Source: http://www.google.com/patents/US7622108?dq=6,455,937
Timestamp: 2016-09-24 23:03:50
Document Index: 781458146

Matched Legal Cases: ['Application No. 60', 'Application No. 2005241008', 'Application No. 05', 'Application No. 05', 'Application No. 05', 'Application No. 200580012739']

Patent US7622108 - Multi-lineage progenitor cells - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsFetal blood multi-lineage progenitor cells that are capable of a wide spectrum of transdifferentiation are described....http://www.google.com/patents/US7622108?utm_source=gb-gplus-sharePatent US7622108 - Multi-lineage progenitor cellsAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS7622108 B2Publication typeGrantApplication numberUS 11/208,873Publication dateNov 24, 2009Priority dateApr 23, 2004Fee statusPaidAlso published asUS20060040392, WO2007025072A2, WO2007025072A3Publication number11208873, 208873, US 7622108 B2, US 7622108B2, US-B2-7622108, US7622108 B2, US7622108B2InventorsDaniel P. Collins, Stacey L. Sprague, Barbara M. TiggesOriginal AssigneeBioe, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (104), Non-Patent Citations (92), Referenced by (9), Classifications (18), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetMulti-lineage progenitor cells
US 7622108 B2Abstract
1. A method of characterizing a population of multi-lineage progenitor cells (MLPC), said method comprising: a) providing a purified population of MLPC, wherein said MLPC are positive for CD9, CD13, CD29, CD44, CD73, CD90 and CD105, and negative for CD10, CD34, CD41, CD45, Stro-1, Stage Specific Embryonic Antigen-3 (SSEA-3) and SSEA-4; and b) assessing expression in said population of MLPC of at least one mRNA selected from the group consisting of CXCR4, FLT3, CD133, ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, ICAM1, CD24, CD34, CD44, CD45, CD58, CD68, CD33, CD37, CD38, TERT and POU5F.
2. The method of claim 1, wherein expression of mRNA for CXCR4, FLT3, and CD133 is assessed.
3. The method of claim 1, wherein expression of mRNA for TERT, KIT, and POU5F is assessed.
4. The method of claim 2 or 3, wherein expression of mRNA for CD34 is assessed.
5. The method of claim 1, wherein said MLPC are further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, CD133, glycophorin-A, stem cell factor, and HLA-DR.
6. The method of claim 1, wherein expression of at least three mRNAs from said group is assessed.
This application is a continuation-in-part and claims benefit under 35 U.S.C. �120 of U.S. application Ser. No. 11/110,299, filed Apr. 20, 2005, which claims benefit under 35 U.S.C. �119(e) of U.S. Application No. 60/564,687, filed on Apr. 23, 2004, both of which are incorporated by reference herein in their entirety.
The invention is based on the identification of a rare undifferentiated cell population from human fetal blood that is capable of self-renewal and has the potential to differentiate into cells representing each of the three embryonic germ layers. These fetal blood-derived cells are referred to as multi-lineage progenitor cells (MLPC). As described herein, fetal blood MLPC are distinguished from bone marrow-derived MSC, HSC, and USSC on the basis of their immunophenotypic characteristics, gene expression profile, morphology, and distinct growth pattern. The invention provides methods for developing monotypic clonal cell lines from individual cells. The invention also provides methods for cryopreserving MLPC (e.g., for cord blood banking) and methods of using MLPC in regenerative therapies.
The invention also features a clonal line of human fetal blood (e.g., cord blood) MLPC, wherein the MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4. The MLPC can display a fibroblast morphology. The MLPC can be further positive for CD13, CD29, CD44, CD73, CD90, and CD105, and can be further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD 14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD41, CD61, CD62E, CD133, glycophorin-A, stem cell factor, SSEA-3, and HLA-DR. The MLPC can adhere to a plastic surface when cultured. The MLPC are capable of differentiating into cells from all three embryonic germ layers, including, for example, cells having an osteocytic phenotype, cells having an adipocytic phenotype, cells having a neural stem cell phenotype, cells having a myocytic phenotype, cells having an endothelial phenotype, cells having a hepatocytic phenotype, and cells having a pancreatic phenotype. The MLPC can include an exogenous nucleic acid (e.g., an exogenous nucleic acid encoding a polypeptide). In some embodiments, the clonal line has undergone at least 5 doublings (e.g., at least 8, at least 10, at least 15, or at least 25 doublings) in culture.
The invention also features a purified population of MLPC or undifferentiated progeny thereof, wherein the MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to a population of MSC. The MLPC further can have an enhanced expression of mRNA for TERT, KIT, and POU5F, or enhanced expression of mRNA for CD34 relative to the population of MSC. The MLPC further can have an enhanced expression of mRNA for CD24, CD44, CD45, CD58, CD68, CD33, CD37, and CD38 relative to the population of MSC, or further have an enhanced expression of the mRNA for ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, and ICAM1 relative to the population of MSC. The MLPC can be obtained from cord blood. The MLPC can be positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4. The MLPC can be capable of differentiating into cells from all three embryonic germ layers, including, for example, cells having an osteocytic phenotype, cells having an adipocytic phenotype, cells having a neural stem cell phenotype, cells having a myocytic phenotype, cells having an endothelial phenotype, cells having a hepatocytic phenotype, and cells having a pancreatic phenotype. The MLPC can include an exogenous nucleic acid, e.g., an exogenous nucleic acid encoding a polypeptide.
In another aspect, the invention features a clonal line of human fetal blood MLPC and undifferentiated progeny thereof, wherein the MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of MSC. The MLPC further can have an enhanced expression of the mRNA for TERT, KIT, and POU5F, or enhanced expression of the mRNA for CD34 relative to the population of MSC. The MLPC further can have an enhanced expression of the mRNA for CD24, CD44, CD45, CD58, CD68, CD33, CD37, and CD38 relative to the population of MSC, or further have an enhanced expression of mRNA for ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, and ICAM1 relative to the population of MSC. The MLPC can be positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4, and can be obtained from cord blood. The MLPC can include an exogenous nucleic acid, e.g., a nucleic acid encoding a polypeptide. In some embodiments, the clonal line has undergone at least 5 doublings (e.g., at least 8, at least 10, at least 15, or at least 25 doublings) in culture.
The invention also features differentiated progeny of a purified population of MLPC or of a clonal line of MLPC. The progeny can have an osteocytic phenotype, an adipocytic phenotype, a neural stem cell phenotype, a myocytic phenotype, an endothelial phenotype, a hepatocytic phenotype, or a pancreatic phenotype.
In yet an another aspect, the invention features an article of manufacture that includes a purified population of MLPC or a clonal line of MLPC. The purified population of MLPC or the clonal line can be housed within a container (e.g., a vial or a bag). The container further can include a cryopreservative. The article of manufacture further can include a label indicating that the MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of MSC. In some embodiments, the article of manufacture further includes a reagent for characterizing the population of MLPC or the clonal MLPC line. The reagent can be selected from the group consisting of a nucleic acid probe for detecting expression of CXCR4, a nucleic acid probe for detecting expression of FLT3, a nucleic acid probe for detecting expression of CD133, a nucleic acid probe for detecting expression of CD34, a nucleic acid probe for detecting expression of TERT, a nucleic acid probe for detecting expression of KIT, a nucleic acid probe for detecting expression of POU5F, an antibody having specific binding affinity for CD9, an antibody having specific binding affinity for CD34, an antibody having specific binding affinity for CD45, and an antibody having specific binding affinity for SSEA-4.
The invention also features a method for purifying a population of MLPC from human fetal blood. The method includes contacting a human fetal blood sample (e.g., cord blood) with a composition that includes dextran, anti-glycophorin A antibody, anti-CD15 antibody, and anti-CD9 antibody; allowing the sample to partition into an agglutinate and a supernatant phase; recovering cells from the supernatant phase; purifying MLPC from the recovered cells by adherence to a solid substrate (e.g., a plastic substrate); and culturing the MLPC such that the MLPC obtain a fibroblast morphology, wherein the MLPC, after obtaining the fibroblast morphology, have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of MSC. The MLPC further can have an enhanced expression of mRNA for TERT, KIT, and POU5F relative to the population of MSC. In some embodiments, the method further includes testing the MLPC for enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to the population of MSC. The method also can include testing for CD9 and/or testing for CD29, CD45, CD73, and CD90.
The invention also features a method of characterizing a population of MLPC. The method includes providing a purified population of MLPC; and assessing expression in the population of MLPC of one or more mRNAs selected from the group consisting of CXCR4, FLT3, CD133, ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, ICAM1, CD24, CD34, CD44, CD45, CD58, CD68, CD33, CD37, CD38, TERT, KIT, and POUF5. In some embodiments, expression of mRNA for CXCR4, FLT3, and CD133 is assessed. In other embodiments, expression of mRNA for TERT, KIT, and POU5F is assessed. In still other embodiments, expression of mRNA for CD34 is assessed.
In another aspect, the invention features a method for characterizing the immaturity of a population of MLPC. The method includes providing a purified population of MLPC; and assessing expression of mRNA for CXCR4, FLT3, and CD133 in the population of MLPC, wherein enhanced expression of CXCR4, FLT3, and CD133 relative to that of a population of MSC is indicative of an immature phenotype.
The invention also features a method of making a clonal line of MLPC. The method includes providing a population of MLPC, culturing a single MLPC in a culture vessel lacking other cells until a plurality of progeny are produced; and culturing the progeny to obtain the clonal line.
FIG. 3 is a table that lists the 631 genes that had >1.4 fold differential expression between MLPC and any one or more of the five cell groups. Shaded text refers to over-expression of the gene in the comparative cell group; bold text refers to over-expression of genes in MLPC. The values represent the ratio of the signal intensity for the comparative cell group to the signal intensity of the MLPC, i.e., for uniq ID 43, signal intensity of B/signal intensity of MLPC is 0.68.
FIG. 4 is a chart that provides examples of the differences between MLPC and MSC in the expression of adhesion molecules.
FIG. 5 is a chart that provides examples of the differences between MLPC and MSC in the expression of growth factors and receptors.
FIG. 6 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes involved in cell cycle, proliferation, and anti-apoptosis.
FIG. 7 is a chart that provides examples of the differences between MLPC and MSC in the expression of transcription factors.
FIG. 8 is a chart that provides examples of the differences between MLPC and MSC in the expression of translation regulators.
FIG. 9 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes for connective tissue, cartilage, and bone.
FIG. 10 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes for extracellular matrix.
FIG. 11 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes for the endothelium.
FIG. 12 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes involved in hematopoiesis and the immune response.
FIG. 13 is a chart that provides examples of the differences between MLPC and MSC in the expression of neural genes.
FIG. 14 is a chart that provides examples of the differences between MLPC and MSC in the expression of hepatic genes.
FIG. 15 is a chart that provides examples of the differences between MLPC and MSC in the expression of muscle, smooth muscle, and cardiac genes.
FIG. 16 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes involved in cell-cell communication.
