGGI12000693, weakly similar to


POLYHOMEOTIC-PROXIMAL


CHROMATIN PROTEIN.


Homo sapiens elongation of very long
0.099979
3.23372
1.50802
2.59E−03


chain fatty acids (FEN1Elo2, SUR4Elo3,


yeast)-like 2 (ELOVL2), mRNA.


/PROD = elongation of very long chain


fatty acids (FEN1Elo2, SUR4Elo3,


yeast)-like 2 /FL = gb: NM_017770.1


Homo sapiens thyrotropin-releasing
−1.65672
1.46994
−0.25839
3.02E−02


hormone (TRH), mRNA.


/PROD = thyrotropin-releasing hormone


/FL = gb: NM_007117.1


ESTs, Weakly similar to A46302 PTB-
0.894051
4.21835
2.69535
1.57E−02


associated splicing factor, long form


(H. sapiens)


Human MLC1emb gene for embryonic
1.3489
5.11354
4.03961
1.61E−02


myosin alkaline light chain, promoter and


exon 1


Homo sapiens microseminoprotein,
−2.37904
1.3101
0.169915
2.46E−02


beta- (MSMB), mRNA.


/PROD = microseminoprotein, beta-


/FL = gb: NM_002443.1


Homo sapiens cDNA FLJ11390 fis,
−1.12944
2.02889
0.364373
0.00E+00


clone HEMBA1000561, weakly similar to


ZINC FINGER PROTEIN 91.


ESTs, Weakly similar to ALU8_HUMAN
2.76608
6.07949
4.57766
1.93E−02


ALU SUBFAMILY SX SEQUENCE


CONTAMINATION WARNING ENTRY


(H. sapiens)


Homo sapiens cDNA FLJ13810 fis,
−2.92389
0.51764
−0.85322
8.08E−03


clone THYRO1000279


Homo sapiens, aminolevulinate, delta-,
1.3099
4.44525
2.76918
7.69E−03


dehydratase, clone MGC: 5057, mRNA,


complete cds. /PROD = aminolevulinate,


delta-, dehydratase /FL = gb: BC000977.1


gb: M13928.1 gb: NM_000031.1


Homo sapiens kidney-specific
1.91929
5.08917
3.44968
6.91E−03


membrane protein NX-17 mRNA,


complete cds. /PROD = kidney-specific


membrane protein NX-17


/FL = gb: AF229179.1


Homo sapiens alpha 2,8-
−1.10548
2.36359
1.05469
4.63E−03


sialyltransferase mRNA, complete cds.


/PROD = alpha 2,8-sialyltransferase


/FL = gb: L43494.1 gb: D26360.1


gb: L32867.1 gb: NM_003034.1


tyrosine 3-monooxygenasetryptophan 5-
2.98486
6.17918
4.60758
8.01E−03


monooxygenase activation protein, eta


polypeptide


Homo sapiens cardiac ankyrin repeat
−0.002291
3.35645
1.98688
3.44E−02


protein (CARP), mRNA. /PROD = cardiac


ankyrin repeat protein


/FL = gb: NM_014391.1


Homo sapiens porcupine (MG61),
1.51754
4.96311
3.68103
9.86E−03


mRNA. /PROD = porcupine


/FL = gb: AF317059.1 gb: AF317058.1


gb: NM_022825.1


collagen, type V, alpha 1
1.5875
3.98844
1.66569
1.17E−03


/FL = gb: D90279.1 gb: NM_000093.1


gb: M76729.1


Homo sapiens forkhead box F2
−1.54469
1.02305
−1.11517
6.95E−03


(FOXF2), mRNA. /PROD = forkhead box


F2 /FL = gb: U13220.1 gb: NM_001452.1


PTPRF interacting protein, binding
−1.92021
3.74378
2.77277
2.10E−02


protein 2 (liprin beta 2)


Homo sapiens PRO1957 mRNA,
1.68941
5.30311
4.22705
1.43E−02


complete cds. /PROD = PRO1957


/FL = gb: AF116676.1


Homo sapiens hypothetical protein
0.564613
5.98527
5.24692
2.85E−02


FLJ23514 (FLJ23514), mRNA.


/PROD = hypothetical protein FLJ23514


/FL = gb: NM_021827.1


Homo sapiens mRNA; cDNA
−1.80897
1.69722
0.523009
2.47E−02


DKFZp586G2120 (from clone


DKFZp586G2120); complete cds.


/PROD = hypothetical protein


/FL = gb: AL136924.1


Cluster Incl. L37033: Human FK-506
0.615619
3.478
1.66132
1.54E−02


binding protein homologue (FKBP38)


mRNA, complete cds /cds = (140, 1207)


/gb = L37033 /gi = 965469 /ug = Hs.173464


/len = 1613


ESTs
−0.004009
3.63657
2.61352
4.29E−02


Homo sapiens apolipoprotein C-I
3.24954
6.71073
5.50997
2.16E−02


(APOC1), mRNA. /PROD = apolipoprotein


C-I precursor /FL = gb: NM_001645.2


ESTs
1.38635
4.93112
3.83641
1.39E−02


Homo sapiens cDNA FLJ34035 fis,
−3.58002
−0.48718
−2.00751
1.46E−02


clone FCBBF2004788.


Homo sapiens, clone IMAGE: 3509274,
0.493686
2.94923
0.794513
1.82E−02


mRNA, partial cds


Homo sapiens putative sterol reductase
1.66857
4.3767
2.47833
5.37E−04


SR-1 (TM7SF2) mRNA, complete cds.


/PROD = putative sterol reductase SR-1


/FL = gb: AF096304.1


nuclear factor of kappa light polypeptide
2.91463
6.48938
5.47812
1.97E−02


gene enhancer in B-cells inhibitor, alpha


/FL = gb: NM_020529.1 gb: BC002601.1


gb: BC004983.1 gb: M69043.1


hypothetical protein FLJ12666
0.215767
3.10249
1.40459
1.91E−02


Homo sapiens ectodermal dysplasia 1,
0.796627
4.22895
3.07833
1.01E−02


anhidrotic (ED1), mRNA.


/PROD = ectodermal dysplasia 1,


anhidrotic /FL = gb: AF060999.1


gb: NM_001399.1 gb: AF040628.1


gb: AF061189.1


glycoprotein M6A /FL = gb: D49958.1
0.274378
3.59045
2.3278
2.64E−02


ESTs
1.04714
3.87608
2.12752
3.75E−02


ESTs
−1.48489
1.371
−0.35043
1.88E−03


ESTs, Weakly similar to KIAA1330
−0.925232
2.41149
1.18187
2.65E−03


protein (H. sapiens)


putative 47 kDa protein
0.156484
2.78771
0.861726
2.60E−02


Homo sapiens hypothetical protein
−0.595622
4.84785
3.94683
3.34E−02


FLJ32835 (FLJ32835), mRNA.


/FL = gb: NM_152506.1


Homo sapiens GS1999full mRNA,
0.137029
2.75189
0.849942
0.00E+00


complete cds. /FL = gb: AB048286.1


Homo sapiens hexabrachion (tenascin
3.96862
5.66572
2.85391
1.39E−02


C, cytotactin) (HXB), mRNA.


/PROD = hexabrachion (tenascin C,


cytotactin) /FL = gb: M55618.1


gb: NM_002160.1


Homo sapiens Pig10 (PIG10) mRNA,
0.129454
2.86013
1.09765
1.16E−03


complete cds. /PROD = Pig10


/FL = gb: AF059611.1 gb: AF010314.1


gb: NM_003633.1 gb: BC000418.1


gb: AF005381.1


Homo sapiens annexin A6 (ANXA6),
3.89026
6.43752
4.49981
8.81E−04


transcript variant 1, mRNA.


/PROD = annexin VI isoform 1


/FL = gb: D00510.1 gb: J03578.1


gb: NM_001155.2


Homo sapiens c-mer proto-oncogene
1.78994
4.92901
3.61665
1.84E−02


tyrosine kinase (MERTK), mRNA.


/PROD = c-mer proto-oncogene tyrosine


kinase /FL = gb: NM_006343.1


gb: U08023.1


Homo sapiens caspase-like apoptosis
0.434199
5.04863
5.2167
2.46E−02


regulatory protein 2 (clarp) mRNA,


alternatively spliced, complete cds.


/PROD = caspase-like apoptosis


regulatory protein 2 /FL = gb: AF005775.1


Human embryonic myosin alkali light
1.94171
5.24959
4.11475
2.11E−02


chain (MLC1) mRNA, complete cds.


/FL = gb: M36172.1 gb: M24121.1


gb: NM_002476.1


Homo sapiens phosphatidylinositol-4-
1.34876
4.12788
2.46657
3.66E−03


phosphate 5-kinase, type I, beta


(PIP5K1B), mRNA.


/PROD = phosphatidylinositol-4-


phosphate 5-kinase, typeI, beta


/FL = gb: NM_003558.1


Homo sapiens mRNA; cDNA
1.36443
4.34866
2.94283
2.92E−03


DKFZp434E082 (from clone


DKFZp434E082)


Homo sapiens dystrophin (muscular
1.40427
4.41881
3.04441
1.83E−02


dystrophy, Duchenne and Becker types),


includes DXS142, DXS164, DXS206,


DXS230, DXS239, DXS268, DXS269,


DXS270, DXS272 (DMD), transcript


variant Dp427p2, mRNA.


/PROD = dystrophin Dp427p2 isoform


/FL = gb: NM_004010.1


Homo sapiens, clone MGC: 14801,
2.03928
3.96694
1.51059
5.65E−04


mRNA, complete cds. /PROD = Unknown


(protein for MGC: 14801)


/FL = gb: BC005997.1


Homo sapiens hypothetical protein
−0.290244
2.2418
0.40157
6.04E−03


BC017868 (LOC159091), mRNA.


/FL = gb: BC017868.1 gb: NM_138819.1


ESTs, Highly similar to AF229172 1
−0.475471
1.94826
0.002417
5.28E−03


class III myosin (H. sapiens)


Homo sapiens solute carrier family 9
0.047487
2.80601
1.19645
2.80E−03


(sodiumhydrogen exchanger), isoform 5


(SLC9A5), mRNA. /PROD = solute carrier


family 9 (sodiumhydrogenexchanger),


isoform 5 /FL = gb: AF111173.1


gb: NM_004594.1


Homo sapiens F37Esophageal cancer-
−0.128814
3.17069
2.10422
1.54E−02


related gene-coding leucine-zipper motif


(FEZ1), mRNA. /PROD = F37Esophageal


cancer-related gene-codingleucine-


zipper motif /FL = gb: AF123659.1


gb: NM_021020.1


Homo sapiens deiodinase,
−0.136937
5.08366
4.22897
1.41E−02


iodothyronine, type III (DIO3), mRNA.


/PROD = thyroxine deiodinase type III


/FL = gb: NM_001362.1 gb: S79854.1


Homo sapiens insulin-like growth factor
2.6126
5.13945
3.30402
9.72E−04


binding protein 6 (IGFBP6), mRNA.


/PROD = insulin-like growth factor binding


protein 6 /FL = gb: BC005007.1


gb: M62402.1 gb: BC003507.1


gb: NM_002178.1


Homo sapiens U2 small nuclear
−0.821533
1.81533
0.096512
4.72E−02


ribonucleoprotein auxiliary factor (65 kD)


(U2AF65), mRNA. /PROD = U2 small


nuclear ribonucleoprotein auxiliaryfactor


(65 kD) /FL = gb: NM_007279.1


Homo sapiens hypothetical protein
−1.11464
1.81375
0.394686
9.09E−03


DKFZp434F0318 (DKFZP434F0318),


mRNA. /PROD = hypothetical protein


DKFZp434F0318 /FL = gb: NM_030817.1


Homo sapiens BACE mRNA for beta-
0.48553
3.13284
1.44588
4.99E−04


site APP cleaving enzyme I-476,


complete cds. /PROD = beta-site APP


cleaving enzyme I-476


/FL = gb: AB050436.1


ESTs, Weakly similar to ALUC_HUMAN
1.07951
4.06134
2.7193
2.11E−02


!!!! ALU CLASS C WARNING ENTRY !!!


(H. sapiens)


Homo sapiens mRNA for KIAA0876
−0.432037
1.97736
0.073246
3.37E−02


protein, partial cds. /PROD = KIAA0876


protein


Homo sapiens mRNA for protein-
0.160986
2.75713
1.04172
2.11E−02


tyrosine kinase, complete cds.


/PROD = protein-tyrosine kinase


/FL = gb: U05682.1 gb: D17517.1


gb: D50479.1


KIAA0418 gene product
−0.677643
1.83134
0.034385
1.45E−04


Homo sapiens phosphofructokinase,
2.039
4.51747
2.70388
1.28E−03


liver (PFKL), mRNA.


/PROD = phosphofructokinase, liver


/FL = gb: NM_002626.1 gb: BC004920.1


gb: X15573.1


Homo sapiens enolase 2, (gamma,
2.60624
5.37458
3.92621
3.17E−03


neuronal) (ENO2), mRNA.


/PROD = enolase 2, (gamma, neuronal)


/FL = gb: NM_001975.1 gb: BC002745.1


gb: M22349.1


Homo sapiens AD036 mRNA, complete
−0.503334
2.65787
1.60278
3.61E−02


cds. /PROD = AD036 /FL = gb: AF260333.1


ESTs
−2.07933
1.00157
−0.1307
2.39E−02


Homo sapiens keratin 19 (KRT19),
3.80582
6.96535
5.91937
2.11E−02


mRNA. /PROD = keratin 19


/FL = gb: NM_002276.1 gb: BC002539.1


pleiomorphic adenoma gene-like 1
−0.646793
1.88692
0.221481
4.21E−03


/FL = gb: U72621.3


Homo sapiens, Similar to lipase protein,
−1.16073
1.9994
0.961706
1.89E−02


clone MGC: 2843, mRNA, complete cds.


/PROD = Similar to lipase protein


/FL = gb: NM_020676.1 gb: BC001698.1


Cluster Incl. AB002344: Human mRNA
2.15679
4.91332
3.47797
7.39E−03


for KIAA0346 gene, partial cds


/cds = (0, 4852) /gb = AB002344


/gi = 2280479 /ug = Hs.103915 /len = 6121


Cluster Incl. N80935: zb07g06.s1 Homo
2.12419
4.48236
2.66555
1.50E−02


sapiens cDNA, 3 end /clone = IMAGE-


301402 /clone_end = 3 /gb = N80935


/gi = 1243636 /ug = Hs.22483 /len = 527


tryptase, alpha
2.99108
5.46095
3.76442
1.31E−03


ESTs, Weakly similar to Z132_HUMAN
1.04865
3.65906
2.12166
2.43E−02


ZINC FINGER PROTEIN 13 (H. sapiens)


Homo sapiens core histone
1.67293
4.73906
3.66212
3.62E−02


macroH2A2.2 (MACROH2A2), mRNA.


/PROD = core histone macroH2A2.2


/FL = gb: AF151534.1 gb: NM_018649.1


ESTs
−0.146413
2.69334
1.39451
1.02E−02


Human rho GDI mRNA, complete cds.
0.019358
1.75075
−0.65155
1.85E−02


/PROD = human rho GDI


/FL = gb: M97579.1 gb: D13989.1


gb: NM_004309.1


heat shock 90 kD protein 1, alpha
1.69225
4.20576
2.60887
2.10E−02


Homo sapiens BTB (POZ) domain
1.10085
3.7628
2.32464
1.85E−02


containing 2 (BTBD2), mRNA.


/PROD = BTB (POZ) domain containing 2


/FL = gb: NM_017797.1


Homo sapiens mandaselin long form
−0.148053
2.58361
1.2296
1.57E−02


mRNA, complete cds.


/PROD = mandaselin long form


/FL = gb: AY048775.1


signal transducer and activator of
−0.837261
1.90254
0.560225
8.83E−04


transcription 3 (acute-phase response


factor)


Homo sapiens lymphocyte antigen 6
0.866364
4.91832
4.92642
2.78E−02


complex, locus E (LY6E), mRNA.


/PROD = lymphocyte antigen 6 complex,


locus E /FL = gb: U42376.1


gb: NM_002346.1 gb: U56145.1


ESTs
−1.24632
1.09042
−0.61316
4.05E−02


Homo sapiens, clone IMAGE: 4047715,
−2.50816
1.81
1.52693
2.63E−02


mRNA.


Cluster Incl. AB002344: Human mRNA
1.89578
4.61333
3.31717
1.12E−02


for KIAA0346 gene, partial cds


/cds = (0, 4852) /gb = AB002344


/gi = 2280479 /ug = Hs.103915 /len = 6121


Homo sapiens singed (Drosophila)-like
4.23317
6.74774
5.25403
5.12E−03


(sea urchin fascin homolog like) (SNL),


mRNA. /PROD = singed (Drosophila)-like


(sea urchin fascinhomolog like)


/FL = gb: BC000521.1 gb: NM_003088.1


gb: U03057.1 gb: U09873.1


Homo sapiens mRNA; cDNA
−2.72593
1.58998
1.27994
4.54E−02


DKFZp586L0120 (from clone


DKFZp586L0120).


Homo sapiens mitogen-activated protein
0.647994
3.46459
2.28644
6.65E−03


kinase 10 (MAPK10), mRNA.


/PROD = mitogen-activated protein kinase


10 /FL = gb: U07620.1 gb: U34819.1


gb: U34820.1 gb: NM_002753.1


Homo sapiens transmembrane tyrosine
−0.006089
2.96035
1.93237
7.69E−03


kinase mRNA, complete cds.


/PROD = tyrosine kinase


/FL = gb: L08961.1


Homo sapiens mRNA; cDNA
2.83663
5.30784
3.79206
6.22E−03


DKFZp762H185 (from clone


DKFZp762H185)


tyrosine 3-monooxygenasetryptophan 5-
3.48271
6.19368
4.92556
2.11E−02


monooxygenase activation protein, eta


polypeptide


Homo sapiens mRNA; cDNA
0.581419
3.38088
2.21428
3.89E−02


DKFZp434K0621 (from clone


DKFZp434K0621); partial cds


ESTs, Weakly similar to ALUF_HUMAN
−1.03247
1.75349
0.587598
2.11E−02


!!!! ALU CLASS F WARNING ENTRY !!!


(H. sapiens)


tudor repeat associator with PCTAIRE 2
2.15771
4.69127
3.27451
2.63E−02


Homo sapiens mRNA; cDNA
4.49405
7.38448
6.33474
1.39E−02


DKFZp564K1672 (from clone


DKFZp564K1672); partial cds.


/PROD = hypothetical protein


ESTs
−0.184212
2.15367
0.554696
1.15E−02


Homo sapiens bHLH factor Hes4
0.47816
3.40725
2.40001
3.16E−02


(LOC57801), mRNA. /PROD = bHLH


factor Hes4 /FL = gb: NM_021170.1


gb: AB048791.1


Homo sapiens guanylate cyclase
−0.714216
1.95339
0.684983
3.38E−03


activator 1B (retina) (GUCA1B), mRNA.


/PROD = guanylate cyclase activator 1B


(retina) /FL = gb: M95174.1


gb: NM_002098.1 gb: M97496.1


KIAA0918 protein
−1.39708
0.961939
−0.57499
1.40E−02


Homo sapiens mRNA for KIAA1161
0.781796
3.32846
1.98143
6.60E−03


protein, partial cds. /PROD = KIAA1161


protein


Homo sapiens, Similar to B9 protein,
1.43881
3.80457
2.29205
8.62E−05


clone MGC: 11339, mRNA, complete


cds. /PROD = Similar to B9 protein


/FL = gb: BC002944.1


hypothetical protein FLJ12666
2.34087
5.11201
4.02837
1.97E−02


Homo sapiens FLICE-like inhibitory
0.135783
4.45631
4.92758
1.96E−02


protein short form mRNA, complete cds.


/PROD = FLICE-like inhibitory protein


short form /FL = gb: U97075.1


Homo sapiens olfactory receptor, family
−0.309574
2.08723
0.636487
4.36E−03


52, subfamily A, member 1 (OR52A1),


mRNA. /PROD = olfactory receptor, family


52, subfamily A, member 1


/FL = gb: NM_012375.1


Human DNA sequence from clone RP4-
2.82768
5.54517
4.43909
2.04E−03


781L3 on chromosome 1p34.3-36.11


Contains a pseudogene similar to


IFITM3 (interferon


inducedntransmembrane protein 3 (1-


8U)), STSs and GSSs


Homo sapiens KIAA0127 gene product
2.05236
4.43794
3.00216
3.12E−02


(KIAA0127), mRNA. /PROD = KIAA0127


gene product /FL = gb: D50917.1


gb: NM_014755.1


Homo sapiens clone HB-2 mRNA
−0.503949
2.27896
1.25171
3.01E−02


sequence


hypothetical protein FLJ23091
−0.76092
3.70916
3.04154
2.45E−02


ESTs
−0.403665
2.01868
0.643615
5.11E−04


ESTs
2.14401
4.70353
3.47114
3.15E−03


Homo sapiens regulator of G protein
3.0091
5.37511
3.95132
1.29E−03


signalling 5 (RGS5) mRNA, complete


cds. /PROD = regulator of G protein


signalling 5 /FL = gb: AF493929.1


endothelin receptor type A
−1.83423
2.54948
1.95432
1.51E−02


/FL = gb: NM_001957.1 gb: L06622.1


H. sapiens skeletal embryonic myosin
0.690058
3.3896
2.36018
3.27E−02


light chain 1 (MLC1) mRNA.


/PROD = myosin light chain 1


Homo sapiens Kruppel-like factor 8
0.326119
2.825
1.63929
3.92E−02


(KLF8), mRNA. /PROD = Kruppel-like


factor 8 /FL = gb: U28282.1


gb: NM_007250.1


hypothetical protein DKFZp434F2322
0.070576
2.58185
1.42121
5.81E−03


Homo sapiens transcription factor 2,
−1.12537
3.45355
2.54347
1.15E−02


hepatic; LF-B3; variant hepatic nuclear


factor (TCF2), transcript variant a,


mRNA. /PROD = transcription factor 2,


isoform a /FL = gb: NM_000458.1


H2A histone family, member X
1.37085
2.48515
−0.04057
1.34E−02


Homo sapiens H1 histone family,
3.21701
5.68105
4.50969
4.29E−03


member X (H1FX), mRNA. /PROD = H1


histone family, member X


/FL = gb: D64142.1 gb: BC000426.1


gb: NM_006026.1


ESTs, Weakly similar to KIAA1399
−0.831241
2.82364
2.79997
4.18E−02


protein (H. sapiens)


Homo sapiens PNAS-145 mRNA,
2.84962
5.38084
4.2868
8.61E−03


complete cds. /PROD = PNAS-145


/FL = gb: U03105.1 gb: NM_006813.1


gb: AF279899.1


Homo sapiens erythrocyte membrane
−0.306603
2.11391
0.923806
4.81E−03


protein band 4.9 (dematin) (EPB49),


mRNA. /PROD = erythrocyte membrane


protein band 4.9 (dematin)


/FL = gb: NM_001978.1 gb: U28389.1


ESTs
0.783545
3.23146
2.07738
2.13E−02


ESTs
1.17515
3.61815
2.46265
1.56E−03


nudix (nucleoside diphosphate linked
2.09472
5.82355
5.68859
3.63E−02


moiety X)-type motif 4


Novel human mRNA from chromosome
−0.533665
3.44679
3.05803
3.99E−02


22. /PROD = hypothetical protein


Homo sapiens partial mRNA for putative
1.55224
3.89366
2.67866
1.53E−03


nuclear factor. /PROD = putative nuclear


factor /FL = gb: NM_017688.1


Homo sapiens BCL2adenovirus E1B
4.51142
6.99506
5.92755
2.25E−03


19 kD-interacting protein 3 (BNIP3),


nuclear gene encoding mitochondrial


protein, mRNA.