FIG. 17 is a chart that provides examples of the differences between MLPC and MSC in the expression of stem cell markers (hematopoietic and mesenchymal).
FIG. 18 is a chart that provides examples of the differences between MLPC and MSC in the expression of epidermal genes.
FIG. 19 is a chart that provides examples of the differences between MLPC and MSC in the expression of adipocytic genes.
FIG. 20 is a chart that provides examples of the differences between MLPC and MSC in the expression of pancreatic genes
FIG. 21 is a chart that provides examples of the differences between MLPC and MSC in the expression of genes involved in development/morphogenesis.
In general, the invention provides purified populations of MLPC from human fetal blood (e.g., umbilical cord blood (“cord blood”), placental blood, or the blood from a fetus) and clonal MLPC lines derived from individual MLPC. Fetal blood provides a source of cells that is more immature than adult bone marrow and has a higher percentage of cells bearing immature cell surface markers. Consequently, there may be advantages in the expansion and differentiation capacity of the progenitor cells from fetal blood. As described herein, MLPC have immunophenotypic characteristics and a gene expression profile distinct from bone marrow derived MSC's, bone marrow-derived HSC, and umbilical cord blood-derived HSC and USSC. The cells described herein have the capacity to self renew and differentiate into diverse tissue types similar to the bone marrow-derived MSC and MAPC cells. MLPC can be used to develop cellular therapies and establish cryopreserved cell banks for future regenerative medicine procedures. MLPC also can be modified such that the cells can produce one or more polypeptides or other therapeutic compounds of interest.
The concentration of anti-CD 15 antibodies in a cell separation composition can range from 0.1 to 15 mg/L (e.g., 0.1 to 10, 1 to 5, or 1 mg/L). Anti-CD15 antibodies can cause homotypic agglutination of granulocytes by crosslinking CD15 molecules that are present on the surface of granulocytes. Anti CD15 antibodies also can cause homotypic and heterotypic agglutination of granulocytes with monocytes, NK-cells and B-cells by stimulating expression of adhesion molecules (e.g., L-selectin and beta-2 integrin) on the surface of granulocytes that interact with adhesion molecules on monocytes, NK-cells and B-cells. Heterotypic agglutination of these cell types can facilitate the removal of these cells from solution along with red cell components. Exemplary monoclonal anti-CD15 antibodies include, without limitation, AHN1.1 (Murine IgM isotype), FMC-10 (Murine IgM isotype), BU-28 (Murine IgM isotype), MEM-157 (Murine IgM isotype), MEM-158 (Murine IgM isotype), 324.3.B9 (Murine IgM isotype; BioE, St. Paul, Minn.), and MEM-167 (Murine IgM isotype). See e.g., Leukocyte typing IV (1989); Leukocyte typing II (1984); Leukocyte typing VI (1995); Solter D. et al., Proc. Natl. Acad. Sci. USA 75:5565 (1978); Kannagi R. et al., J. Biol. Chem. 257:14865 (1982); Magnani, J. L. et al., Arch. Biochem. Biophys 233:501 (1984); Eggens I. et al., J. Biol. Chem. 264:9476 (1989).
In some embodiments, a cell separation composition contains antibodies against CD41, which can selectively agglutinate platelets. In some embodiments, a cell separation composition contains antibodies against CD3, which can selectively agglutinate T-cells. In some embodiments, a cell separation composition contains antibodies against CD2, which can selectively agglutinate T-cells and NK cells. In some embodiments, a cell separation composition contains antibodies against CD72, which can selectively agglutinate B-cells. In some embodiments, a cell separation composition contains antibodies against CD16, which can selectively agglutinate NK cells and neutrophilic granulocytes. The concentration of each of these antibodies can range from 0.01 to 15 mg/L. Exemplary anti-CD41 antibodies include, without limitation, PLT-1 (Murine IgM isotype), CN19 (Murine IgG1 isotype), and 8.7.C3 (Murine IgG1 isotype). Non-limiting examples of anti-CD3 antibodies include OKT3 (Murine IgG1), HIT3a (Murine IgG2a isotype), SK7 (Murine IgG1) and BC3 (Murine IgG2a). Non-limiting examples of anti-CD2 antibodies include 7A9 (Murine IgM isotype), T11(Murine IgG1 isotype), and Leu5b (Murine IgG2a Isotype). Non-limiting examples of anti-CD72 antibodies include BU-40 (Murine IgG1 isotype) and BU-41 (Murine IgG1 isotype). Non-limiting examples of anti-CD16 antibodies include 3G8 (Murine IgG).
MLPC also can be characterized based on the expression of one or more genes. Methods for detecting gene expression can include, for example, measuring levels of the mRNA or protein of interest (e.g., by Northern blotting, reverse-transcriptase (RT)-PCR, microarray analysis, Western blotting, ELISA, or immunohistochemical staining). As described in Example 12, the gene expression profile of MLPC is significantly different than other cell types. Microarray analysis indicated that the MLPC lines have an immature phenotype that differs from the phenotypes of, for example, CD133+ HSC, lineage negative cells (Forrz et al., Stem Cells, 22(1):100-108 (2004)), and MSC (catalog #PT-2501, Cambrex, Walkersville; Md., U.S. Pat. No. 5,486,359), which demonstrate a significant degree of commitment down several lineage pathways.
Comparison of the gene expression profile of MLPC and MSC demonstrates MSC are more committed to connective tissue pathways. There are 80 genes up-regulated in MSC, and 152 genes up-regulated in MLPC. In particular, the following genes were up-regulated in MLPC when compared with MSC, i.e., expression was decreased in MSC relative to MLPC: ITGB2, ARHGAP9, CXCR4, INTEGRINB7, PECAM1, PRKCB—1, PRKCB—3, IL7R, AIF1, CD45_EX10-11, PLCG2, CD37, PRKCB—2, TCF2—1, RNF138, EAAT4, EPHA1, RPLP0, PTTG, SERPINA12, ITGAX, CD24, F11R, RPL4, ICAM1, LMO2, HMGB2, CD38, RPL7A, BMP3, PTHR2, S100B, OSF, SNCA, GRIK1, HTR4, CHRM1, CDKN2D, HNRPA1, IL6R, MUSLAMR, ICAM2, CSK, ITGA6, MMP9, DNMT1, PAK1, IKKB, TFRC_MIDDLE, CHI3L2, ITGA4, FGF20, NBR2, TNFRSF1B, CEBPA—3, CDO1, NFKB1, GATA2, PDGFRB, ICSBP1, KCNE3, TNNC1, ITGA2B, CCT8, LEFTA, TH, RPS24, HTR1F, TREM1, CCNB2, SELL, CD34, HMGIY, COX7A2, SELE, TNNT2, SEM2, CHEK1, CLCN5, F5, PRKCQ, ITGAL, NCAM2, ZNF257-MGC12518-ZNF92-ZNF43-ZNF273-FLJ90430, CDK1, RPL6, RPL24, IGHA1-IGHA2_M, PUM2, GJA7, HTR7, PTHR1, MAPK14, MSI2—1, KCNJ3, CD133, SYP, TFRC—5PRIME, TDGF1-TDGF3—2, FLT3, HPRT, SEMA4D, ITGAM, KIAA0152—3, ZFP42, SOX20, FLJ21190, CPN2, POU2F2, CASP8—1, CLDN10, TREM2, TERT, OLIG1, EGR2, CD44_EX3-5, CD33, CNTFR, OPN, COL9A1—2, ROBO4, HTR1D1, IKKA, KIT, NPPA, PRKCH, FGF4, CD68, NUMB, NRG3, SALL2, NOP5, HNF4G, FIBROMODULIN, CD58, CALB1, GJB5, GJA5, POU5F—1, GDF5, POU6F1, CD44_EX16-20, BCAN, PTEN1-PTEN2, AGRIN, ALB, KCNQ4, DPPA5, EPHB2, TGFBR2, and ITGA3.
MLPC express a number of genes associated with “sternness,” which refers to the ablility to self-renew undifferentiated and ability to differentiate into a number of different cell types. Genes associated with “stemness” include the genes known to be over-expressed in human human embryonic stem cells, including, for example, POU5F (October 4), TERT, and ZFP42. For example, 65 genes associated with protein synthesis are down-regulated, 18 genes linked with phosphate metabolism are down-regulated, 123 genes regulating proliferation and cell cycling are down-regulated, 12 different gene clusters associated with differentiation surface markers are down-regulated, e.g., genes associated with connective tissue, including integrin alpha-F, laminin and collagen receptor, ASPIC, thrombospondins, endothelium endothelin-1 and -2 precursors, epidermal CRABP-2, and genes associated with adipocytes, including, for example, the leptin receptor, and 80 genes linked to nucleic acid binding and regulation of differentiation are up-regulated. Thus, the immaturity of a population of MLPC can be characterized based on the expression of one or more genes (e.g., one or more of CXCR4, FLT3, TERT, KIT, POU5F, or hematopoietic CD markers such as CD9, CD34, and CD133).
MLPC can be induced to differentiate into cells having a myocytic phenotype using an induction medium (e.g., SkGM™, catalog # CC-3160, from Cambrex) containing EGF, insulin, Fetuin, dexamethasone, and FGF-basic (Wernet, et al., U.S. patent publication 20020164794 A1). Cells having a myocytic phenotype express fast skeletal muscle myosin and alpha actinin.
MLPC can be induced to differentiate into cells having an endothelial phenotype using an endothelial growth medium (e.g., EGM™-MV, catalog # CC-3125, from Cambrex) containing heparin, bovine brain extract, epithelial growth factor (e.g., human recombinant epithelial growth factor), and hydrocortisone. Endothelial differentiation can be confirmed by expression of E-selectin (CD62E), ICAM-2 (CD102), CD34, and STRO-1.
MLPC can be induced to differentiate into cells having a hepatocyte/pancreatic precursor cell phenotype using an induction medium (e.g., HCM™—hepatocyte culture medium, catalog # CC-3198, from Cambrex) containing ascorbic acid, hydrocortisone, transferrin, insulin, EGF, hepatocyte growth factor, FGF-basic, fibroblast growth factor-4, and stem cell factor. Liver and pancreas cells share a common progenitor. Hepatocyte differentiation can be confirmed by expression of hepatocyte growth factor and human serum albumin. Pancreatic cell differentiation can be confirmed by production of insulin and pro-insulin.
Purified populations of MLPC or clonal MLPC lines can be combined with packaging material and sold as a kit. The packaging material included in a kit typically contains instructions or a label describing how the purified populations of MLPC or clonal lines can be grown, differentiated, or used. A label also can indicate that the MLPC have enhanced expression of, for example, CXCR4, FLT3, or CD133 relative to a population of MSC. Components and methods for producing such kits are well known.
An article of manufacture or kit also can include one or more reagents for characterizing a population of MLPC or a clonal MLPC line. For example, a reagent can be a nucleic acid probe or primer for detecting expression of a gene such as CXCR4, FLT3, CD133, CD34, TERT, KIT, POU5F, ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, ICAM1, CD24, CD44, CD45, CD58, CD68, CD33, CD37, or CD38. Such a nucleic acid probe or primer can be labeled, (e.g., fluorescently or with a radioisotope) to facilitate detection. A reagent also can be an antibody having specific binding affinity for a cell surface marker such as CD9, CD45, SSEA-4, CD34, CD13, CD29, CD41, CD44, CD73, CD90, CD105, stem cell factor, STRO-1, SSEA-3, CD133, CD15, CD38, glycophorin A (CD235a), CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD16, CD19, CD20, CD21, CD22, CD29, CD33, CD36, CD41, CD61, CD62E, CD72, CD73, CD90, HLA-DR, CD102, or CD105. An antibody can be detectably labeled (e.g., fluorescently or enzymatically).
Anti-human glycophorin A (murine IgM monoclonal
antibody, clone 2.2.2.E7)
0.25 mg/L)
Anti-CD15 (murine IgM monoclonal antibody, clone
324.3.B9)
2.0 mg/L)
Anti-CD9 (murine IgM monoclonal antibody, clone
8.10.E7)
Briefly, 50-150 ml of CPDA anti-coagulated umbilical cord blood (<48 hours old) was gently mixed with an equal volume of cell separation composition described in Table 3 for 30 minutes. After mixing was complete, the container holding the blood/cell separation composition mixture was placed in an upright position and the contents allowed to settle by normal 1�g gravity for 30 minutes. After settling was complete, the non-agglutinated cells were collected from the supernatant. The cells were recovered from the supernatant by centrifugation then washed with PBS. Cells were resuspended in complete MSCGM™ (Mesenchymal stem cell growth medium, catalog # PT-3001, Cambrex, Walkersville, Md.) and adjusted to 2-9�106 cells/ml with complete MSCGM™. Cells were plated in a standard plastic tissue culture flask (e.g., Corning), chambered slide, or other culture device and allowed to incubate overnight at 37� C. in a 5% CO2 humidified atmosphere. All subsequent incubations were performed at 37� C. in a 5% CO2 humidified atmosphere unless otherwise noted. MLPC attached to the plastic during this initial incubation. Non-adherent cells (T-cells, NK-cells and CD34+ hematopoietic stem cells) were removed by vigorous washing of the flask or well with complete MSCGM™.
In order to determine the surface markers present on MLPC, freshly isolated cells were plated in 16 well chamber slides and grown to confluency. At various times during the culture (from 3 days post plating to post confluency), cells were harvested and stained for the following markers: CD45-FITC (BD/Pharmingen), CD34-PE (BD/Pharmingen), CD4-PE (BioE), CD8-PE (BioE), anti-HLA-DR-PE (BioE), CD41-PE (BioE), CD9-PE (Ancell), CD105-PE (Ancell), CD29-PE (Coulter), CD73-PE (BD/Pharmingen), CD90-PE (BD/Pharmingen), anti-hu Stem Cell Factor-FITC (R&D Systems), CD14-PE (BD/Pharmingen), CD 15-FITC (Ancell), CD38-PE (BD/Pharmingen), CD2-PE (BD/Pharmingen), CD3-FITC (BD/Pharmingen), CD5-PE (BD/Pharmingen), CD7-PE (BD/Pharmingen), CD16-PE (BD/Pharmingen), CD20-FITC (BD/Pharmingen), CD22-FITC (BD/Pharmingen), CD19-PE (BD/Pharmingen), CD33-PE (BD/Pharmingen), CD10-FITC (BD/Pharmingen), CD61-FITC (BD/Pharmingen), CD133-PE (R&D Systems), anti-STRO-1 (R&D Systems) and Goat anti-mouse IgG(H+L)-PE (BioE), SSEA-3 (R&D Systems) and goat anti-rat IgG (H+L)-PE (BioE), SSEA-4 (R&D Systems) and goat anti-mouse IgG(H+L)-PE (BioE). The cell surface markers also were assessed in bone marrow MSC (Cambrex, Walkersville, Md.) and cord blood HSC (obtained from the non-adherent cells described above).
At least 52 clonal cell lines have been established using this procedure and were designated: UM081704-1-E2, UM081704-1-B6, UM081704-1-G11, UM081704-1-G9, UM081704-1-E9, UM081704-1-E11, UM081704-1-G8, UM081704-1-H3, UM081704-1-D6, UM081704-1-H11, UM081704-1-B4, UM081704-1-H4, UM081704-1-C2, UM081704-1-G1, UM081704-1-E10, UM081704-1-B7, UM081704-1-G4, UM081704-1-F12, UM081704-1-H1, UM081704-1-D3, UM081704-1-A2, UM081704-1-B11, UM081704-1-D5, UM081704-1-E4, UM081704-1-C10, UM081704-1-A5, UM081704-1-E8, UM081704-1-C12, UM081704-1-E5, UM081704-1-A12, UM081704-1-C5, UM081704-1-A4, UM081704-1-A3, MH091404-2 #1-1.G10, UM093004-1-A3, UM093004-1-B7, UM093004-1-F2, UM093004-1-A12, UM093004-1-G11, UM093004-1-G4, UM093004-1-B12, UM093004-2-A6, UM093004-2-A9, UM093004-2-B9, UM093004-2-C5, UM093004-2-D12, UM093004-2-H3, UM093004-2-H11, UM093004-2-H4, UM093004-2-A5, UM093004-2-C3, and UM093004-2-C10. The surface markers of clonal cell line UM081704-1-E8 were assessed according to the procedure outlined in Example 4 and found to be the same as the “mature MLPC” having fibroblast morphology, as shown in Table 4.
A population of MLPC and clonal cell line UM081704-1-E8 each were cultured in complete MSCGM™ and grown under logarithmic growth conditions outlined above. Cells were harvested by treatment with PBS+0.1% EGTA and replated at 5�103 to 2�104/ml in complete MSCGM™. The cells were allowed to adhere overnight and then the medium was replaced with Osteogenic Differentiation Medium (catalog # PT-3002, Cambrex) consisting of complete MSCGM™ supplemented with dexamethasone, L-glutamine, ascorbate, and β-glycerophosphate. Cells were cultured at 37� C. in a 5% CO2 atmosphere and fed every 3-4 days for 2-3 weeks. Deposition of calcium crystals was demonstrated by using a modification of the Alizarin red procedure and observing red staining of calcium mineralization by phase contrast and fluorescent microscopy.
MLPC (both a population and clonal cell line UM081704-1-E8) were plated in complete MSCGM™ at a concentration of 1.9�104 cells/well within a 4-chamber fibronectin pre-coated slide and allowed to attach to the plate for 24-48 hr at 37� C. in a 5% CO2 atmosphere. Medium was removed and replaced with 10 μM 5-azacytidine (catalog #A1287, Sigma Chemical Co.) and incubated for 24 hours. Cells were washed twice with PBS and fed with SkGM™ Skeletal Muscle Cell Medium (catalog # CC-3160, Cambrex) containing recombinant human epidermal growth factor (huEGF), human insulin, Fetuin, dexamethasone, and recombinant human basic fibroblast growth factor (100 ng/mL) (huFGF-basic, catalog # F0291, Sigma Chemical Co., St. Louis, Mo.). Cells were fed every 2-3 days for approximately 21 days. Control wells were fed with MSCGM™ while experimental wells were fed with SkGM™ (as described above).
Bone marrow derived hMSC (Cambrex), cord blood MLPC, and MLPC clonal cell line were grown under logarithmic growth conditions described above. Cells were harvested as described above and replated at 0.8�104 cells per chamber in 4-chamber slides that were pre-coated with poly-D-lysine and laminin (BD Biosciences Discovery Labware, catalog #354688) in 0.5 mL of NPMM™ (catalog #CC-3209, Cambrex) containing huFGF-basic, huEGF, brain-derived neurotrophic factor, neural survival factor-1, fibroblast growth factor-4 (20 ng/mL), and 200 mM GlutaMax I Supplement (catalog #35050-061, Invitrogen, Carlsbad, Calif.). The medium was changed every 2-3 days for 21 days. Neurospheres developed after 4 to 20 days. Transformation of MLPC to neural lineage was confirmed by positive staining for nestin (monoclonal anti-human nestin antibody, MAB1259, clone 196908, R&D Systems), class III beta-tubulin (tubulin b-4) (monoclonal anti-neuron-specific class III beta-tubulin antibody, MAB 1195, Clone TuJ-1, R&D Systems), glial fibrillary acidic protein (GFAP) (monoclonal anti-human GFAP, HG2b-GF5, clone GF5, Advanced immunochemical, Inc.), and galactocerebroside (GalC) (mouse anti-human GalC monoclonal antibody MAB342, clone mGalC, Chemicon).
MLPC were plated at 1.9�104 cells per well within a 4-chamber slide (2 cm2). Cells were fed with 1 ml of endothelial growth medium-microvasculature (EGM™-MV, catalog #CC-3125, Cambrex) containing heparin, bovine brain extract, human recombinant epithelial growth factor and hydrocortisone. The cells were fed by changing the medium every 2-3 days for approximately 21 days. Morphological changes occurred within 7-10 days. Differentiation of MLPC's to endothelial lineage was assessed by staining for CD62E [E-selectin, mouse anti-human CD62E monoclonal antibody, catalog #551145, clone 68-5H11, BD Pharmingen] and CD102 [ICAM-2, monoclonal anti-human ICAM-2, MAB244, clone 86911, R&D Systems], CD34 [BD Pharmingen] and STRO-1 (R&D Systems]. Control MLPC cultures grown in MSCGM for 14 days were negative for CD62E staining and CD102, CD34 and STRO-1, while differentiated cultures were positive for both CD62E, CD102, CD34, and STRO-1.
Comparative Gene Expression of MLPC
In order to determine the relationship between MLPC and various other cell types at various stages of lineage commitment, the relative expression of 942 genes associated with “stemness” and differentiating capacity was assessed by high definition microarray. The phenotype of MLPC (E8 clone, passage 8, see Example 5) was compared to human umbilical cord mononuclear cells (MNC), PrepaCyte�-MLPC isolated cord blood cells, CD133+ positively selected cord blood progenitor cells, lineage negative cord blood cells, and bone marrow-derived mesenchymal stem cells (MSC, Cambrex, Walkersville, Md.). The MNC were isolated by density gradient centrifugation method on Ficoll. Cells were mature terminally differentiated lymphocytes and monocytes with some minor contamination with platelets and granulocytes. The PrepaCyte isolated cells were human umbilical cord blood cells isolated by PrepaCyte-MLPC method. Cells were mainly lymphocytes, hematopoietic stem cells, and MLPC with some minor contamination with monocytes, granulocytes, and platelets. CD133+ selected cells were human umbilical cord blood cells separated by Ficoll density gradient separation then positively selected using anti-CD133 coated paramagnetic particles (Miltenyi). These cells are consistent with hematopoietic stem cells. Lineage Negative cells were isolated from human umbilical cord blood cells by Ficoll density gradient separation then negatively selected by depletion with anti-CD45, anti-CD7, and anti-glycophorin A-coated paramagnetic particles. These cells have been demonstrated to have some multi-lineage differentiating capacity. See, Forrz et al., Stem Cells, 22(1):100-108 (2004). MSC were purchased from Cambrex (catalog # PT-2501).