/PROD = BCL2adenovirus E1B 19 kD-


interacting protein 3 /FL = gb: AF002697.1


gb: NM_004052.2 gb: U15174.1


Homo sapiens bone morphogenetic
−0.6099
1.76351
0.614791
6.46E−03


protein 5 (BMP5), mRNA. /PROD = bone


morphogenetic protein 5


/FL = gb: M60314.1 gb: NM_021073.1


Homo sapiens nuclear receptor
−0.213822
4.14059
3.29782
3.42E−02


subfamily 0, group B, member 1


(NR0B1), mRNA. /PROD = adrenal


hypoplasia protein /FL = gb: NM_000475.2


Homo sapiens mRNA; cDNA
1.76846
4.196
3.11284
1.23E−02


DKFZp434P228 (from clone


DKFZp434P228)


Homo sapiens pilin-like transcription
3.14467
5.51066
4.38212
3.80E−03


factor (PILB), mRNA. /PROD = pilin-like


transcription factor /FL = gb: AF122004.1


gb: NM_012228.1


Homo sapiens complement component
−1.89639
1.65637
1.59643
1.74E−02


5 (C5), mRNA. /PROD = complement


component 5 /FL = gb: M57729.1


gb: NM_001735.1


Homo sapiens adaptor-related protein
−0.208283
2.17404
1.07398
1.39E−02


complex 3, beta 2 subunit (AP3B2),


mRNA. /PROD = adaptor-related protein


complex 3, beta 2subunit


/FL = gb: AF022152.1 gb: NM_004644.1


gb: U37673.1


Homo sapiens dynein, axonemal, light
0.458271
2.88755
1.8494
3.66E−03


polypeptide 4 (DNAL4), mRNA.


/PROD = dynein, axonemal, light


polypeptide 4 /FL = gb: BC002968.1


gb: NM_005740.1


Homo sapiens, Similar to RIKEN cDNA
0.087468
2.45867
1.42701
3.35E−03


C330013D18 gene, clone MGC: 11226,


mRNA, complete cds


Homo sapiens cDNA: FLJ22731 fis,
−2.30948
1.62817
2.24143
4.15E−02


clone HSI15841.


ESTs
0.532996
4.6172
3.79228
2.76E−02


Homo sapiens CXCR4 gene encoding
3.11086
7.24917
6.36084
3.13E−02


receptor CXCR4


stanniocalcin 1
2.48686
6.20245
5.70081
4.94E−02


KIAA0761 protein
−2.09591
1.57935
1.05907
1.86E−02


ESTs
−1.16374
2.24964
2.52108
3.83E−02


Homo sapiens cDNA FLJ36116 fis,
3.02587
6.7146
6.15456
3.88E−02


clone TESTI2022338.


Homo sapiens, clone MGC: 24252
−1.53346
2.53854
1.59251
3.91E−03


IMAGE: 3932604, mRNA, complete cds.


/PROD = Unknown (protein for


MGC: 24252) /FL = gb: BC014364.1


Homo sapiens cDNA FLJ13392 fis,
−1.90184
1.76129
1.16856
1.47E−02


clone PLACE1001280


Homo sapiens hypothetical protein
1.70617
5.11419
4.73391
2.71E−02


FLJ12538 similar to ras-related protein


RAB17 (FLJ12538), mRNA.


/PROD = hypothetical protein FLJ12538


similar toras-related protein RAB17


/FL = gb: AL136645.1 gb: NM_022449.1


Human DNA sequence from clone
−0.889422
2.90076
2.11955
3.52E−02


RP11-446H13 on chromosome 10.


Contains the 3 end of the gene for a


novel protein similar to KIAA1059


(ortholog of mouse VPS10 domain


receptor protein SORCS), an RPL23A


(60S ribosmal protein 23A) pseudogene,


ESTs, STSs an


Homo sapiens calmegin (CLGN),
−0.104278
2.97817
3.11092
1.89E−02


mRNA. /PROD = calmegin


/FL = gb: NM_004362.1 gb: D86322.1


Homo sapiens testican 3 (HSAJ1454),
−2.81226
0.7939
1.55068
3.99E−02


mRNA. /PROD = testican 3


/FL = gb: NM_016950.1 gb: BC000460.1


gb: BC003017.1


Homo sapiens gelsolin (amyloidosis,
1.6198
4.54047
4.45008
1.07E−02


Finnish type) (GSN), mRNA.


/PROD = gelsolin (amyloidosis, Finnish


type) /FL = gb: NM_000177.1


Homo sapiens endothelin receptor type
0.763007
4.05983
3.55203
3.99E−02


A (EDNRA), mRNA. /PROD = endothelin


receptor type A /FL = gb: NM_001957.1


gb: L06622.1


Homo sapiens cDNA: FLJ22808 fis,
−0.367449
2.7584
2.41394
4.31E−02


clone KAIA2925.


Homo sapiens hypothetical protein
1.59498
4.6335
4.89157
4.85E−02


FLJ10312 (FLJ10312), mRNA.


/PROD = hypothetical protein FLJ10312


/FL = gb: NM_030672.1


Homo sapiens lipase mRNA, complete
−2.2131
1.0208
0.55849
3.57E−02


cds. /PROD = lipase /FL = gb: AF225418.1


Homo sapiens clone FLC1492 PRO3121
−0.300037
−0.46894
−3.39203
1.80E−02


mRNA, complete cds. /PROD = PRO3121


/FL = gb: AF130082.1


protease, serine, 4 (trypsin 4, brain)
−0.345569
2.86256
3.35886
4.65E−02


Homo sapiens ret finger protein-like 2
−2.15511
1.1797
0.503917
1.94E−02


(RFPL2), mRNA. /PROD = ret finger


protein-like 2 /FL = gb: NM_006605.1


Homo sapiens beta3GalNAcT-1 mRNA
−2.73257
0.21269
−0.0984
2.48E−02


for globoside synthase, complete cds,


clone: type 2. /PROD = globoside


synthase /FL = gb: AB050856.1


Homo sapiens hypothetical protein
−3.34565
−0.42738
−0.71255
3.07E−02


FLJ11155 (FLJ11155), mRNA.


/PROD = hypothetical protein FLJ11155


/FL = gb: NM_018342.1


Homo sapiens, dual specificity
2.11487
5.16681
5.60057
3.11E−02


phosphatase 4, clone MGC: 3713,


mRNA, complete cds. /PROD = dual


specificity phosphatase 4


/FL = gb: NM_001394.2 gb: BC002671.1


gb: U48807.1 gb: U21108.1


Homo sapiens MAD (mothers against
0.917516
4.29847
3.5309
2.44E−02


decapentaplegic, Drosophila) homolog 6


(MADH6), mRNA. /PROD = MAD


(mothers against


decapentaplegic, Drosophila) homolog 6


/FL = gb: U59914.1 gb: NM_005585.1


secreted frizzled-related protein 1
1.64933
4.41286
4.21799
4.12E−02


/FL = gb: AF056087.1 gb: NM_003012.2


gb: AF017987.1 gb: AF001900.1


Homo sapiens secreted apoptosis
3.18446
5.9316
5.74251
3.31E−02


related protein 2 (SARP2) mRNA,


complete cds. /PROD = secreted


apoptosis related protein 2


/FL = gb: AF056087.1 gb: NM_003012.2


gb: AF017987.1 gb: AF001900.1


KIAA0882 protein
2.05587
4.63577
4.67451
4.89E−02


Homo sapiens nudix (nucleoside
3.91519
6.54632
6.42767
4.63E−02


diphosphate linked moiety X)-type motif


4 (NUDT4), mRNA. /PROD = nudix


(nucleoside diphosphate linked


moietyX)-type motif 4


/FL = gb: NM_019094.1 gb: AF191653.1


gb: AF191649.1 gb: AF191650.1


Homo sapiens cDNA FLJ31061 fis,
−1.30452
2.06218
1.20749
1.72E−02


clone HSYRA2000927.


Homo sapiens diphosphoinositol
2.62612
5.53515
5.12935
3.96E−02


polyphosphate phosphohydrolase type 2


beta (NUDT4) mRNA, complete cds.


/PROD = diphosphoinositol


polyphosphate phosphohydrolasetype 2


beta /FL = gb: NM_019094.1


gb: AF191653.1 gb: AF191649.1


gb: AF191650.1


Homo sapiens titin (TTN), mRNA.
−0.466154
2.46856
2.00309
4.18E−03


/PROD = titin /FL = gb: NM_003319.1


carboxypeptidase E
2.72316
5.80072
5.15668
2.99E−02


/FL = gb: NM_001873.1


ESTs
−1.80443
0.692933
0.613229
1.46E−03


Homo sapiens hypothetical protein
−1.60435
1.6149
2.4204
2.10E−02


FLJ39502 (FLJ39502), mRNA.


/FL = gb: NM_173648.1


Homo sapiens renal tumor antigen
−0.305406
2.4556
2.09136
3.30E−02


(RAGE), mRNA. /PROD = renal tumor


antigen /FL = gb: NM_014226.1


gb: AB022694.1


T-box 3 (ulnar mammary syndrome)
−1.70952
1.31576
0.679584
1.29E−02


Homo sapiens mitogen-activated protein
−1.14241
1.82177
1.23928
2.48E−02


kinase kinase kinase 8 (MAP3K8),


mRNA. /PROD = mitogen-activated


protein kinase kinase kinase8


/FL = gb: NM_005204.1 gb: D14497.1


Homo sapiens enoyl-Coenzyme A,
0.226733
3.18231
2.59938
6.22E−03


hydratase3-hydroxyacyl Coenzyme A


dehydrogenase (EHHADH), nuclear


gene encoding mitochondrial protein,


mRNA. /PROD = enoyl-Coenzyme A,


hydratase3-hydroxyacylCoenzyme A


dehydrogenase /FL = gb: NM_001966.1


gb: L07077.1


ESTs
1.65525
3.9984
3.98375
1.97E−02


Homo sapiens secreted frizzled-related
3.91678
7.18429
6.24313
2.35E−02


protein 1 (SFRP1), mRNA.


/PROD = secreted frizzled-related protein


1 /FL = gb: AF056087.1 gb: NM_003012.2


gb: AF017987.1 gb: AF001900.1


ESTs
2.28659
5.21675
4.59884
3.11E−02


Homo sapiens selenoprotein P, plasma,
2.73332
5.13625
5.04138
1.75E−02


1 (SEPP1), mRNA.


/PROD = selenoprotein P precursor


/FL = gb: NM_005410.1


hypothetical protein FLJ12838
2.21522
5.4785
4.50835
4.55E−02


/FL = gb: NM_024641.1


Homo sapiens mRNA; cDNA
−2.59761
0.092924
−0.31569
4.19E−02


DKFZp761M1216 (from clone


DKFZp761M1216)


Homo sapiens KPL1 (KPL1) mRNA,
−0.08886
2.51294
2.15967
1.97E−02


complete cds. /PROD = KPL1


/FL = gb: AF081583.1 gb: U89715.1


gb: NM_021200.1


Homo sapiens, synovial sarcoma, X
−1.57209
1.39869
2.12662
6.84E−03


breakpoint 1, clone MGC: 5162, mRNA,


complete cds. /PROD = synovial


sarcoma, X breakpoint 1


/FL = gb: BC001003.2 gb: NM_005635.1


Homo sapiens, Similar to testican 3,
−2.778
0.563149
1.66935
1.87E−02


clone MGC: 8506, mRNA, complete cds.


/PROD = Similar to testican 3


/FL = gb: NM_016950.1 gb: BC000460.1


gb: BC003017.1


ESTs
0.502949
3.0682
2.72874
2.02E−02


Homo sapiens cDNA FLJ32963 fis,
1.49644
3.87336
3.68478
6.89E−03


clone TESTI2008405.


triadin /FL = gb: U18985.1
−1.61408
0.713083
0.566175
1.87E−02


gb: NM_006073.1


Homo sapiens adlican mRNA, complete
0.872786
3.20404
3.02406
4.21E−02


cds. /PROD = adlican


/FL = gb: AF245505.1


Human midkine mRNA, complete cds.
3.55275
5.93819
5.68363
1.89E−02


/PROD = midkine /FL = gb: NM_002391.1


gb: M69148.1


Homo sapiens, synovial sarcoma, X
−0.806785
1.90972
2.5215
4.17E−02


breakpoint 4, clone MGC: 12411, mRNA,


complete cds. /PROD = synovial


sarcoma, X breakpoint 4


/FL = gb: BC005325.1


ESTs
−3.61193
−0.9408
−1.53439
2.43E−03


ESTs
−0.619714
1.93617
1.44843
4.14E−02


ESTs
0.446983
3.14315
2.48676
1.98E−02


Homo sapiens flavin containing
−0.476203
1.99948
1.55491
3.73E−02


monooxygenase 5 (FMO5), mRNA.