Methods: Total RNA was extracted from each cell population using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, Calif.). Complementary DNAs (cDNA) were derived from the total RNA samples and the cDNA labeled with Cy3 (MLPC) or Cy5 (other cell types) according to the PIQOR™ Instruction Manual. The labeled samples then were hybridized to the PIQOR™ 942 gene Stem Cell Human Antisense Microarray platform (Miltenyi Biotec) according to the PIQOR™ Instruction Manual. Thus, a total of five hybridizations were done: MLPC and PrepaCyte cells (designated hybridization B), MLPC and MNC (designated hybridization C), MLPC and CD133+ selected cells (designated hybridization D), MLPC and MSC (designated hybridization E), and MLPC and LinNeg cells (designated hybridization F). After hybridization, the intensity of the Cy3 and Cy5 labels was assessed on each microarray. Comparative over-expression of genes in MLPC resulted in a green spot, over-expression of genes in the comparative cell group resulted in a red spot. Equal expression resulted in a yellow spot. The ratio of green to red fluorescence allowed the determination of over- or under-expression. Analysis of the microarrays was performed by the Memorec division of Miltenyi Biotec in a blinded analysis.
Genes that did not reach an at least 1.4-fold differential expression in at least one experiment were excluded from further analyses. The resulting dataset included 631 genes that were >1.4 fold up- or down-regulated between MLPC and any one or more of the other five cell groups (see FIG. 3; shaded text refers to over-expression of the gene in the comparative cell group; bold text refers to over-expression of genes in MLPC; the values represent the ratio of the signal intensity for the comparative cell group to the signal intensity of the MLPC, i.e., for uniq ID 43, signal intensity of B/signal intensity of MLPC is 0.68). To identify the discriminatory genes, genes were selected that had their maximum expression value in hybridizations B, C, D, E, or F. Genes were removed where the ‘maximum expression’ was just due to a less pronounced down-regulation relative to the other experiments. The difference between the highest and the second-best expression value was determined for each gene and used as an indicated for its suitability to separate between the different conditions. A corresponding procedure was performed to identify the down-regulated genes most suitable for the discrimination of the different cell populations.
Pathway and signal network analysis delineated numerous biological pathways characterizing MLPCs unique features. During this procedure, each group was screened for significant enrichment of genes belonging to a common pathway or sharing other biological properties.
MLPC vs. PrepaCyte Cells
Sixty-five (65) genes were up-regulated in the PrepaCyte cells relative to the MLPC: PF4-PF4V1, CLDN5, IL7R, CHI3L2, CD38, RBL2, MAD1, INTEGRINB7, CD7, DRD3, HIST1H2AC, PBXIP1, ITGAL, FKHR, CDKN2B, RPL13A, MMRN, KCNQ2—2, MFHI, BMP6, ABCC8, MUSLAMR, ALCAM, CDKN1B, ANGPT2, RPL24, FLJ10884, ICAM2, FOXG1A-FOXG1B, PLCG1, ERBB2, DAB1, RPL6, RPLP0, MMP21-22-23, RPL4, RPL7A, SMAD2, RPS24, ATM, CDO1, SELE, SELL, GRIK1, VEGFB, KCNQ4, TGFBR1, TFP1, KIAA0152—3, COL4A5, MAP-2, FAST1, TBX3, PRKCH, HNF4A, SEMA3C, TEAD1, NFKB2, COL4A6, HNF4G, DPPA5, LEFTA, FGF20, OSF, RPSA, and ITGA2B. Among the 65 genes, 8 were annotated as ribosomal subunits (RPLP0 (genno. 19690), RPL6 (genno. 22801), RPS24 (genno. 27255), RPL24 (genno. 28658), RPL13A (genno. 31144), RPL4 (genno. 32719), RPSA (genno. 3929), and RPL7A (genno. 7951). This enrichment may point to an enhancement of protein synthesis relative to the MLPC and also shows that the PrepaCyte cells are a heterogeneous group of cells, many of which are mature.
Fifty-five (55) genes were down-regulated relative to MLPC, including PROX1, DLK1, CRABP1, HNF3G, CSPG2—1, TIMP2, CD44_EX10-12, ANPEP, ZNF117, IFNGR1, COL4A3—1, ATF4, LAMB3, ELAVL4, IFNGR2, CTNNA1, CDC25C, S100A11, CRABP2, MMP11, PAFAH1B1, MTHFD2, BRACHYURY, BMPRIA, KCNA4, TUBA, TTR, ACVR2B, KCNJ6, ADH4, VLDLR, GAPD, RACK17, MCAM, HNF3B, TNFR1, JAM2, IL3RA, FGF1, VGR3, ORP150, SNA12, GPC4, KIF4A, TC10-PIGF, RAC1, CYP3A4, THBD, CHEK2, VEGF 1, AMBP, TCF3, KCNQ5, DDX21, and LAPTM4B. Thus, MLPC and PrepaCyte cells have different profiles.
MLPC vs. MNC
MLPC and MNC also have different profiles. One hundred and eight (108) genes were up-regulated in the MNC, including: PROX1, KCNJ15, IL1R2, SNCA, HBZ, F7, MYL4, CDKN2D, BMPR1B, TCF2—1, RXRA, CLCN5, SOX6, SLC2A1, TAL1, CLCN3, SALL2, POU6F1, IGHA1-IGHA2, MAT1A, EGR2, HDAC2, FGFR3, TREM1, MAP3K3, PRKCZ, PUM2, BMP4, PTEN1-PTEN2, KCNQ3, SYP, CD44_EX8-10, IL4R, ACVRL1, CDC25B, ODC1, SLC16A1, PGH2, EDNRA, TRK-C, CDH5, COL10A1, PIK3CG, NEUROG1, AKT2, ITGA3, CXCR4, DLX2, IL6R, KCNJ1, SLIT-1, IKKA, GDF5, ITGB3, CASP8—1, FIBROMODULIN, AGRIN, TENASCINX, KIT, CD44_EX7-9, TERT, ZFP42, MS12—1, ACVR2B, CDC42—1, POU5F—1, CPN2, RARA1, TNFSF4, ROBO4, SEM2, CLDN1, TDGF1-TDGF3—2, PRKCQ, COL9A1—2, ELAVL4, EPHB2, ELOVL6, PAK1, SOX20, BRACHYURY, NOTCH1, LRRN1, SEMA4D, ARL8, HGF, PRKCB—2, BMP3, WNT10B, BCRP, NKX2-2, FGFR2, HAND1, PLAUR, CSK, PDGFRB, COL18A1—1, CCNG2, ITGA3-5PRIME, CD45_EX10-11, PTHR1, SEMAL, MAPK3, TNNT2, NBR2, APC3, PTHLH, and HTR6. Table 5 lists the 23 genes identified as belonging to a particular pathway or family (“A” refers to a kinase and “B” refers to phosphate metabolism).
Eighteen genes were identified as belonging to the family of protein kinases (ACVR2B, ACVRL1, BMPR1B, EPHB2, PRKCZ, PRKCQ, MAPK3, AKT2, PPKCB, PAK1, MAP3K3, CSK, KIT, PDGFRB, IKKA, FGFR2, NTRK3, and FGFR3). Five genes (CD45, PIK3CG, PTEN1-PTEN2, CDC25B, and TDGF1-TDGF3) were identified as being involved in phosphate metabolism in general, e.g., as phosphatases. The PIQOR probes PTEN1-PTEN2 or TDGF1-TDGF3 are specific for two different, highly similar genes each. Among these kinases, growth factor receptors like, e.g., FGFR2, FGFR3, PDGFRB, KIT, and NTRK3, and two activin receptors, ACVR2B and ACVRL1 can be found.
Pathway analysis for up-regulated genes in MNC
genno.
TDGF1-TDGF3_2_HUMAN: (TDGF1 OR CRIPTO) TERATOCARCINOMA-DERIVED
GROWTH FACTOR 1 (EPIDERMAL GROWTH FACTOR-LIKE CRIPTO PROTEIN CR1)
(CRIPTO-1 GROWTH FACTOR) (CRGF) (TDGF3 OR TDGF2) TERATOCARCINOMA-
DERIVED GROWTH FACTOR 2 (EPIDERMAL GROWTH FACTOR-LIKE CRIPTO PROTEIN
CR3) (CRIPTO-3 GROWTH FACTOR).
ACVR2B: (ACVR2B) ACTIVIN RECEPTOR TYPE IIB PRECURSOR (EC 2.7.1.—) (ACTR-IIB).
ACVRL1: (ACVRL1 OR ACVRLK1 OR ALK1) SERINE/THREONINE-PROTEIN KINASE
RECEPTOR R3 PRECURSOR (EC 2.7.1.37) (SKR3) (ACTIVIN RECEPTOR-LIKE KINASE 1)
(ALK-1) (TGF-B SUPERFAMILY RECEPTOR TYPE I) (TSR-I).
BMPR1B: (BMPR1B OR ACVRLK6) BONE MORPHOGENETIC PROTEIN RECEPTOR TYPE
IB PRECURSOR (EC 2.7.1.37) (SERINE/THREONINE-PROTEIN KINASE RECEPTOR R6)
(SKR6) (ACTIVIN RECEPTOR-LIKE KINASE 6) (ALK-6).
EPHB2: (EPHB2 OR EPTH3 OR ERK OR DRT OR HEK5) EPHRIN TYPE-B RECEPTOR 2
PRECURSOR (EC 2.7.1.112) (TYROSINE-PROTEIN KINASE RECEPTOR EPH-3) (DRT)
(RECEPTOR PROTEIN-TYROSINE KINASE HEK5) (ERK).
PRKCZ: (PRKCZ OR PKC2) PROTEIN KINASE C, ZETA TYPE (EC 2.7.1.—) (NPKC-ZETA).
CD45_EX10-11: (PTPRC OR CD45) LEUKOCYTE COMMON ANTIGEN PRECURSOR (EC
3.1.3.48) (L-CA) (CD45 ANTIGEN) (T200).
PRKCQ: (PRKCQ OR PRKCT) PROTEIN KINASE C, THETA TYPE (EC 2.7.1.—) (NPKC-
MAPK3: (MAPK3 OR PRKM3 OR ERK1) MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC
2.7.1.—) (EXTRACELLULAR SIGNAL-REGULATED KINASE 1) (ERK-1) (INSULIN-
STIMULATED MAP2 KINASE) (MAP KINASE 1) (MAPK 1) (P44-ERK1) (ERT2) (P44-MAPK)
(MICROTUBULE-ASSOCIATED PROTEIN-2 KINASE).
AKT2: (AKT2) RAC-BETA SERINE/THREONINE PROTEIN KINASE (EC 2.7.1.—) (RAC-PK-
BETA) (PROTEIN KINASE AKT-2) (PROTEIN KINASE B, BETA) (PKB BETA).
PRKCB_2: (PRKCB1 OR PRKCB OR PKCB) PROTEIN KINASE C, BETA TYPE (EC 2.7.1.37)
(PKC-BETA) (PKC-B).
PAK1: (PAK1) SERINE/THREONINE-PROTEIN KINASE PAK 1 (EC 2.7.1.—) (P21-
ACTIVATED KINASE 1) (PAK-1) (P65-PAK) (ALPHA-PAK).
MAP3K3: (MAP3K3 OR MAPKKK3 OR MEKK3) MITOGEN-ACTIVATED PROTEIN
KINASE KINASE KINASE 3 (EC 2.7.1.—) (MAPK/ERK KINASE KINASE 3) (MEK KINASE 3)
(MEKK 3).
CSK: (CSK) TYROSINE-PROTEIN KINASE CSK (EC 2.7.1.112) (C-SRC KINASE) (PROTEIN-
TYROSINE KINASE CYL).
PIK3CG: (PIK3CG) PHOSPHATIDYLINOSITOL 3-KINASE CATALYTIC SUBUNIT,
GAMMA ISOFORM (EC 2.7.1.137) (PI3-KINASE P110 SUBUNIT GAMMA) (PTDINS-3-
KINASE P110) (PI3K).
KIT: (KIT OR SL) MAST/STEM CELL GROWTH FACTOR RECEPTOR PRECURSOR (EC
2.7.1.112) (SCFR) (PROTO-ONCOGENE TYROSINE-PROTEIN KINASE KIT) (C-KIT) (CD117
ANTIGEN) (C-KIT RECEPTOR TYROSINE KINASE).
PDGFRB: (PDGFRB OR PDGFR) BETA PLATELET-DERIVED GROWTH FACTOR
RECEPTOR PRECURSOR (EC 2.7.1.112) (PDGF-R-BETA) (CD140B ANTIGEN).
IKKA: (IKK ALPHA OR CHUK) INHIBITOR OF NUCLEAR FACTOR KAPPA-B KINASE
ALPHA SUBUNIT (EC 2.7.1.—) (I KAPPA-B KINASE ALPHA) (IKBKA) (IKK-ALPHA) (IKK-
A) (IKAPPAB KINASE) (I-KAPPA-B KINASE 1) (IKK1) (CONSERVED HELIX-LOOP-HELIX
UBIQUITOUS KINASE) (NUCLEAR FACTOR NFKAPPAB INHIBITOR KINASE ALPHA)
(NFKBIKA).
PTEN1-PTEN2: (PTEN OR MMAC1 OR TEP1) PROTEIN-TYROSINE PHOSPHATASE PTEN
(EC 3.1.3.48) (MUTATED IN MULTIPLE ADVANCED CANCERS 1). (PTEN2)
HYPOTHETICAL 39.9 KDA PROTEIN (EC 3.1.3.48).
FGFR2: (FGFR2 OR ECT1 OR BEK) FIBROBLAST GROWTH FACTOR RECEPTOR 2
PRECURSOR (FGFR-2) (EC 2.7.1.112) (KERATINOCYTE GROWTH FACTOR RECEPTOR).
(K-SAM). (BFR2) FIBROBLAST GROWTH FACTOR RECEPTOR BFR-2 PRECURSOR (EC
2.7.1.112).
NTRK3: (NTRK3 OR TRKC) NT-3 GROWTH FACTOR RECEPTOR PRECURSOR (EC
2.7.1.112) (TRKC TYROSINE KINASE) (GP145-TRKC) (TRK-C).
CDC25B: (CDC25B OR CDC25HU2) M-PHASE INDUCER PHOSPHATASE 2 (EC 3.1.3.48).
FGFR3: (FGFR3 OR JTK4) FIBROBLAST GROWTH FACTOR RECEPTOR 3 PRECURSOR
(FGFR-3) (EC 2.7.1.112).
The following genes were down-regulated in MNC relative to MLPC: ID1, EDN1, CALU, UPA, CDK4, CTNNB1, NME2, SERPINH1-SERPINH2, PSMA3, HSPA4—1, TIMP1, FGF2—1, KRT14, RTN4, LAMG1, HSPA9, PCK2, IGFBP2, GFAP—1, ANXA2, EPRS, EED, ITGA5, OB—2, COL4A5, CRM1, HSPC150, PAX5, IDH1, KRAS2A-KRAS2B, TUBB1-TUBB5, ATF2, MMP6, NMYC, EGFR-SHORT, FGF19, PEG1-MEST, CEBPG, KRT18, GRIA1, CACNA1B, BAMACAN, CITED2, FKHL16, SDF2, CRYL1, MGST1, MMRN, VCAM1, FBXL13, IGF1, LAMB1, TFCOUP1, CCCAP, KIR2.4, KCNJ8, BUB1B, GPS1—3PRIME, PSMA2, VEGFB, FGF7, RAMP1, BMP11, SST, IL1R1, PAI2, ENG, NGN3, OTX2, SMAD2, COL9A1—1, NPM1, TUBB4, ID2, SNRPF, RAMP3, CD164, COL14A1, BUB3, KS, DPYSL3, RARG1, FLN1, KCNK2, CLCN4, INTEGRINB5, SEMA3C, MAD2L2, VAP-A, KPNA2, KCNK5, TEAD1, PTN, VTN, AKT, MTHFD1, GGH, KIAA0152—1, MYH7, OSP, WNT5A, ITGB1, DTYMK, CLDN6, FGF5, GADi—1, MAPK6, DLX1, CD47, BRIX, FABE, L30, FAB1, SMS, VEGC, NFATCB—1, DAB1, GATA6, SELPLG, CTNNA2, ISL1, ENO2, and EIF4A1.
MLPC vs. CD133
The following genes were up-regulated in CD133+ cells relative to MLPC: ELAVL2, FZD3, TFRC—3PRIME, HSPC150, CDK4, TFRC MIDDLE, KCNH2, NOP5, SNRPF, BUB1B, CLDN10, MAD2L1, PTTG, MTHFD1, CNTF-ZFP91—1, CRM1, HMGB2, LMO2, BAMACAN, DTYMK, FLT3, TK1, CEBPA 3, MGST1, EED, E21G3, HTR7, KPNA2, DNMT1, FGF19, MAD2L2, HMGIY, FGF16, MAP3K5, MYB, PLCB4, L30, NMYC, LIN-28, FABE, HNRPA1, BUB3, ENOS, C200RF1, GGH, PSMA3, PPAT, FLJ21190, CCNB2, SELPLG, CDK1, CDC25C, HSPA4—1, HOXA2, CHEK2, MAWBP, ANKRD17—1, OB—2, CCNE2, CCT8, NPM1, GAL, DLX1, BRIX, SMS, CHEK1, TUBB1-TUBB5, GATA5, TUBA, EAAT2—1, CD133, NPPA, FBXL13, HPRT, KCNJ8, IGHA1-IGHA2_M, RBL1, NGN3, RNF138, RAMP3, KIF4A, ITGA6, PSMA2, ALB, FGF4, KCNJ9, BEX2-BEX1, ILlRI, GPS1—3PRIME, KCNJ3, NCAM2, LDHB, IMPDH2, P53, TNFSF11, CRYL1, ZNF257-MGC12518-ZNF92-ZNF43-ZNF273-FLJ90430, GRIA1, KIAA1573, SST, CLCN4, VAP-A, TFRC—5PRIME, LAPTM4B, SCARB1, ZNF117, ICSBP1, HNF3G, DLK1, CD164, NME2, NFKB1, COL9A1—1, KIAA0152—1, CNTFR, EAAT4, BCAN, EGFR-SHORT, PAI2, HSPA9, BUB1, SOX2, PPP2R1B—1, TCF4, ATF4, TRK-B, LIF, and NRG3. Table 6 lists 42 genes identified as belonging to a particular pathway or family. In Table 6, “A” refers to cell cycle, DNA repair, DNA metabolism, as determined by Memorec's annotation system (ME), “B” refers to M phase as determined by gene ontology (GO), “C” refers to cell cycle as determined by GO, “F” refers to regulation of mitosis as determined by GO, “H” refers to mitotic cell cycle as determined by GO, “I” refers to cell cycle checkpoint as determined by GO, “L” refers to nucleotide synthesis as determined by GO, “P” refers to nucleotide/nucleoside metabolism as determined by GO, and “S” refers to microtubule cytoskeleton as determined by GO.
Pathway analysis for up-regulated genes in CD133+ cells
CHEK1: (CHEK1 OR CHK1) SERINE/THREONINE-PROTEIN KINASE CHK1 (EC
2.7.1.—) CHECKPOINT KINASE 1.
CHEK2: (CHEK2 OR CHK2) SERINE/THREONINE-PROTEIN KINASE CHK2 (EC
2.7.1.—) (CDS1).
HMGB2: (HMGB2 OR HMG2) HIGH MOBILITY GROUP PROTEIN HMG2 (HMG-2).
CCNB2: (CCNB2) CYCLIN B2 G2/MITOTIC SPECIFIC CYCLIN B2.
ELAVL2: (ELAVL2 OR HUB) ELAV-LIKE PROTEIN 2 (HU-ANTIGEN B) (HUB)
(ELAV-LIKE NEURONAL PROTEIN 1) (NERVOUS SYSTEM-SPECIFIC RNA
BINDING PROTEIN HEL-N1).
CCNE2: (CCNE2) G1/S-SPECIFIC CYCLIN E2.
BUB1: (BUB1 OR BUB1L) MITOTIC CHECKPOINT SERINE/THREONINE-
PROTEIN KINASE BUB1 (EC 2.7.1.—) (HBUB1) (BUB1A).
DNMT1: (DNMT1 OR DNMT OR AIM) DNA (CYTOSINE-5)-
METHYLTRANSFERASE HSAI (EC 2.1.1.37) (DNA METHYLTRANSFERASE
HSAI) (DNA MTASE HSAI) (MCMT) (M.HSAI).
HMGIY: (HMGIY OR HMGA1 OR HMGI) HIGH MOBILITY GROUP PROTEIN
HMG-Y (HIGH MOBILITY GROUP AT-HOOK 1).
GPS1_3PRIME: (GPS1 OR COPS1) COP9 SIGNALOSOME COMPLEX SUBUNIT 1
(G PROTEIN PATHWAY SUPPRESSOR 1) (GPS1 PROTEIN) (MFH PROTEIN).
HNRPA1: (HNRPA1) HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1
(HELIX-DESTABILIZING PROTEIN) (SINGLE-STRAND BINDING PROTEIN)
(HNRNP CORE PROTEIN A1).
BAMACAN: (BAM OR SMCD OR HCAP OR CSPG6 OR SMC3 OR SMC3L1 OR
BMH) STRUCTURAL MAINTENANCE OF CHROMOSOME 3 (CHONDROITIN
SULFATE PROTEOGLYCAN 6) (CHROMOSOME SEGREGATION PROTEIN
SMCD) (BAMACAN) BASEMENT MEMBRANE-ASSOCIATED CHONDROITIN
PROTEOGLYCAN) (HCAP).