/PROD = flavin containing


monooxygenase 5 /FL = gb: L37080.1


gb: NM_001461.1


Homo sapiens aminopeptidase A
−0.758987
2.07596
1.20831
3.00E−02


mRNA, complete cds.


/PROD = aminopeptidase A


/FL = gb: L12468.1 gb: NM_001977.1


gb: L14721.1


Homo sapiens hypothetical protein
−0.713164
2.03145
1.24823
2.17E−03


DKFZp761H1710 (DKFZP761H1710),


mRNA. /PROD = hypothetical protein


DKFZp761H1710 /FL = gb: NM_031297.1


ESTs
−1.94194
0.608912
0.013238
1.72E−02


Homo sapiens sialyltransferase 8 (alpha-
−1.07243
1.83889
0.855205
2.16E−02


2,8-polysialytransferase) D (SIAT8D),


mRNA. /PROD = sialyltransferase 8


(alpha-2,8-polysialytransferase) D


/FL = gb: NM_005668.1 gb: L41680.1


Homo sapiens, clone IMAGE: 5194204,
−2.13256
0.383746
1.02639
4.55E−02


mRNA.


hypothetical protein MGC4342
−0.403018
2.01722
1.41263
1.97E−02


/FL = gb: NM_024329.1 gb: BC003033.1


Homo sapiens PHD finger protein 1
0.620171
2.95191
2.42296
2.32E−02


(PHF1), transcript variant 2, mRNA.


/PROD = PHD finger protein 1, isoform b


/FL = gb: NM_024165.1 gb: AF052205.1


actinin, alpha 4
0.436893
3.11051
2.23625
5.44E−03


Homo sapiens PCTAIRE protein kinase
2.38171
4.87071
4.16957
2.90E−03


1 (PCTK1), mRNA. /PROD = PCTAIRE


protein kinase 1 /FL = gb: NM_006201.1


Human DNA sequence from clone
0.167347
2.56439
1.93736
3.93E−02


RP11-165F24 on chromosome 9.


Contains the 3 end of the gene for a


novel protein (similar to Drosophila


CG6630 and CG11376, KIAA1058, rat


TRG), an RPL12 (60S ribosomal protein


L12) pseudogene, ESTs, STSs, GSSs


and a C . . .


insulin-like growth factor binding protein
0.921068
3.60279
2.67905
4.80E−02


3 /FL = gb: NM_000598.1


Homo sapiens, clone IMAGE: 3840937,
3.59778
6.12936
6.92649
8.62E−05


mRNA, partial cds. /PROD = Unknown


(protein for IMAGE: 3840937)


Homo sapiens phosphoglucomutase 1
4.74364
7.09524
6.43056
2.21E−02


(PGM1), mRNA.


/PROD = phosphoglucomutase 1


/FL = gb: NM_002633.1 gb: BC001756.1


gb: M83088.1


apolipoprotein C-I
2.77701
5.23054
4.44049
3.39E−02


Homo sapiens insulin induced gene 1
2.11462
4.44128
3.77765
3.21E−02


(INSIG1), mRNA. /PROD = insulin


induced gene 1 /FL = gb: NM_005542.1


Human a6 (IV) collagen (COL4A6)
0.798849
3.23623
2.41372
1.38E−03


mRNA, complete cds. /PROD = A type IV


collagen /FL = gb: U04845.1


Homo sapiens mRNA for alpha 1,6-
1.31479
3.6699
2.90742
3.48E−02


fucosyltransferase, complete cds.


/PROD = alpha 1,6-fucosyltransferase


/FL = gb: AB049740.2


ESTs, Moderately similar to Six5
0.434259
2.81512
1.9556
1.23E−02


(M. musculus)


solute carrier family 2 (facilitated glucose
5.23156
7.63734
6.74444
1.99E−02


transporter), member 3


/FL = gb: NM_006931.1 gb: M20681.1


Human acid sphingomyelinase (ASM)
1.03965
3.45689
2.52125
2.62E−02


mRNA, complete cds. /PROD = acid


sphingomyelinase /FL = gb: NM_000543.1


gb: M59916.1


ESTs, Weakly similar to unnamed
3.0131
5.41021
4.46614
4.79E−03


protein product (H. sapiens)


Homo sapiens cDNA FLJ33178 fis,
0.137476
−1.9718
−4.73502
2.23E−03


clone ADRGL2002753.


Homo sapiens, parathyroid hormone-like
2.46289
−0.34647
−1.96791
3.05E−02


hormone, clone MGC: 14611, mRNA,


complete cds. /PROD = parathyroid


hormone-like hormone


/FL = gb: BC005961.1


Homo sapiens colon carcinoma related
2.50614
−0.01889
−1.04926
2.52E−02


protein (LOC51159), mRNA.


/PROD = colon carcinoma related protein


/FL = gb: NM_016206.1 gb: AF099505.1


KIAA0036 gene product
3.05174
−0.66523
−2.2068
2.62E−02


Homo sapiens cDNA FLJ13536 fis,
4.27968
1.86625
1.70965
1.11E−02


clone PLACE1006521


Homo sapiens cDNA: FLJ22463 fis,
3.68063
1.24806
1.39728
1.42E−02


clone HRC10126


Homo sapiens UDP-N-
2.71164
0.270906
0.433883
1.98E−02


acetylglucosamine:a-1,3-D-mannoside


beta-1,4-N-


acetylglucosaminyltransferase IV-


homolog (HGNT-IV-H), mRNA.


/PROD = UDP-N-acetylglucosamine:a-


1,3-D-mannosidebeta-1,4-N-


acetylglucosaminyltransferase IV-


homolog /FL = gb: AB024729.1 gb: NM_0


tissue factor pathway inhibitor 2
4.19529
1.63281
1.58393
7.53E−03


/FL = gb: D29992.1 gb: L27624.1


gb: NM_006528.1 gb: BC005330.1


ESTs
2.8924
0.521234
0.762594
2.82E−02


putative gene product
−0.518738
−0.66615
1.82038
4.91E−02


Homo sapiens mRNA for KIAA1758
2.42082
−0.03923
0.213473
1.25E−02


protein, partial cds. /PROD = KIAA1758


protein


prostaglandin E receptor 4 (subtype
2.58497
0.196846
0.527172
1.42E−02


EP4) /FL = gb: L25124.1 gb: D28472.1


gb: NM_000958.1 gb: L28175.1


Homo sapiens cDNA: FLJ22463 fis,
2.85068
0.473875
0.830447
1.97E−02


clone HRC10126


Homo sapiens heparan sulfate
3.18462
0.571052
0.709206
4.54E−02


(glucosamine) 3-O-sulfotransferase 2


(HS3ST2), mRNA. /PROD = heparan


sulfate D-glucosaminyl3-O-


sulfotransferase 2 /FL = gb: AF105375.1


gb: AF105374.1 gb: NM_006043.1


Homo sapiens schwannomin interacting
5.5957
3.21054
3.57915
2.11E−02


protein 1 (SCHIP-1), mRNA.


/PROD = schwannomin interacting protein


1 /FL = gb: AF145713.1 gb: NM_014575.1


Human lysyl oxidase (LOX) gene, exon 7
2.11886
−0.24936
0.138069
2.55E−03


Human nephropontin mRNA, complete
7.82555
5.27856
5.06542
4.61E−02


cds. /PROD = nephropontin


/FL = gb: M83248.1


Human mRNA for KIAA0386 gene,
3.9213
1.56305
1.13663
1.93E−02


complete cds. /FL = gb: AB002384.1


ESTs
3.22146
0.851355
0.404487
3.41E−03


Human glioma pathogenesis-related
3.20187
0.502872
0.348681
1.34E−02


protein (GliPR) mRNA, complete cds.


/PROD = glioma pathogenesis-related


protein /FL = gb: NM_006851.1


gb: U16307.1


ESTs
2.96697
0.440894
0.773495
1.97E−02


Homo sapiens cDNA FLJ13384 fis,
5.9221
3.48419
3.9531
3.38E−02


clone PLACE1001062, highly similar to


Homo sapiens mRNA for lysine-


ketoglutarate reductasesaccharopine


dehydrogenase.


Homo sapiens mRNA; cDNA
6.06894
3.31919
3.4798
4.97E−02


DKFZp761I1912 (from clone


DKFZp761I1912)


ESTs
4.12419
1.53997
1.88239
5.68E−03


Homo sapiens fibroblast growth factor 2
5.49297
2.64534
2.76054
4.71E−02


(basic) (FGF2), mRNA.


/PROD = fibroblast growth factor 2 (basic)


/FL = gb: NM_002006.1 gb: M27968.1


ribosomal protein L34 pseudogene 1
3.45116
2.78236
5.10812
2.42E−02


HIV-1 rev binding protein 2
4.4245
1.47599
1.52824
2.10E−02


H. sapiens FGF gene, exon 3
2.04849
−0.34703
−0.95745
3.33E−02


/FL = gb: NM_000800.1 gb: M13361.1


ESTs
2.03304
−0.9164
−0.83676
4.19E−02


ESTs
2.87393
0.428858
1.02011
9.72E−04


Homo sapiens clone 23700 mRNA
4.60148
1.64243
1.72052
4.74E−02


sequence


ESTs, Highly similar to S21424 nestin
3.08579
0.725853
1.40345
1.99E−02


(H. sapiens)


Homo sapiens actin, gamma 2, smooth
7.17155
4.15208
4.18551
4.72E−02


muscle, enteric (ACTG2), mRNA.


/PROD = actin, gamma 2 propeptide


/FL = gb: NM_001615.2


hypothetical protein FLJ22833
2.80785
0.480636
1.25154
2.43E−02


Homo sapiens LIM homeobox protein 6
2.52571
−0.06036
0.459727
4.37E−02


(LHX6), mRNA. /PROD = LIM homeobox


protein 6 /FL = gb: AB031041.1


gb: NM_014368.1 gb: AL136570.1


Homo sapiens, clone IMAGE: 5271039,
3.05576
0.189935
0.452681
4.05E−02


mRNA.


Homo sapiens cAMP response element-
2.90971
0.487264
1.21334
4.24E−02


binding protein CRE-BPa


(H_GS165L15.1), mRNA. /PROD = cAMP


response element-binding protein CRE-


BPa /FL = gb: NM_004904.1 gb: L05911.1


G protein-coupled receptor 1
1.15804
−1.24345
−0.49352
2.28E−03


/FL = gb: NM_005279.1


Homo sapiens neuropilin (NRP) and
3.25525
0.20233
0.301354
2.83E−02


tolloid (TLL)-like 1 (NETO1), transcript


variant 3, mRNA. /PROD = neuropilin-


and tolloid-like protein 1 isoform


3precursor /FL = gb: AF448838.1


gb: NM_138966.2


Homo sapiens mRNA for KIAA0559
2.37088
−0.29358
0.199028
2.81E−02


protein, partial cds. /PROD = KIAA0559


protein


Human 65-kilodalton phosphoprotein
3.25522
0.620942
1.15317
2.88E−02


(p65) mRNA, complete cds.


/PROD = phosphoprotein p65


/FL = gb: M22300.1 gb: NM_002298.2


gb: J02923.1


Human DNA sequence from clone
4.77522
2.18971
2.77459
4.04E−02


RP11-31K16 on chromosome 9.


Contains a snoRNA binding domain


pseudogene, the ELAVL2 gene for


ELAV (embryonic lethal, abnormal


vision, Drosophila)-like 2, ESTs, STSs,


GSSs and a CpG island


hypothetical protein FLJ11006
1.75389
−0.66646
0.100591
7.78E−04


Homo sapiens mRNA; cDNA
3.97344
1.32598
1.8683
3.19E−02


DKFZp566A1046 (from clone


DKFZp566A1046)


ESTs
4.13356
1.60769
2.27415
1.85E−02


H. sapiens gene from PAC 106H8.
3.05906
−0.08711
−0.13845
4.34E−02


Human T-cell receptor rearranged beta-
3.43862
0.94127
1.64514
5.31E−03


chain V-region (V-D-J) mRNA, complete


cds. /FL = gb: M15564.1


peptidylprolyl isomerase A (cyclophilin
1.76352
0.908832
3.26124
2.19E−02


A)


Homo sapiens mRNA for KIAA1597
6.20675
3.06104
3.12403
1.78E−02


protein, partial cds. /PROD = KIAA1597


protein


HIV-1 rev binding protein 2
3.54001
0.617783
0.325316
3.52E−02


Homo sapiens nidogen 2 (NID2), mRNA.
3.39549
0.915898
1.65422
4.70E−03


/PROD = nidogen 2 /FL = gb: NM_007361.1


gb: D86425.1


Homo sapiens mRNA; cDNA
3.49826
0.859233
1.46022
3.01E−02


DKFZp566A1046 (from clone


DKFZp566A1046)


Homo sapiens muscleblind (Drosophila)-
3.53962
0.508166
0.298438
3.90E−02


like (MBNL), mRNA.


/PROD = muscleblind (Drosophila)-like


/FL = gb: NM_021038.1 gb: AB007888.1


hypothetical protein FLJ20163
5.26708
2.17148
2.01643
3.33E−02


KIAA0455 gene product
3.25374
0.057742
−0.01612
2.66E−02


hypothetical protein FLJ22833
2.93229
0.533105
1.40606
3.19E−02


/FL = gb: NM_022837.1


ESTs
2.19313
−0.32822
0.427128
1.87E−02


Homo sapiens NADPH oxidase 4
5.57794
2.61602
2.28462
4.04E−02


(NOX4), mRNA. /PROD = NADPH


oxidase 4 /FL = gb: AF261943.1


gb: NM_016931.1 gb: AF254621.1


gb: AB041035.1


ESTs
3.3266
0.840887
1.69605
0.00E+00


ESTs
1.99635
−0.56359
0.236236
1.39E−02


Homo sapiens serumglucocorticoid
4.81433
2.46478
3.47797
2.02E−02


regulated kinase (SGK), mRNA.


/PROD = serumglucocorticoid regulated


kinase /FL = gb: BC001263.1


gb: NM_005627.1 gb: AF153609.1


A kinase (PRKA) anchor protein 2
6.03149
3.65135
4.63469
1.17E−02


ESTs
3.8522
1.17894
0.483367
3.08E−02


Homo sapiens similar to rat
1.74077
−1.45032
−1.24979
5.69E−04


tricarboxylate carrier-like protein


(BA108L7.2), mRNA. /PROD = similar to


rat tricarboxylate carrier-likeprotein


/FL = gb: NM_030971.1


Homo sapiens, lectin, galactoside-
4.53668
1.85893
2.59596
3.84E−03


binding, soluble, 3 (galectin 3), clone


MGC: 2058, mRNA, complete cds.


/PROD = lectin, galactoside-binding,


soluble, 3 (galectin 3)


/FL = gb: NM_002306.1 gb: M35368.1


gb: BC001120.1 gb: M36682.1


gb: M57710.1 gb: AB006780.1


ESTs
1.06738
−1.51321
−0.67814
7.68E−03


Homo sapiens heptacellular carcinoma
5.55437
3.02649
3.91576
2.22E−02


novel gene-3 protein (LOC51339),


mRNA. /PROD = heptacellular carcinoma


novel gene-3 protein


/FL = gb: NM_016651.2 gb: AF251079.2


Homo sapiens, Similar to transforming
4.82642
2.07249
2.74019
1.03E−02


growth factor beta 1 induced transcript 1,


clone MGC: 4078, mRNA, complete cds.


/PROD = Similar to transforming growth


factor beta 1induced transcript 1


/FL = gb: NM_015927.1 gb: BC001830.1


gb: AF116343.1


ESTs, Highly similar to FXD3_HUMAN
3.81523
1.18624
1.98102
4.17E−02


FORKHEAD BOX PROTEIN D3


(H. sapiens)


ESTs
4.52094
1.70664
2.3196
2.78E−02


Homo sapiens potassium
3.68048
0.897369
1.5433
1.32E−02


intermediatesmall conductance calcium-


activated channel, subfamily N, member


2 (KCNN2), mRNA. /PROD = potassium


intermediatesmall conductancecalcium-


activated channel, subfamily N, member


2 /FL = gb: NM_021614.1 gb: AF239613.1


Homo sapiens transporter similar to
5.68324
3.35483
4.46553
3.00E−03


yeast MRS2 (MRS2L), mRNA.


/PROD = transporter similar to yeast


MRS2 /FL = gb: AF288288.1


gb: NM_020662.1


Homo sapiens a disintegrin-like and
4.54713
2.07152
1.10726
5.91E−03


metalloprotease (reprolysin type) with


thrombospondin type 1 motif, 5


(aggrecanase-2) (ADAMTS5), mRNA.


/PROD = a disintegrin and


metalloprotease withthrombospondin


motifs-5 preproprotein


/FL = gb: NM_007038.1 gb: AF14209


HIV-1 rev binding protein 2
3.85003
0.823769
0.407316
3.76E−02


Human, parathyroid-like protein
1.73564
−1.5631
−1.41799
4.04E−02


(associated with humoral hypercalcemia


of malignancy) mRNA, complete cds.


/FL = gb: J03580.1


ESTs
5.27633
2.76577
3.71542
1.39E−02


ESTs
1.7022
−1.29085
−0.81236
1.72E−02


pyruvate dehydrogenase phosphatase
6.62966
4.26189
5.37384
1.04E−03


/FL = gb: NM_018444.1 gb: AF155661.1


ESTs
3.90566
1.11378
1.80307
1.57E−02


Homo sapiens DRM (DRM) mRNA,
6.16645
3.12369
3.57831
1.59E−02


complete cds. /PROD = DRM


/FL = gb: NM_013372.1 gb: AF110137.2


gb: AF045800.1 gb: AF154054.1


Homo sapiens cDNA: FLJ22769 fis,
2.36483
−0.5543
0.025213
1.45E−04


clone KAIA1316


ESTs
4.31647
1.44801
2.11226
2.19E−02


ESTs
1.24891
−1.42983
−0.57145
1.29E−02


Homo sapiens mRNA; cDNA
2.85031
0.231102
1.16439
1.08E−02


DKFZp586N012 (from clone


DKFZp586N012)


jagged 1 (Alagille syndrome)
2.54951
0.151382
1.31733
4.46E−03


ESTs, Weakly similar to I38588 reverse
1.16209
−1.26828
−0.1187
3.40E−02


transcriptase homolog (H. sapiens)


KIAA1339 protein
4.41803
2.09451
3.35786
3.90E−02


ESTs
3.83407
0.892884
1.56759
8.98E−03


DKFZP434D156 protein
4.42983
2.05154
3.28988
1.60E−02


Homo sapiens mRNA expressed only in
3.84408
0.705031
1.20856
2.25E−02


placental villi, clone SMAP41.


RAS guanyl releasing protein 2 (calcium
2.7724
0.235409
1.34452
1.96E−02


and DAG-regulated)


KIAA0164 gene product
4.99867
2.5327
3.72535
2.85E−02


/FL = gb: NM_014739.1 gb: D79986.1


Homo sapiens mRNA; cDNA
5.14752
2.61217
3.76652
3.78E−02


DKFZp761M0111 (from clone


DKFZp761M0111)


Homo sapiens NPD009 mRNA,
2.15383
−0.27664
0.990295
2.09E−03


complete cds. /PROD = NPD009


/FL = gb: NM_020686.1 gb: AF237813.1


muscleblind (Drosophila)-like
3.23823
0.467215
1.39628
7.60E−03


/FL = gb: NM_021038.1 gb: AB007888.1


ESTs
4.49318
1.35004
1.91395
3.79E−02


muscleblind (Drosophila)-like
1.84877
−0.57315
0.732468
8.06E−03


/FL = gb: NM_021038.1 gb: AB007888.1


ESTs
6.068
3.42334
4.51189
1.89E−02


ESTs, Weakly similar to unnamed
3.24528
0.740266
1.98306
3.89E−04


protein product (H. sapiens)


Human complement cytolysis inhibitor
6.50766
4.03647
5.33533
1.77E−02


(CLI) mRNA, complete cds.


/FL = gb: J02908.1 gb: NM_001831.1


gb: M64722.1 gb: M25915.1


ESTs
5.36759
2.42249
3.24765
3.23E−03


ESTs
0.957239
−1.43055
−0.04796
5.55E−04


transporter similar to yeast MRS2
3.77395
1.18311
2.36844
1.83E−02


Homo sapiens, Similar to regulator for
5.09957
2.72179
4.14116
2.72E−02


ribosome resistance homolog (S. cerevisiae),


clone MGC: 2755, mRNA,


complete cds. /PROD = Similar to


regulator for ribosome


resistancehomolog (S. cerevisiae)


/FL = gb: BC001811.1


SWISNF related, matrix associated,
2.54229
−0.29226
0.695255
1.89E−02


actin dependent regulator of chromatin,


subfamily a, member 2


/FL = gb: NM_003070.1 gb: D26155.1


Homo sapiens mRNA; cDNA
3.34446
0.795373
2.07147
1.03E−02


DKFZp586L2424 (from clone


DKFZp586L2424)


ESTs
4.63117
2.08252
3.36946
2.25E−03


Homo sapiens bicarbonate transporter-
3.17407
0.745852
2.17046
1.71E−03


related protein BTR1 mRNA, complete


cds. /PROD = bicarbonate transporter-


related protein BTR1


/FL = gb: AF336127.1


ESTs
1.08377
−1.27197
0.228305
4.76E−02


ESTs
2.94657
0.127188
1.20032
1.43E−02


Homo sapiens mRNA; cDNA
2.47798
−0.26439
0.894731
4.51E−03


DKFZp434B2016 (from clone


DKFZp434B2016).


ESTs
2.39983
−1.04089
−0.57976
3.37E−02


Homo sapiens synaptotagmin interacting
2.11009
−0.57109
0.653181
1.78E−02


protein STIP1 mRNA, partial cds.


/PROD = synaptotagmin interacting


protein STIP1


Homo sapiens connexin 26 (GJB2)
2.86049
−0.57377
−0.09328
4.27E−04


mRNA, complete cds. /PROD = connexin


26 /FL = gb: NM_004004.1 gb: M86849.2


Homo sapiens brain-derived
1.71599
−0.79412
0.616733
3.09E−02


neurotrophic factor (BDNF), mRNA.


/PROD = brain-derived neurotrophic


factor /FL = gb: NM_001709.1


ESTs, Weakly similar to unknown
2.33677
−0.04066
1.5217
1.87E−03


(H. sapiens)


ESTs
2.57265
0.177794
1.74876
2.74E−04


ESTs
2.6809
0.209095
1.70624
1.21E−02


Homo sapiens DKC1 gene, exons 1 to
4.18307
1.70283
3.20863
2.00E−02


11


Homo sapiens T cell receptor beta chain
4.45228
1.38318
2.30031
3.27E−03


(TCRBV13S1-TCRBJ2S1) mRNA,


complete cds. /PROD = T cell receptor


beta chain /FL = gb: AF043179.1


Homo sapiens PRO0066 mRNA,
0.466715
−1.87679
−0.22665
4.25E−03


complete cds. /PROD = PRO0066


/FL = gb: AF113007.1


axonal transport of synaptic vesicles
5.20385
2.74124
4.27648
3.33E−03


Homo sapiens, Similar to cyclin M2,
0.139313
−2.31405
−0.76333
1.46E−02


clone MGC: 12933 IMAGE: 4308662,


mRNA, complete cds. /PROD = Similar to


cyclin M2 /FL = gb: BC021222.1


Homo sapiens clone CDABP0095
3.87201
2.7936
5.73958
9.63E−03


mRNA sequence


H. sapiens mRNA for ribosomal protein
2.00401
0.354674
2.73013
4.70E−04


L18a homologue. /PROD = ribosomal


protein L18a homologue


ESTs, Weakly similar to ALU7_HUMAN
3.43639
0.843826
2.28468
3.37E−02


ALU SUBFAMILY SQ SEQUENCE


CONTAMINATION WARNING ENTRY


(H. sapiens)


Homo sapiens, clone IMAGE: 5242616,
−0.525567
−2.07832
0.406964
2.23E−02


mRNA.


Homo sapiens C1orf24 mRNA, complete
5.17968
2.58724
4.04213
1.98E−04


cds. /PROD = C1orf24


/FL = gb: AF288391.1 gb: AB050477.1


gb: NM_022083.1


Homo sapiens clone IMAGE: 451939,
5.15776
2.22786
3.40438
2.60E−02


mRNA sequence


Homo sapiens cDNA FLJ14942 fis,
0.682724
−2.08716
−0.74074
1.45E−02


clone PLACE1011185, highly similar to


INSERTION ELEMENT IS1 PROTEIN


INSB.


hypothetical protein FLJ20425
4.23689
1.90847
3.69988
1.15E−02


/FL = gb: NM_017816.1 gb: AL136750.1


Homo sapiens matrix metalloproteinase
2.28308
−0.65903
0.521586
8.87E−03


10 (stromelysin 2) (MMP10), mRNA.