C20ORF1: (C20ORF1 OR C20ORF2 OR DIL2 OR TPX2) RESTRICTED EXPRESSION
PROLIFERATION ASSOCIATED PROTEIN 100 (P100) (DIFFERENTIALLY
EXPRESSED IN LUNG CELLS 2) (DIL-2) (TARGETING PROTEIN FOR XKLP2)
(C20ORF1 PROTEIN) (C20ORF2 PROTEIN) (PROTEIN FLS353).
VAP-A: (VAP-A OR VAP33) VAMP-ASSOCIATED PROTEIN A (VAPA).
NME2: (NME2 OR NM23B) NUCLEOSIDE DIPHOSPHATE KINASE B (EC 2.7.4.6)
(NDK B) (NDP KINASE B) (P18).
PTTG_HUMAN: (PTTG) PITUITARY TUMOR TRANSFORMING GENE,
PITUITARY TUMOR TRANSFORMING GENE PROTEIN 1 (SECURIN HOMOLOG)
(HPTTG OR PTTG1 OR TUTR1) (PTTG2) PITUITARY TUMOR TRANSFORMING
GENE 2 PROTEIN (PTTG3) PITUITARY TUMOR TRANSFORMING GENE
MAD2L1: (MAD2L1 OR MAD2 OR MAD2A) MITOTIC SPINDLE ASSEMBLY
CHECKPOINT PROTEIN MAD2A (MAD2-LIKE 1).
KPNA2: (KPNA2 OR RCH1 OR SRP1) IMPORTIN ALPHA-2 SUBUNIT
(KARYOPHERIN ALPHA-2 SUBUNIT) (SRP1-ALPHA) (RAG COHORT PROTEIN
LAPTM4B: (LAPTM4B OR LAPTM4BETA OR DKFZP586E1124) LYSOSOMAL-
ASSOCIATED TRANSMEMBRANE PROTEIN 4 BETA (NT2RM1000066) (LC27)
(INTEGRAL MEMBRANE TRANSPORTER) (HYPOTHETICAL PROTEIN
PSEC0001).
HPRT: (HPRT1 OR HPRT) HYPOXANTHINE-GUANINE
PHOSPHORIBOSYLTRANSFERASE (EC 2.4.2.8) (HGPRT) (HGPRTASE).
SNRPF: (SNRPF OR PBSCF) SMALL NUCLEAR RIBONUCLEOPROTEIN F
(SNRNP-F) (SM PROTEIN F) (SM-F) (SMF).
BUB1B: (BUB1B OR MAD3L OR BUBR1) MITOTIC CHECKPOINT
SERINE/THREONINE-PROTEIN KINASE BUB1 BETA (EC 2.7.1.—) (HBUBR1)
(MAD3/BUB1-RELATED PROTEIN KINASE) (MITOTIC CHECKPOINT KINASE
MAD3L).
BUB3: (BUB3) MITOTIC CHECKPOINT PROTEIN BUB3.
DTYMK: (DTYMK OR TYMK OR TMPK OR CDC8) THYMIDYLATE KINASE (EC
2.7.4.9) (DTMP KINASE).
MAD2L2: (MAD2L2 OR MAD2B OR REV7) MITOTIC SPINDLE ASSEMBLY
CHECKPOINT PROTEIN MAD2B (MAD2-LIKE 2) (HREV7) (2310033C13RIK).
PPP2R1B_1: (PPP2R1B) SERINE/THREONINE PROTEIN PHOSPHATASE 2A, 65
KDA REGULATORY SUBUNIT A, BETA ISOFORM (PP2A, SUBUNIT A, PR65-
BETA ISOFORM) (PP2A, SUBUNIT A, R1-BETA ISOFORM) (TRANSCRIPT
VARIANT 1).
NPM1: (NPM1 OR NPM) NUCLEOPHOSMIN (NPM) (NUCLEOLAR
PHOSPHOPROTEIN B23) (NUMATRIN) (NUCLEOLAR PROTEIN NO38).
IMPDH2: (IMPDH2 OR IMPD2) INOSINE-5′-MONOPHOSPHATE
DEHYDROGENASE 2 (EC 1.1.1.205) (IMP DEHYDROGENASE 2) (IMPDH-II)
(IMPD 2).
KIAA1573: (KIAA1573) HYPOTHETICAL PROTEIN KIAA1573 (B430218L07RIK)
(DKFZP686L04115) (FLJ12509) (FLJ14194).
KIF4A: (KIF4A OR KIF4) CHROMOSOME-ASSOCIATED KINESIN KIF4A
(CHROMOKINESIN).
LIN-28: (LIN28 OR LIN-28) HYPOTHETICAL PROTEIN FLJ12457 (RNA-BINDING PROTEIN
LIN-28).
MTHFD1: (MTHFD1 OR MTHFD OR MTHFC) C-1-TETRAHYDROFOLATE
SYNTHASE, CYTOPLASMIC (C1-THF SYNTHASE).
PPAT: (PPAT OR GPAT) AMIDOPHOSPHORIBOSYLTRANSFERASE PRECURSOR
(EC 2.4.2.14) (GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE
AMIDOTRANSFERASE) (ATASE) (GPAT).
TUBA_HUMAN: (TUBA3) (TUBA6) TUBULIN ALPHA-UBIQUITOUS CHAIN
(ALPHA-TUBULIN UBIQUITOUS) (TUBULIN K-ALPHA-1) (TUBULIN ALPHA-6
CHAIN) (ALPHA-TUBULIN 6) (TUBULIN ALPHA-3 CHAIN) (ALPHA-TUBULIN 3)
(TUBULIN B-ALPHA-1).
MYB: (MYB) MYB PROTO-ONCOGENE PROTEIN (C-MYB).
CDK1: (CDC2) CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2.7.1.—)
(P34 PROTEIN KINASE) (CYCLIN-DEPENDENT KINASE 1) (CDK1).
CDK4: (CDK4) CELL DIVISION PROTEIN KINASE 4 (EC 2.7.1.—) (CYCLIN-
DEPENDENT KINASE4) (PSK-J3).
TUBB1-TUBB5_HUMAN: (TUBB1) TUBULIN BETA-1 CHAIN. (TUBB5) TUBULIN BETA-5
CDC25C: (CDC25C) M-PHASE INDUCER PHOSPHATASE 3 (EC 3.1.3.48).
TK1: (TK1) THYMIDINE KINASE, CYTOSOLIC (EC 2.7.1.21).
P53: (TP53 OR P53) CELLULAR TUMOR ANTIGEN P53 (TUMOR SUPPRESSOR
P53) (PHOSPHOPROTEIN P53).
RBL1: (RBL1) RETINOBLASTOMA-LIKE PROTEIN 1 (107 KDA
RETINOBLASTOMA-ASSOCIATED PROTEIN) (PRB1) (P107).
Many of the genes up-regulated in the CD133+ cells belong to different protein families that are involved in cell cycle regulation, indicating that the CD133+ cell population was in strong proliferation before harvesting. Among the up-regulated genes are cyclin-dependent kinases (CDK2 and CDK4), checkpoint proteins (CHEK1, BUB1, MAD2L1, BUB1B, BUB3, and MAD2L2), cyclins (CCNB2 and CCNE2), enzymes involved in nucleotide metabolism (NME2, HPRT, DTYMK, IMPDH2, MTHFD1, PPAT, and DNMT1), P53 and proteins involved in re-modelling of the cytoskeleton (BAMACAN, C200RF1, VAP-A, PPP2R1B—1, NPM1, KIF4A, TUBA_HUMAN, and TUBB1-TUBB5_HUMAN).
The following genes were down-regulated in CD133+ cells relative to MLPC: CDKN1A, MAPK13, ALCAM, CHI3L1, BMP6, VIM, COL15A1, COL7A1, LAMA2, CD44_EX8-10, CD9, IL6, THROMBOSPONDIN1, S100A10, CDKN2A—1, STX1A, TPA, EDNRA, CEBPB, LAMA3, MAP-2, ACTB, IL4R, SMAD7, COL1A1, BDNF, INHBA, RXRA, MAPK3, WISP3, M6PR, CCNG2, ITGA1—2, PLCG1, MYL4, VEGFD, ABCC8, COL4A1, CD44_EX13-15, SLIT-1, LAMA4, CLDN1, KCNK4, KRT8, LXR-ALPHA, INTEGRINA8, KCNK1, COL11A1, GBP2, CDH5, CTGF, PAI1, NT5, CD7, TRK-C, FN1_REPEAT-B, ACVR1, ITGB3, SLC2A1, PDGFRA, RHOA, SOX9, MAT1A, GCK, THY1, DRD3, IGHA1-IGHA2, PRRX1, AGGRECAN1, MMP2, IL6ST, COL16A1, HAND1, FOXG1A-FOXG1B, ASCL1, FN1, HB-EGF, SMAD5, BMP1—1, MMP12, INTEGRINB6, and CD44_EX7-9. Table 7 lists the 38 genes identified as belonging to a particular pathway or family. In Table 7, “B” refers to extracellular matrix as determined by ME, “C” refers to LAM_G_DOMAIN as determined by PS, “D” refers to cell adhesion as determined by GO, and “E” refers to structural protein as determined by ME.
Pathway analysis for down-regulated genes in CD133+ cells
KRT8: (KRT8 OR CYK8) KERATIN, TYPE II CYTOSKELETAL 8 (CYTOKERATIN 8) (K8)
(CK 8) (KRT2-8).
CD44_EX13-15_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
GLYCOPROTEIN I) (PGP-1) (HUTCH-I) (EXTRACELLULAR MATRIX RECEPTOR-III)
(ECMR-III) (GP90 LYMPHOCYTE HOMING/ADHESION RECEPTOR) (HERMES
ANTIGEN) (HYALURONATE RECEPTOR) (HEPARAN SULFATE PROTEOGLYCAN)
(EPICAN) (CDW44).
CD44_EX7-9_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
CD44_EX8-10_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
CTGF: (CTGF OR HCS24) CONNECTIVE TISSUE GROWTH FACTOR PRECURSOR
(HYPERTROPHIC CHONDROCYTE-SPECIFIC PROTEIN 24).
SLIT1: (SLIT1 or KIAA0813 or MEGF4) Slit homolog 1 protein precursor (Slit-1) (Multiple
epidermal growth factor-like domains 4).
MYL4: (MYL4 OR MLC1) MYOSIN LIGHT CHAIN 1, EMBRYONIC MUSCLE/ATRIAL
ISOFORM (PRO1957). MYOSIN LIGHT CHAIN ALKALI, GT-1 ISOFORM (FRAGMENT).
ACTB: (ACTB) BETA1, CYTOPLASMIC (BETA-ACTIN) ACTIN, CYTOPLASMIC 1.
VIM: (VIM) VIMENTIN.
COL1A1: (COL1A1) COLLAGEN ALPHA 1(I) CHAIN PRECURSOR.
COL11A1: (COL11A1) COLLAGEN ALPHA 1(XI) CHAIN PRECURSOR.
COL15A1: (COL15A1) COLLAGEN ALPHA 1(XV) CHAIN PRECURSOR.
COL16A1: (COL16A1) COLLAGEN ALPHA 1(XVI) CHAIN PRECURSOR.
COL4A1: (COL4A1) COLLAGEN ALPHA 1(IV) CHAIN PRECURSOR (ARRESTEN).
COL7A1: (COL7A1) COLLAGEN ALPHA 1(VII) CHAIN PRECURSOR (LONG-CHAIN
COLLAGEN) (LC COLLAGEN).
ITGA8: (ITGA8) INTEGRIN ALPHA-8 (INTEGRINA8).
ITGB6: (ITGB6) INTEGRIN BETA-6 PRECURSOR (INTEGRINB6).
CLDN1: (CLDN1 OR CLD1 OR SEMP1) CLAUDIN-1 (SENESCENCE-ASSOCIATED
EPITHELIAL MEMBRANE PROTEIN).
PAI1: (SERPINE1 OR PAI1 OR PLANH1) PLASMINOGEN ACTIVATOR INHIBITOR-1
PRECURSOR (PAI-1) (ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR) (PAI).
TPA: (PLAT) TISSUE-TYPE PLASMINOGEN ACTIVATOR PRECURSOR (EC 3.4.21.68)
(TPA) (T-PA) (T-PLASMINOGEN ACTIVATOR) (ALTEPLASE) (RETEPLASE).
LAMA2: (LAMA2 OR LAMM) LAMININ ALPHA-2 CHAIN PRECURSOR (LAMININ M
CHAIN) (MEROSIN HEAVY CHAIN).
LAMA3: (LAMA3) LAMININ ALPHA-3 CHAIN PRECURSOR (EPILIGRIN 170 KDA
SUBUNIT) (E170).
LAMA4: (LAMA4) LAMININ ALPHA-4 CHAIN PRECURSOR.
AGGRECAN1: (AGC1 OR CSPG1 OR AGC) AGGRECAN CORE PROTEIN PRECURSOR
(CARTILAGE-SPECIFIC PROTEOGLYCAN CORE PROTEIN) (CSPCP) (CHONDROITIN
SULFATE PROTEOGLYCAN CORE PROTEIN 1).
BMP1_1: (BMP1 OR PCP-3) BONE MORPHOGENETIC PROTEIN 1 PRECURSOR (EC
3.4.24.—) (BMP-1) PROCOLLAGEN C-PROTEINASE 3.
FN1: (FN1 OR FN) FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE GLOBULIN)
MMP12: (MMP12 OR HME) MACROPHAGE METALLOELASTASE PRECURSOR (EC
3.4.24.65) (HME) (MATRIX METALLOPROTEINASE-12) (MMP-12).
MMP2: (MMP2 OR CLG4A) 72 KDA TYPE IV COLLAGENASE PRECURSOR (EC
3.4.24.24) (72 KDA GELATINASE) (MATRIX METALLOPROTEINASE-2) (MMP-2)
(GELATINASE A) (TBE-1).
THBS1: (THBS1 OR TSP1 OR TSP) THROMBOSPONDIN 1 PRECURSOR
(THROMBOSPONDIN1).
FN1_REPEAT-B: (FN1 OR FN) FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE
GLOBULIN) (CIG).
CDH5: (CDH5) VASCULAR ENDOTHELIAL-CADHERIN PRECURSOR (VE-CADHERIN)
(CADHERIN-5) (7B4 ANTIGEN) (CD144 ANTIGEN) (CDH5).
FOXG1A-FOXG1B: (FOXG1B OR FKHL1) FORKHEAD PROTEIN G1B (FORKHEAD-
RELATED PROTEIN FKHL1) (TRANSCRIPTION FACTOR BF-1) (BRAIN FACTOR 1)
(BF1) (HFK1) (FOXG1A OR FKHL2) FORKHEAD BOX PROTEIN G1A (FORKHEAD-
RELATED PROTEIN FKHL2) (TRANSCRIPTION FACTOR BF-2).
ALCAM: (ALCAM) CD166 ANTIGEN PRECURSOR (ACTIVATED LEUKOCYTE-CELL
ADHESION MOLECULE) (ALCAM) (MEMD PROTEIN) (HB2) (KG-CAM) (MEMD
PROTEIN) (HB2) (KG-CAM).
MAP-2: (MAP2) MICROTUBULE-ASSOCIATED PROTEIN 2 (MAP2B) [CONTAINS:
MAP2C].
ITGB3: (ITGB3 OR GP3A) INTEGRIN BETA-3 PRECURSOR (PLATELET MEMBRANE
GLYCOPROTEIN IIIA) (GPIIIA) (CD61 ANTIGEN).
ITGA1_2: (ITGA1) INTEGRIN ALPHA-1 (LAMININ AND COLLAGEN RECEPTOR) (VLA-
1) (CD49A).
CD9: (CD9 OR MIC3) CD9 ANTIGEN (P24) (LEUKOCYTE ANTIGEN MIC3) (MOTILITY-
RELATED PROTEIN) (MRP-1).
RHOA: (ARHA OR ARH12 OR RHOA OR RHO12) TRANSFORMING PROTEIN RHOA
(H12).
Many extracellular matrix genes were down-regulated in the CD133+ cells, including collagens (COL11A1, COL15A1, COL16A1, COL1A1, COL4A1, and COL7A1), laminins (LAMA2, LAMA3, and LAMA4), and integrins (ITGA1—2, ITGA8, ITGB3, ITGB6). Interestingly, this subset of genes overlaps with those up-regulated in the MSC cells (see below).
MLPC vs. MSC
The following genes were up-regulated in MSC cells relative to MLPC: ACTC, ACTA2, SEMA3B, COL16A1, LUMICAN, COL12A1, COL8A1, COL5A2, FGF7, COL1A2, INHBA, DCN—1, TPM1, THROMBOSPONDIN5, TIMP3, ITGB8, THROMBOSPONDIN2, BDNF, LAMA2, INTEGRINB5, SERPINH1-SERPINH2, COL6A2—1, COL6A1, FKHL16, LAMA1, LAMG1, GATA4, COL1A1, KRT14, CTGF, HESR1, FN1, PAI1, NCAD, FLN1, COL4A1, PRKCM, INTEGRINA7, TNC, CXCL12, NFKB3, MCAM, BMP1—1, CALU, VLDLR, PRRX1, IL6, SMAD7, ACTG2, PRDC, ID3, LAMA4, FN1_REPEAT-B, DPYSL3, RARG1, EN1, ANXA2, INTEGRINB6, ITGA1—2, FZD4, VEGC, WNT3, IGFBP2, EDN1, PEG1-MEST, LAMB1, THROMBOSPONDIN1, ITGB1, COL7A1, FGF21, CDKN1A, RTN4, WNT5A, FN1_REPEAT-A, KRT18, MAPK6, EGFR-LONG, IGF2—1, COL11A1, HERMES, SNAI1, ATF2, VEGFD, ITGA5, NGFB, HJ1, EDN2, HNF3B, SOX9, DLX5, HSCDGF, MMP2, FN1_EIIIA, CRABP2, COL15A1, GPC4, THY1, COL14A1, TUBB4, CTNNA1, FZD1—2, COL4A3—1, WISP3, GATA6, HIF1A, SMAD5, CDKN2A—1, BMP15, ISL1, EBCTF, NRG1, PCK2, M6PR, EAAT1, RARB2—1, ALPL, RXRG, MMP13, CLDN6, TFCOUP1, AGGRECAN1, MMP19, EDN3, ENO2, BFGFR—1, KRT17, ASCL1, BMPR1A, VEGF—2, KCNK2, ASPIC1, JAM2, DNMT3B, SNAI2, TC10-PIGF, KS, MMP11, ENG, FGF6, LEPR, EPHB4, CEBPG, TCF3, EPRS, NTF3, VTN, MYH11, VEGF—1, TTR, PLCE, KRT8, CDH1, CNP, KCNQ5, INTEGRINA8, SDF2, FGF5, TIMP1, RELN, TPA, PTN, FGF1, SMAD3, IL6ST, ACRP, PMX2B, IGF1R, KRAS2A-KRAS2B, BMP11, IGF1, KCNK1, SMAD1, ORP150, NFATCB—1, LXR-ALPHA, ACVR1, KCNK4, PAFAHIB1, RACK17, BMP7, DDX21, SEMA4C, DJ924G13.1, VCAM1, CTNNA2, ADH4, RHOA, ACVR2, FABI, TRF1, LAMB3, CCCAP, MAPT, EMX-2, ACTB, AKT, VGR3, CYP3A4, STX1A, CITED2, PDGB, PDGFRA, LAMA5, GAPD, RYUDOCAN, SHH, AMBP, and GFAP—1. The MSC had a high amount of strongly up-regulated genes. Table 8 lists the genes identified as belonging to a particular pathway or family. In Table 8, “A” refers to extracellular matrix as determined by GO, “C” refers to structural protein as determined by ME/GO, “D” refers to extracellular matrix as determined by ME, “E” refers to extracellular region as determined by GO, “I” refers to histogenesis as determined by GO, “J” refers to collagen as determined by ME, “K” refers to laminin as determined by ME, “L” refers to adherens junction as determined by the Kegg pathway (KE), and “R” refers to hormone, growth factor, secreted factor as determined by ME.
Pathway analysis for genes up-regulated in MSC
KRT18: (KRT18 OR CYK18) KERATIN, TYPE I CYTOSKELETAL 18
(CYTOKERATIN 18) (K18) (CK 18).
KRT8: (KRT8 OR CYK8) KERATIN, TYPE II CYTOSKELETAL 8 (CYTOKERATIN
8) (K8) (CK 8) (KRT2-8).
TPM1: (TPM1 OR TPMA OR TMSA) TROPOMYOSIN ALPHA CHAIN.
ACVR2: (ACVR2) ACTIVIN RECEPTOR TYPE II PRECURSOR (EC 2.7.1.—) (ACTR-
II) (ACTRIIA).
BMP15: (BMP15 OR GDF9B) BONE MORPHOGENETIC PROTEIN 15 PRECURSOR
(BMP-15) (GROWTH/DIFFERENTIATION FACTOR 9B) (GDF-9B).
BMPR1A: (BMPR1A OR ACVRLK3) BONE MORPHOGENETIC PROTEIN
RECEPTOR TYPE IA PRECURSOR (EC 2.7.1.—) (SERINE/THREONINE-PROTEIN
KINASE RECEPTOR R5) (SKR5) (ACTIVIN RECEPTOR-LIKE KINASE 3) (ALK-3).
GATA4_HUMAN: (GATA4) TRANSCRIPTION FACTOR GATA-4 (GATA BINDING
FACTOR-4).
SHH: (SHH) SONIC HEDGEHOG PROTEIN PRECURSOR (SHH) (HHG-1).
FZD4: (FZD4) WNT RECEPTOR FRIZZLED-4, FRIZZLED 4 PRECURSOR
(FRIZZLED-4) (FZ-4) (HFZ4) (FZE4) (MFZ4) (RFZ4).