/PROD = matrix metalloproteinase 10


preproprotein /FL = gb: BC002591.1


gb: NM_002425.1


ESTs
3.67167
2.20619
4.86553
3.16E−03


Homo sapiens prominin (mouse)-like 1
5.46002
2.79737
4.29571
1.38E−03


(PROML1), mRNA. /PROD = prominin


(mouse)-like 1 /FL = gb: NM_006017.1


gb: AF027208.1


Homo sapiens mRNA; cDNA
2.40278
−0.68409
0.399469
1.48E−02


DKFZp434M2415 (from clone


DKFZp434M2415).


Homo sapiens hypothetical protein
2.1471
0.312211
2.65409
2.28E−04


FLJ20701 (FLJ20701), mRNA.


/PROD = hypothetical protein FLJ20701


/FL = gb: NM_017933.1


Homo sapiens cDNA FLJ35259 fis,
6.79033
3.86627
5.12737
1.23E−03


clone PROST2004251.


Homo sapiens hypothetical protein
4.0369
1.34318
2.84336
7.69E−03


FLJ39553 (FLJ39553), mRNA.


/FL = gb: NM_173549.1


Human microfibril-associated
3.89326
0.527618
1.35764
1.81E−03


glycoprotein-2 MAGP-2 mRNA,


complete cds. /PROD = microfibril-


associated glycoprotein-2 MAGP-2


/FL = gb: NM_003480.1 gb: U37283.1


Homo sapiens proprotein convertase
3.76897
0.368734
1.17396
1.38E−02


subtilisinkexin type 5 (PCSK5), mRNA.


/PROD = proprotein convertase


subtilisinkexin type 5 /FL = gb: U56387.2


gb: NM_006200.1


Cluster Incl. AI735391:at10e09.x1 Homo
2.59941
0.203564
2.03637
1.12E−04


sapiens cDNA, 3 end /clone = IMAGE-


2354728 /clone_end = 3 /gb = AI735391


/gi = 5056915 /ug = Hs.20137 /len = 567


ESTs
2.30031
−1.01596
−0.08964
1.46E−02


Kruppel-like factor 4 (gut)
3.50041
1.09954
2.95915
1.22E−02


Homo sapiens full length insert cDNA
−1.40946
−3.04877
−0.39378
3.32E−02


YI25A03


ESTs
0.806666
−2.01081
−0.53076
1.91E−02


Homo sapiens mRNA; cDNA
3.1892
0.012768
1.13562
1.35E−03


DKFZp761G02121 (from clone


DKFZp761G02121); partial cds


Homo sapiens TMEFF2 mRNA,
2.28638
−0.89508
0.225546
1.21E−03


complete cds. /FL = gb: AB017269.1


gb: NM_016192.2 gb: AF179274.2


gb: AF242222.1


Homo sapiens, clone MGC: 3328,
3.54625
0.772486
2.30168
4.16E−02


mRNA, complete cds. /PROD = Unknown


(protein for MGC: 3328)


/FL = gb: BC001745.1 gb: NM_014392.1


ESTs
2.18149
−0.58303
0.984522
2.68E−02


synaptojanin 2
4.166
1.83143
3.84199
0.00E+00


KIAA0367 protein
1.5999
−1.50555
−0.26286
1.58E−02


ESTs, Moderately similar to
0.868185
−1.46804
0.573078
1.69E−02


ALU8_HUMAN ALU SUBFAMILY SX


SEQUENCE CONTAMINATION


WARNING ENTRY (H. sapiens)


bromodomain adjacent to zinc finger
1.60872
−1.12544
0.518361
2.11E−02


domain, 1B


Human complement cytolysis inhibitor
6.61592
3.91665
5.596
1.66E−02


(CLI) mRNA, complete cds.


/FL = gb: J02908.1 gb: NM_001831.1


gb: M64722.1 gb: M25915.1


forkhead box O1A (rhabdomyosarcoma)
5.04975
2.59802
4.53545
1.60E−03


/FL = gb: NM_002015.2 gb: AF032885.1


gb: U02310.1


Homo sapiens cDNA: FLJ22727 fis,
4.88391
2.09093
3.68875
1.91E−02


clone HSI15054


cadherin 6, type 2, K-cadherin (fetal
3.27371
0.243335
1.61199
2.74E−04


kidney) /FL = gb: D31784.1


gb: NM_004932.1


ESTs
3.97749
0.786845
1.99803
1.73E−02


endonuclease G-like 1
1.11519
−2.09018
−0.88324
1.69E−02


/FL = gb: AB020523.1 gb: NM_005107.1


Homo sapiens cDNA FLJ13034 fis,
4.59641
0.64328
1.1121
1.66E−02


clone NT2RP3001232


Human DNA sequence from clone RP4-
2.91607
0.209555
1.92644
1.93E−02


614O4 on chromosome 20q11.1-12


Contains the 3 part of the MMP24


(matrix metalloproteinase 24


(membrane-inserted)) gene, the


ITGB4BP (integrin beta 4 binding


protein) gene, the 3 end of a novel gene,


the 3 end o . . .


Homo sapiens, collapsin response
0.672753
−1.75265
0.250803
8.65E−03


mediator protein-5; CRMP3-associated


molecule, clone MGC: 11247, mRNA,


complete cds. /PROD = collapsin


response mediator protein-5; CRMP3-


associated molecule


/FL = gb: BC002874.1


ESTs, Moderately similar to
2.91576
0.289656
2.10334
2.57E−03


ALU7_HUMAN ALU SUBFAMILY SQ


SEQUENCE CONTAMINATION


WARNING ENTRY (H. sapiens)


ESTs, Moderately similar to CA1C RAT
6.2681
1.94518
1.82747
4.12E−02


COLLAGEN ALPHA 1 (XII) CHAIN


(R. norvegicus)


Homo sapiens alpha-aminoadipate
5.2604
2.72549
4.6685
4.33E−03


semialdehyde synthase mRNA,


complete cds. /PROD = alpha-


aminoadipate semialdehyde synthase


/FL = gb: AF229180.1


Homo sapiens Wnt inhibitory factor-1
1.03265
−1.51207
0.433159
8.62E−05


(WIF-1), mRNA. /PROD = Wnt inhibitory


factor-1 /FL = gb: AF122922.1


gb: NM_007191.1


ribosomal protein S11
4.95358
2.25158
4.04683
2.98E−04


Homo sapiens tumor necrosis factor,
2.42729
−0.73843
0.613222
1.93E−02


alpha-induced protein 6 (TNFAIP6),


mRNA. /PROD = tumor necrosis factor,


alpha-induced protein 6


/FL = gb: NM_007115.1


Homo sapiens hairyenhancer-of-split
2.3848
−0.44154
1.27956
3.16E−03


related with YRPW motif 2 (HEY2),


mRNA. /PROD = hairyenhancer-of-split


related with YRPW motif2


/FL = gb: NM_012259.1 gb: AF311884.1


gb: AB044755.1 gb: AF232238.1


gb: AF237949.1 gb: AF173901.1


Homo sapiens semenogelin I (SEMG1),
0.585816
−2.46893
−0.95223
3.05E−02


mRNA. /PROD = semenogelin I


/FL = gb: J04440.1 gb: NM_003007.1


Homo sapiens, Similar to cadherin 6,
2.38648
−0.02212
2.14359
2.59E−03


type 2, K-cadherin (fetal kidney), clone


MGC: 1470, mRNA, complete cds.


/PROD = Similar to cadherin 6, type 2, K-


cadherin (fetalkidney)


/FL = gb: BC000019.1


Homo sapiens mRNA; cDNA
2.16899
−0.56794
1.27546
3.16E−03


DKFZp434H0350 (from clone


DKFZp434H0350); partial cds.


/PROD = hypothetical protein


Homo sapiens growth factor receptor-
4.2346
1.17569
2.73613
4.80E−03


bound protein 14 (GRB14), mRNA.


/PROD = growth factor receptor-bound


protein 14 /FL = gb: L76687.1


gb: NM_004490.1


Homo sapiens, Similar to TAF5-like RNA
2.10954
−0.1313
2.24853
8.24E−04


polymerase II, p300CBP-associated


factor (PCAF)-associated factor, 65 kDa,


clone MGC: 46101 IMAGE: 5551246,


mRNA, complete cds. /PROD = Similar to


TAF5-like RNA polymerase II, p300CBP-


associated factor (PCAF)-associat


Homo sapiens mRNA; cDNA
1.42404
−1.99222
−0.77126
3.21E−02


DKFZp761J1324 (from clone


DKFZp761J1324)


solute carrier family 30 (zinc
2.82535
0.288493
2.38889
0.00E+00


transporter), member 1


antizyme inhibitor
3.65887
0.37346
1.74912
1.86E−02


Homo sapiens GRO1 oncogene
2.51473
0.107263
2.38874
7.16E−04


(melanoma growth stimulating activity,


alpha) (GRO1), mRNA. /PROD = GRO1


oncogene (melanoma growth


stimulatingactivity, alpha)


/FL = gb: NM_001511.1


Homo sapiens proprotein convertase
4.90999
0.871309
1.52441
1.71E−02


subtilisinkexin type 5 (PCSK5), mRNA.


/PROD = proprotein convertase


subtilisinkexin type 5 /FL = gb: U56387.2


gb: NM_006200.1


ESTs
0.254736
−1.96122
0.51777
4.00E−02


G-protein gamma-12 subunit
3.93653
1.06178
2.88889
5.11E−04


/FL = gb: NM_018841.1 gb: AF119663.1


platelet-derived growth factor alpha
3.50343
0.651246
2.50207
3.99E−03


polypeptide


clusterin (complement lysis inhibitor, SP-
2.88196
−0.42677
0.9712
8.48E−03


40,40, sulfated glycoprotein 2,


testosterone-repressed prostate


message 2, apolipoprotein J)


ESTs
2.82081
−1.69816
−1.89985
4.10E−02


Homo sapiens insulinoma-associated 1
2.94347
0.120494
2.02248
1.43E−03


(INSM1), mRNA. /PROD = insulinoma-


associated 1 /FL = gb: NM_002196.1


gb: M93119.1


LBP protein 32 /FL = gb: NM_014552.1
2.68546
−0.16678
1.70626
1.87E−03


gb: AF198489.1


ESTs, Highly similar to T42654
2.84504
0.515703
2.95379
7.92E−04


hypothetical protein DKFZp434G1115.1


(H. sapiens)


Homo sapiens Friend leukemia virus
1.7744
−2.28064
−1.56765
3.14E−02


integration 1 (FLI1), mRNA.


/PROD = Friend leukemia virus


integration 1 /FL = gb: BC001670.1


gb: NM_002017.2 gb: M98833.3


ESTs
0.846429
−2.42046
−0.91891
1.57E−02


Homo sapiens oxidative 3 alpha
1.30153
−1.64227
0.196394
3.20E−03


hydroxysteroid dehydrogenase; retinol


dehydrogenase; 3-hydroxysteroid


epimerase (RODH), mRNA.


/PROD = oxidative 3 alpha hydroxysteroid


dehydrogenase;retinol dehydrogenase;


3-hydroxysteroid epimerase


/FL = gb: AF016509.1 gb: A


minor histocompatibility antigen HA-1
1.89498
−0.37136
2.15157
4.61E−02


UDP-glucose pyrophosphorylase 2
4.26327
0.968939
2.4776
6.20E−03


Homo sapiens regulator of G-protein
5.42952
3.05504
5.49711
1.21E−03


signalling 2, 24 kD (RGS2), mRNA.


/PROD = regulator of G-protein signalling


2, 24 kD /FL = gb: L13463.1


gb: NM_002923.1


ESTs
4.49987
1.01623
2.35659
1.86E−03


ESTs
0.623422
−1.94907
0.317138
8.95E−04


ESTs
0.326084
−1.69499
1.13586
1.10E−02


ESTs
3.82952
0.673296
2.37112
3.92E−02


Homo sapiens cDNA FLJ10160 fis,
2.80876
−0.24405
1.56853
1.39E−02


clone HEMBA1003545, highly similar to


INSULIN GENE ENHANCER PROTEIN


ISL-2.


thyroid hormone receptor-associated
4.66717
2.01883
4.2391
4.63E−03


protein, 150 kDa subunit


/FL = gb: NM_005119.1 gb: AF117756.1


ESTs
2.4548
−0.3811
1.65212
4.08E−03


Homo sapiens mRNA; cDNA
3.13835
0.605922
2.95511
1.53E−03


DKFZp667D095 (from clone


DKFZp667D095)


Homo sapiens synaptojanin 2 mRNA,
2.94452
0.23595
2.4148
3.63E−04


complete cds. /PROD = synaptojanin 2


/FL = gb: AF318616.1


Homo sapiens procollagen C-
3.37999
0.343624
2.21149
1.46E−02


endopeptidase enhancer (PCOLCE),


mRNA. /PROD = procollagen C-


endopeptidase enhancer


/FL = gb: BC000574.1 gb: NM_002593.2


gb: AB008549.1 gb: L33799.1


ESTs
3.24495
0.925809
3.52818
1.70E−04


Homo sapiens mRNA for KIAA0930
3.65772
0.911608
3.09513
8.62E−05


protein, partial cds. /PROD = KIAA0930


protein


Homo sapiens forkhead transcription
1.51993
−2.14186
−0.86243
3.75E−02


factor FOXL2 (BPES), mRNA.


/PROD = forkhead transcription factor


FOXL2 /FL = gb: AF301906.1


gb: NM_023067.1


ESTs
1.9867
−1.5948
−0.2171
2.81E−03


ESTs, Highly similar to AF174600 1 F-
3.06175
0.202953
2.35691
2.45E−02


box protein Fbx20 (H. sapiens)


Homo sapiens ankyrin 3, node of
3.11644
0.101116
2.09865
2.90E−03


Ranvier (ankyrin G) (ANK3), transcript


variant 2, mRNA. /PROD = ankyrin 3,


isoform 2 /FL = gb: NM_001149.1


gb: U43965.1


Homo sapiens tumor necrosis factor
2.39086
−1.04254
0.558779
1.62E−02


receptor superfamily, member 6


(TNFRSF6), mRNA. /PROD = apoptosis


(APO-1) antigen 1 /FL = gb: NM_000043.1


gb: M67454.1


Homo sapiens thyrotropin-releasing
1.30849
−1.50688
0.822517
4.91E−02


hormone degrading ectoenzyme


(TRHDE), mRNA. /PROD = thyrotropin-


releasing hormone


degradingectoenzyme


/FL = gb: AF126372.1 gb: NM_013381.1


ESTs
4.65711
0.915563
2.32327
6.29E−03


Homo sapiens mRNA; cDNA
2.36072
−2.02594
−1.25286
1.50E−02


DKFZp564N1116 (from clone


DKFZp564N1116)


Homo sapiens placental protein 13-like
3.55894
0.434263
2.47085
3.48E−03


protein (LOC56891), mRNA.


/PROD = placental protein 13-like protein


/FL = gb: NM_020129.1 gb: AF267852.1


Homo sapiens, Similar to receptor
2.05578
0.178992
3.5007
4.46E−04


tyrosine kinase-like orphan receptor 1,


clone MGC: 12687, mRNA, complete


cds. /PROD = Similar to receptor tyrosine


kinase-like orphanreceptor 1


/FL = gb: BC006374.1


ESTs
0.159066
−2.96514
−0.84976
3.25E−02


Homo sapiens, Similar to v-ets avian
2.22285
−1.58562
−0.1074
2.63E−02


erythroblastosis virus E26 oncogene


homolog 1, clone MGC: 29755


IMAGE: 3946751, mRNA, complete cds.


/PROD = Similar to v-ets avian


erythroblastosis virusE26 oncogene


homolog 1 /FL = gb: BC017314.1


ESTs, Highly similar to dedicator of cyto-
0.83871
−2.29829
−0.12951
1.85E−02


kinesis 1 (H. sapiens)


ESTs
1.1465
−1.7403
0.682768
3.99E−02


ESTs
1.7466
−1.67509
0.283174
2.97E−03


ESTs, Weakly similar to PMXB_HUMAN
1.03192
−2.44485
−0.51457
1.87E−03


PAIRED MESODERM HOMEOBOX


PROTEIN 2B (H. sapiens)


ESTs
3.22222
−0.29339
1.59904
7.36E−03


Homo sapiens cDNA FLJ10561 fis,
2.295
−1.60823
−0.08369
3.93E−03


clone NT2RP2002672


Homo sapiens, Similar to KIAA0441
1.38545
−1.60426
0.86909
2.33E−03


gene product, clone MGC: 45124


IMAGE: 5578893, mRNA, complete cds.


/PROD = Similar to KIAA0441 gene


product /FL = gb: BC036731.1


Homo sapiens, clone IMAGE: 4067166,
0.195869
−2.50507
0.276993
1.16E−03


mRNA


ESTs
1.5857
−2.61787
−1.30022
1.12E−02


Homo sapiens methyl-CpG binding
0.490598
−3.45465
−1.70163
3.90E−03


protein MBD2 (MBD2) mRNA, complete


cds. /PROD = methyl-CpG binding protein


MBD2 /FL = gb: NM_003927.2


gb: AF072242.1


KIAA1151 protein
0.642527
−2.04288
1.00885
4.09E−03


H. sapiens mRNA for B-HLH DNA
5.43034
1.56039
3.45122
1.12E−04


binding protein. /PROD = B-HLH DNA


binding protein /FL = gb: NM_000474.1


Homo sapiens, Similar to hypothetical
0.520711
−1.99011
1.26899
2.63E−02


protein FLJ32001, clone MGC: 39559


IMAGE: 4828136, mRNA, complete cds.


/PROD = Similar to hypothetical protein


FLJ32001 /FL = gb: BC036200.1


Homo sapiens Wilms tumor 1 (WT1),
2.40462
−2.96375
−2.43546
1.41E−02


transcript variant D, mRNA.


/PROD = Wilms tumor 1 isoform D


/FL = gb: NM_024424.1 gb: NM_024426.1


ESTs
1.53104
−1.54553
1.37309
8.14E−03


Homo sapiens, Similar to cadherin 6,
2.54746
−0.69811
2.13419
1.45E−04


type 2, K-cadherin (fetal kidney), clone


MGC: 1470, mRNA, complete cds.


/PROD = Similar to cadherin 6, type 2, K-


cadherin (fetalkidney)


/FL = gb: BC000019.1


ESTs
0.133612
−3.77953
−1.56636
1.40E−02


paternally expressed 3
2.87581
−1.67669
−0.0118
2.17E−02


Homo sapiens Charot-Leyden crystal
3.30086
−0.41644
2.1096
2.08E−02


protein (CLC), mRNA. /PROD = Charot-


Leyden crystal protein


/FL = gb: NM_001828.3 gb: L01664.1


ESTs
3.26198
−0.72777
1.5682
3.16E−03


Homo sapiens tachykinin, precursor 1
2.77265
−1.02081
1.48319
4.07E−04


(substance K, substance P, neurokinin


1, neurokinin 2, neuromedin L,


neurokinin alpha, neuropeptide


K, neuropeptide gamma) (TAC1),


transcript variant beta, mRNA.


/PROD = tachykinin 2 precursor, isoform


beta /FL = gb: U3


Homo sapiens PNAS-123 mRNA,
1.59659
−1.46656
1.84663
3.90E−03


complete cds


Homo sapiens hypothetical protein
−0.359245
−3.46654
−0.06528
2.55E−03


FLJ20075 (FLJ20075), mRNA.


/PROD = hypothetical protein FLJ20075


/FL = gb: NM_017655.1


Human platelet-derived growth factor
2.44464
−0.5846
2.90484
3.63E−04


alpha-receptor (PDGFRA) mRNA, exons


13-16


Homo sapiens cDNA: FLJ22547 fis,
4.29365
−0.10882
2.09548
1.54E−02


clone HSI00356


Homo sapiens HSPC156 protein
0.786188
−2.32161
1.29619
8.62E−05


(HSPC156), mRNA. /PROD = HSPC156


protein /FL = gb: NM_014178.1


gb: AF161505.1


Homo sapiens mRNA; cDNA
0.485642
−4.8524
−3.09356
2.02E−02


DKFZp586P1124 (from clone


DKFZp586P1124)


caldesmon 1 /FL = gb: M64110.1
7.09236
2.67863
5.63436
7.47E−03


gb: NM_004342.2


Homo sapiens, Similar to hypothetical
1.69808
−2.74939
0.215566
1.92E−02


protein FLJ10058, clone MGC: 34305


IMAGE: 5167647, mRNA, complete cds.


/PROD = Similar to hypothetical protein


FLJ10058 /FL = gb: BC034293.1


Homo sapiens, Similar to LIM homeobox
−0.062884
−4.30121
−1.10961
1.16E−02


protein 8, clone IMAGE: 4839343,


mRNA.


Homo sapiens chorionic
0.812166
−2.21846
2.21843
3.60E−03


somatomammotropin hormone 2


(CSH2), transcript variant 4, mRNA.


/PROD = chorionic somatomammotropin


hormone 2, isoform4


/FL = gb: NM_022646.1


Homo sapiens HES-related repressor
2.80599
−1.19396
2.40562
2.44E−03


protein 1 HERP1 mRNA, complete cds.


/PROD = HES-related repressor protein 1


HERP1 /FL = gb: NM_012259.1


gb: AF311884.1 gb: AB044755.1


gb: AF232238.1 gb: AF237949.1


gb: AF173901.1


Homo sapiens cadherin 6, type 2, K-
0.824752
−3.83509
−0.87634
2.49E−02


cadherin (fetal kidney) (CDH6), mRNA.


/PROD = cadherin 6, type 2, K-cadherin


(fetal kidney) /FL = gb: D31784.1


gb: NM_004932.1


paternally expressed 3
2.87398
−4.12925
−3.49232
2.19E−02


Homo sapiens cDNA FLJ11398 fis,
2.5268
−1.71619
1.84868
4.46E−04


clone HEMBA1000637.


Homo sapiens BCL2-interacting killer
4.30042
0.023414
3.56237
1.94E−03


(apoptosis-inducing) (BIK), mRNA.


/PROD = BCL2-interacting killer


/FL = gb: NM_001197.2 gb: BC001599.1


gb: U34584.1 gb: U49730.1


Homo sapiens testis expressed
3.51154
−2.84891
−1.3824
2.18E−02


sequence 14 (TEX14), mRNA.


/PROD = testis expressed sequence 14


/FL = gb: NM_031272.1


Homo sapiens growth hormone 2 (GH2),
0.922569
−2.77587
1.4679
1.60E−03


transcript variant 2, mRNA.


/PROD = growth hormone 2, isoform 2


precursor /FL = gb: J03756.1


gb: NM_022557.1


Homo sapiens, clone IMAGE: 4828836,
0.972907
−4.24111
−1.48398
1.63E−02


mRNA.


Homo sapiens galanin receptor 1
2.50745
−3.13141
−0.33802
3.38E−03


(GALR1), mRNA. /PROD = galanin


receptor 1 /FL = gb: NM_001480.2


gb: U23854.1 gb: L34339.1 gb: U53511.1


Homo sapiens, serine (or cysteine)
1.96851
−4.13943
−1.56568
3.06E−02


proteinase inhibitor, clade B (ovalbumin),


member 3, clone MGC: 12244, mRNA,


complete cds. /PROD = serine (or


cysteine) proteinase inhibitor, cladeB


(ovalbumin), member 3


/FL = gb: NM_006919.1 gb: U19556.1


gb: BC005224.1


DKFZP566K1924 protein
2.88225
−2.59632
1.39062
0.00E+00


ESTs, Moderately similar to
4.24074
−0.69782
3.87444
1.12E−04


ALU2_HUMAN ALU SUBFAMILY SB


SEQUENCE CONTAMINATION


WARNING ENTRY (H. sapiens)


Human mRNA upregulated during
0.494505
−4.62912
−0.18915
4.38E−02


camptothecin-induced apoptosis of U937


cells.


nuclear receptor subfamily 1, group I,
1.77902
−4.02806
−0.19682
4.34E−02


member 3


ESTs
−1.3795
−6.08172
−0.94606
6.91E−04


Homo sapiens chorionic
1.12437
−3.12138
2.61605
1.12E−04


somatomammotropin hormone 2


(CSH2), transcript variant 1, mRNA.


/PROD = chorionic somatomammotropin


hormone 2, isoform 1precursor


/FL = gb: NM_020991.2 gb: BC002717.1


Homo sapiens mRNA for SCCA2b,
3.32911
−3.16288
0.568045
1.93E−02


complete cds. /PROD = SCCA2b


/FL = gb: AB046400.1


Homo sapiens KIAA0469 gene product
4.03097
−1.94826
2.56518
2.49E−04


(KIAA0469), mRNA. /PROD = KIAA0469


gene product /FL = gb: AB007938.1


gb: NM_014851.1


ESTs
4.45389
−2.40636
5.33359
5.65E−04
















TABLE VII







THE EFFECT OF WNT-3A TREAMENT ON CYTOKINE EXPRESSION IN POPULATIONS


OF CELLS FROM THE HUMAN EMBRYONIC STEM CELL LINE H9.










Cell lysate
Cell conditioned media
















w/o wnt 1
w/o wnt 2
w/wnt 1
w/wnt 2
w/o wnt 1
w/o wnt 2
w/wnt 1
w/wnt 2



















POS
62,842.33
67,606.06
49,758.34
50,702.57
96,585.15
109,721.82
199,709.04
195,889.94


NEG
23.38
34.49
370.82
400.66
58.06
34.45
216.90
105.20


Angiogenin
38.50
132.91
619.40
750.85
13,726.35
11,749.55
57,442.48
54,969.04


BDNF
167.50
167.82
763.51
951.45
689.22
563.39
770.08
715.86


BLC
2.00
48.99
742.24
476.36
107.69
512.77
337.12
249.80


BMP-4
247.00
273.70
348.44
304.48
583.40
541.83
1,158.54
771.19


BMP-6
8.50
141.92
571.23
641.05
354.91
355.28
985.70
670.60


CK beta 8-1
1.00
1.13
22.48
21.54
17.79
25.31
42.78
75.44


CNTF
1.00
55.19
1,382.51
1,060.82
282.81
211.86
787.19
541.51


EGF
32.50
82.22
742.24
984.39
1,681.85
1,406.12
23,331.57
22,768.53


Eotaxin
1.00
7.32
301.87
253.80
207.89
199.67
622.91
291.71


Eotaxin-2
262.00
336.77
1,155.31
960.31
890.56
744.31
2,156.21
1,445.14


Eotaxin-3
184.50
284.96
934.12
847.56
975.77
665.56
2,140.81
1,480.35


FGF-6
629.00
379.57
1,327.92
964.54
1,239.85
947.73
3,283.94
2,519.78


FGF-7
31.00
222.45
1,075.02
941.73
449.49
352.47
1,086.66
861.72


Flt-3 Ligand
44.00
180.21
857.45
795.19
315.58
277.47
511.67
792.98


Fractalkine
23.50
62.51
792.02
456.51
120.80
145.30
359.37
367.15


GCP-2
1.00
45.05
593.71
538.86
203.21
166.86
451.78
362.12


GDNF
84.00
292.84
436.75
444.68
176.99
194.98
443.22
509.66


GM-CSF
433.00
636.37
1,343.17
1,161.75
777.25
722.75
1,808.82
1,446.82


I-309
152.50
246.10
708.52
743.25
1,828.87
179.05
318.30
288.36


IFN-gamma
349.00
441.52
1,202.67
1,037.60
283.74
559.64
1,238.97
1,435.08


IGFBP-1
1.50
3.38
59.41
85.73
33.71
24.37
227.60
155.91


IGFBP-2
3,785.50
5,554.45
2,671.90
2,414.72
27,656.85
28,473.95
42,215.53
43,934.33


IGFBP-4
5,553.00
6,043.27
4,459.85
4,173.19
10,091.09
6,029.45
18,988.34
13,380.14


IGF-I
1.00
2.25
558.38
208.19
88.96
148.11
254.98
187.77


IL-10
158.00
422.93
1,222.75
1,096.30
722.93
564.32
1,625.71
1,129.96


IL-13
428.50
555.84
1,486.89
1,407.96
1,114.36
786.49
2,522.42
1,807.27


IL-15
599.50
808.13
1,677.16
1,601.79
1,140.59
858.67
2,495.04
1,981.62


IL-16
2.00
137.97
651.11
668.50
259.39
214.67
436.38
405.71


IL-1alpha
1,236.00
1,216.99
1,612.93
1,421.89
10,155.70
5,842.90
3,999.26
3,545.79


IL-1beta
1.00
1.13
352.05
312.08
117.99
75.93
316.59
278.30


IL-1ra
33.00
25.91
302.68
198.48
142.34
93.74
354.23
581.75


IL-2
210.50
681.99
966.23
1,133.88
188.22
363.72
758.10
881.84


IL-3
3,146.50
1,976.13
2,119.94
1,497.48
4,598.86
5,260.77
11,345.78
12,174.73


IL-4
17.50
74.34
625.42
638.94
357.72
239.04
804.30
673.95


IL-5
400.50
555.84
1,245.23
1,167.24
1,056.31
791.18
2,120.27
1,713.38


IL-6
828.50
516.98
1,121.19
842.07
7,377.28
9,527.88
9,774.82
9,991.93


IL-7
190.00
313.12
1,212.31
531.68
456.98
430.27
681.09
1,017.63


Leptin
900.50
617.79
1,234.39
1,091.65
2,999.42
2,016.38
7,238.71
4,886.99


LIGHT
10.50
232.02
1,081.04
888.10
619.92
453.71
1,346.78
1,108.17


MCP-1
242.50
282.71
1,022.03
1,076.03
572.17
500.58
2,015.89
1,567.53


MCP-2
24.00
109.25
909.63
1,152.46
129.23
351.53
694.78
694.07


MCP-3
1.00
168.38
1,184.61
1,255.50
401.73
169.67
701.62
583.42


MCP-4
7.50
46.74
1,380.51
1,606.86
123.61
61.87
231.02
276.62


M-CSF
1.00
63.64
852.63
867.83
529.09
269.04
740.98
684.01


MDC
1.00
2.25
269.36
280.41
157.32
52.50
280.65
132.44


MIG
291.00
226.39
1,113.16
1,137.26
272.50
486.52
617.77
1,222.17


MIP-1-delta
1.50
1.13
491.75
322.22
144.21
66.56
474.02
291.71


MIP-3-alpha
1.00
5.63
1,602.09
1,399.93
55.25
0.00
208.78
184.41


NAP-2
1.00
6.19
465.66
177.37
97.39
110.61
371.35
343.68


NT-3
1.00
1.13
316.73
275.76
97.39
171.55
571.57
427.51


PARC
1.00
2.25
352.85
353.04
170.43
81.56
474.02
363.80


PDGF-BB
437.00
1,644.99
517.84
597.56
4,729.96
8,521.09
2,635.37
2,005.09


RANTES
1.00
89.54
724.98
637.25
393.31
347.78
682.80
855.01


SCF
74.50
148.67
1,152.50
1,021.13
691.09
487.46
1,394.69
2,155.98


SDF-1
23.50
51.25
940.14
1,294.78
382.07
123.74
326.85
616.95


TARC
1.50
5.07
372.93
343.33
206.95
71.24
277.23
1,642.97


TGF-beta 1
362.00
230.33
1,426.67
1,240.30
752.90
740.56
1,827.65
1,849.18


TGF-beta 3
3.00
25.91
377.74
1,130.93
111.44
115.30
294.34
343.68


TNF-alpha
431.00
579.49
2,851.74
1,285.91
1,205.20
929.91
2,609.70
2,167.71


TNF-beta
241.00
342.40
1,049.33
1,026.62
675.17
671.19
1,701.01
1,637.94


Internal Control
18,642.67
18,642.67
18,642.67
18,642.67
18,642.67
18,642.67
18,642.67
18,642.67


POS
60,794.83
48,106.85
35,652.42
47,391.58
94,013.71
90,889.16
141,321.30
149,125.43


NEG
11.13
5.53
11.22
30.45
24.93
17.75
36.22
33.27


Acrp30
1,036.00
744.50
601.61
690.66
1,797.52
1,319.88
2,795.71
2,302.99


AgRP
11.00
153.99
77.82
89.00
378.17
195.90
651.26
482.21


Angiopoietin-2
28.50
10.43
357.97
771.04
655.82
632.60
65,959.24
71,664.21


Amphiregulin
17.50
15.44
73.63
154.62
250.52
151.01
619.07
782.60


Axl
637.50
795.83
88.59
139.03
401.31
324.05
610.40
748.34


bFGF
59,820.50
54,057.29
11,722.95
21,398.52
7,948.03
4,993.83
1,226.99
1,241.09
















TABLE VIII







CHANGES IN THE PERCENTAGE OF INSULIN-


EXPRESSING CELLS AND SYNAPTOPHYSIN-


EXPRESSING CELLS DERIVED FROM HUMAN


EMBRYONIC STEM CELLS WITH DIFFERENT


CONCENTRATION OF GLUCOSE.











5 mM
10 mM
20 mM
















Insulin
8.9%
10.9%
16%



Synaptophysin
19.2%
  21%
36%

## Claims

1. A method for increasing insulin in a patient having Type 1 diabetes comprising:
and differentiating pancreatic endoderm cells into a population of pancreatic endocrine cells by culturing pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treating with a factor selected from the group consisting of: a gamma secretase inhibitor, Exendin-4, and a combination of Exendin-4 and hepatocyte growth factor; and
providing the population of pancreatic endocrine cells to the patient, wherein the cells are provided by implanting encapsulated cells into the liver or by infusing the cells into the hepatic portal vein, wherein cells in the population of pancreatic endocrine cells express insulin.

2. A method for increasing insulin in a patient having Type 1 diabetes comprising:
differentiating human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent stem cells with activin A;
differentiating the definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor;
differentiating the pancreatic endoderm cells into a population of pancreatic endocrine cells by culturing the pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treating with a factor selected from the group consisting of: a gamma secretase inhibitor, Exendin-4, and a combination of Exendin-4 and hepatocyte growth factor; and
providing the population of pancreatic endocrine cells to the patient, wherein the population is provided by implanting encapsulated cells into the liver or by infusing the cells into the hepatic portal vein, and wherein cells in the population express insulin.

3. The method of claim 2, wherein the human pluripotent embryonic stem cells are human embryonic stem cells.

4. The method of claim 3, wherein the human pluripotent embryonic stem cells are derived from a cell line of the group consisting of H1 and H9.

5. The method of claim 1, wherein the human pluripotent embryonic stem cells are human embryonic stem cells.

6. The method of claim 5, wherein the human pluripotent embryonic stem cells are derived from a cell line of the group consisting of H1 and H9.

7. The method of claim 1, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with a gamma secretase inhibitor.

8. The method of claim 1, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with Exendin-4.

9. The method of claim 1, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with a combination of Exendin-4 and hepatocyte growth factor.

10. The method of claim 2, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with a gamma secretase inhibitor.

11. The method of claim 2, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with Exendin-4.

12. The method of claim 2, wherein the pancreatic endocrine cells are produced by culturing pancreatic endoderm cells with a combination of Exendin-4 and hepatocyte growth factor.

13. A method for increasing insulin in a patient having Type 1 diabetes comprising:
generating a population of human pancreatic endocrine cells in which cells express insulin by culturing pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treating with a factor selected from the group consisting of: a gamma secretase inhibitor, Exendin-4, and a combination of Exendin-4 and hepatocyte growth factor; and
providing the population of endocrine cells to the patient by implanting encapsulated cells into the liver or by infusing cells into the hepatic portal vein.

14. The method of claim 13, wherein the pancreatic endoderm cells are cultured in medium supplemented with a gamma secretase inhibitor and glucose at a concentration from about 10 mM to about 20 mM.

15. The method of claim 13, wherein the pancreatic endoderm cells are cultured in medium supplemented with Exendin-4 and glucose at a concentration from about 10 mM to about 20 mM.

16. The method of claim 13, wherein the pancreatic endoderm cells are cultured in medium supplemented with Exendin-4, hepatocyte growth factor and glucose at a concentration from about 10 mM to about 20 mM.