PRKCM: (PRKCM) PROTEIN KINASE C, MU TYPE (EC 2.7.1.—) (NPKC-MU).
NTF3: (NTF3) NEUROTROPHIN-3 PRECURSOR (NT-3) (NEUROTROPHIC
FACTOR) (HDNF) (NERVE GROWTH FACTOR 2) (NGF-2).
BDNF: (BDNF) BRAIN-DERIVED NEUROTROPHIC FACTOR PRECURSOR
PDGB: (PDGFB OR C-SIS OR PDGF2 OR SIS) PLATELET-DERIVED GROWTH
FACTOR, B CHAIN PRECURSOR (PDGF B-CHAIN) (PDGF-2) (BECAPLERMIN) (C-
MAPK6: (MAPK6 OR PRKM6 OR ERK3) MITOGEN-ACTIVATED PROTEIN
KINASE 6 (EC 2.7.1.—) (EXTRACELLULAR SIGNAL-REGULATED KINASE 3)
(ERK3) (P55-MAPK).
VEGF_1: (VEGF OR VEGFA) VASCULAR ENDOTHELIAL GROWTH FACTOR
PRECURSOR (VEGF) (VASCULAR PERMEABILITY FACTOR) (VPF)(VEGF A).
WISP3: (WISP3 OR CCN6 OR DJ142L7.3 OR LIBC) WNT1 INDUCIBLE SIGNALING
PATHWAY PROTEIN 3 PRECURSOR (WISP-3) (CONNECTIVE TISSUE GROWTH
FACTOR (NOV, GIG) LIKE PROTEIN (WISP3) (CONNECTIVE TISSUE GROWTH
FACTOR RELATED PROTEIN WISP-3) (LOST IN INFLAMMATORY BREAST
CANCER TUMOR SUPPRESSOR PROTEIN).
VEGFD: (FIGF OR VEGF-D) VASCULAR ENDOTHELIAL GROWTH FACTOR D (C-
FOS INDUCED GROWTH FACTOR).
VEGF_2: (VEGF OR VEGFA) VASCULAR ENDOTHELIAL GROWTH FACTOR
PRECURSOR (VEGF) (VASCULAR PERMEABILITY FACTOR) (VPF) (VEGF A).
EPHB4: (EPHB4 OR HTK) EPHRIN TYPE-B RECEPTOR 4 PRECURSOR (EC
2.7.1.112) (TYROSINE-PROTEIN KINASE RECEPTOR HTK).
MAPT: (MAPT OR MTBT1 OR TAU) MICROTUBULE-ASSOCIATED PROTEIN
TAU (NEUROFIBRILLARY TANGLE PROTEIN) (PAIRED HELICAL FILAMENT-
TAU) (PHF-TAU).
IGFBP2: (IGFBP2 OR BP2) INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN
2 PRECURSOR (IGFBP-2) (IBP-2) (IGF-BINDING PROTEIN 2).
SEMA3B: (SEMA3B OR SEMA5) SEMAPHORIN 3B PRECURSOR (SEMAPHORIN
V) (SEMA V). (SEMA3B OR SEMAA OR SEMA) SEMAPHORIN 3B PRECURSOR
(SEMAPHORIN A) (SEMA A).
SEMA4C: (SEMA4C OR KIAA1739) SEMAPHORIN 4C PRECURSOR (SEMAI)
(SEMACL1) (SEMAPHORIN C-LIKE 1) KIAA1739 PROTEIN (FRAGMENT).
ASPIC1: (ASPIC1 OR CEP-68) ASPIC PRECURSOR (CHONDROCYTE EXPRESSED
PROTEIN 68 KDA) ((2810454P21RIK OR CRTAC1) (CRTAC1-B PROTEIN)
(CARTILAGE ACIDIC PROTEIN 1) (FLJ10320).
INHBA: (INHBA) INHIBIN BETA A CHAIN PRECURSOR (ACTIVIN BETA-A
CHAIN) (ERYTHROID DIFFERENTIATION PROTEIN) (EDF).
VTN: (VTN) VITRONECTIN PRECURSOR (SERUM SPREADING FACTOR) (S-
VEGC: (VEGFC) VASCULAR ENDOTHELIAL GROWTH FACTOR C PRECURSOR
(VEGF-C) (VASCULAR ENDOTHELIAL GROWTH FACTOR RELATED PROTEIN)
(VRP) (FLT4 LIGAND) (FLT4-L).
COL12A1: (COL12A1) COLLAGEN ALPHA 1(XII) CHAIN PRECURSOR.
COL14A1: (COL14A1) EXTRACELLULAR MATRIX PROTEIN COLLAGEN TYPE
XIV, C-TERMINUS (FRAGMENT) (UNDULIN).
COL6A1: (COL6A1) COLLAGEN (VI) ALPHA-1 CHAIN (FRAGMENT) COLLAGEN
ALPHA 1(VI) CHAIN PRECURSOR.
COL7A1: (COL7A1) COLLAGEN ALPHA 1(VII) CHAIN PRECURSOR (LONG-
CHAIN COLLAGEN) (LC COLLAGEN).
COL8A1: (COL8A1) COLLAGEN ALPHA 1(VIII) CHAIN PRECURSOR
(ENDOTHELIAL COLLAGEN).
COL1A2: (COL1A2) COLLAGEN ALPHA 2(I) CHAIN PRECURSOR.
COL5A2: (COL5A2) COLLAGEN ALPHA 2(V) CHAIN PRECURSOR.
COL6A2_1: (COL6A2) COLLAGEN ALPHA 2(VI) CHAIN PRECURSOR.
COLLAGEN VI ALPHA-2 C-TERMINAL GLOBULAR DOMAIN (FRAGMENT).
(DKFZP586E1322).
COL4A3_1: (COL4A3) COLLAGEN ALPHA 3(IV) CHAIN PRECURSOR.
ITGA7: (ITGA7) INTEGRIN ALPHA-7 (INTEGRIN ALPHA 7 CHAIN) (INTEGRIN
ALPHA-7) (INTEGRINA7).
ITGB5: (ITGB5) INTEGRIN BETA-5 PRECURSOR (INTEGRINB5).
ITGB8: (ITGB8) INTEGRIN BETA-8 PRECURSOR.
CLDN6: (CLDN6) CLAUDIN-6 (SKULLIN 2).
PAI1: (SERPINE1 OR PAI1 OR PLANH1) PLASMINOGEN ACTIVATOR
INHIBITOR-1 PRECURSOR (PAI-1) (ENDOTHELIAL PLASMINOGEN ACTIVATOR
INHIBITOR) (PAI).
TIMP1: (TIMP1 OR TIMP OR CLGI) METALLOPROTEINASE INHIBITOR 1
PRECURSOR (TIMP-1) (ERYTHROID POTENTIATING ACTIVITY) (EPA) (TISSUE
INHIBITOR OF METALLOPROTEINASES) (FIBROBLAST COLLAGENASE
INHIBITOR) (COLLAGENASE INHIBITOR).
TIMP3: (TIMP3) METALLOPROTEINASE INHIBITOR 3 PRECURSOR (TIMP-3)
(TISSUE INHIBITOR OF METALLOPROTEINASES-3) (MIG-5 PROTEIN).
TPA: (PLAT) TISSUE-TYPE PLASMINOGEN ACTIVATOR PRECURSOR (EC
3.4.21.68) (TPA) (T-PA) (T-PLASMINOGEN ACTIVATOR) (ALTEPLASE)
(RETEPLASE).
BMP7: (BMP7 OR BMP-7 OR OP1) BONE MORPHOGENETIC PROTEIN 7
PRECURSOR (BMP-7) (OSTEOGENIC PROTEIN 1) (OP-1).
LAMA1: (LAMA1 OR LAMA) LAMININ ALPHA-1 CHAIN PRECURSOR (LAMININ
A CHAIN).
LAMA2: (LAMA2 OR LAMM) LAMININ ALPHA-2 CHAIN PRECURSOR (LAMININ
M CHAIN) (MEROSIN HEAVY CHAIN).
LAMA5: (KIAA0533 OR LAMA5) KIAA0533 PROTEIN (LAMININ ALPHA 5
CHAIN) (FRAGMENT).
LAMB1: (LAMB1) LAMININ BETA-1 CHAIN PRECURSOR (LAMININ B1 CHAIN).
LAMB3: (LAMB3) LAMININ BETA-3 CHAIN PRECURSOR (LAMININ B1K
CHAIN) (KALININ B1 CHAIN).
LAMG1: (LAMC1 OR LAMB2) LAMININ GAMMA-1 CHAIN PRECURSOR
(LAMININ B2 CHAIN).
AGGRECAN1: (AGC1 OR CSPG1 OR AGC) AGGRECAN CORE PROTEIN
PRECURSOR (CARTILAGE-SPECIFIC PROTEOGLYCAN CORE PROTEIN)
(CSPCP) (CHONDROITIN SULFATE PROTEOGLYCAN CORE PROTEIN 1).
BMP1_1: (BMP1 OR PCP-3) BONE MORPHOGENETIC PROTEIN 1 PRECURSOR
(EC 3.4.24.—) (BMP-1) PROCOLLAGEN C-PROTEINASE 3.
FN1: (FN1 OR FN) FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE
LUMICAN: (LDC) LUMICAN PRECURSOR (LUM) (KERATAN SULFATE
PROTEOGLYCAN).
MMP11: (MMP11 OR STMY3) STROMELYSIN-3 PRECURSOR (EC 3.4.24.—)
(MATRIX METALLOPROTEINASE-11) (MMP-11) (ST3) (SL-3).
MMP13: (MMP13) COLLAGENASE 3 PRECURSOR (EC 3.4.24.—) (MATRIX
METALLOPROTEINASE-13) (MMP-13).
DCN_1: (DCN) BONE PROTEOGLYCAN II PRECURSOR (PG-S2) (DECORIN)
(PG40) (PGS2).
RYUDOCAN: (SDC4) SYNDECAN-4 PRECURSOR (AMPHIGLYCAN) (SYND4)
(RYUDOCAN CORE PROTEIN).
TNC: (TNC OR HXB) TENASCIN PRECURSOR (TN) (HEXABRACHION)
(CYTOTACTIN) (NEURONECTIN) (GMEM) (JI) (MIOTENDINOUS ANTIGEN)
(GLIOMA-ASSOCIATED-EXTRACELLULAR MATRIX ANTIGEN) (GP 150-225)
(TENASCIN-C) (TN-C).
KCNK4: (KCNK4 OR TRAAK) POTASSIUM CHANNEL SUBFAMILY K MEMBER 4
(TWIK-RELATED ARACHIDONIC ACID-STIMULATED POTASSIUM CHANNEL
PROTEIN) (TRAAK) MECHANOSENSITIVE TANDEM PORE POTASSIUM
THBS2: (THBS2 OR TSP2) THROMBOSPONDIN 2 PRECURSOR
(THROMBOSPONDIN2).
COMP: (COMP) CARTILAGE OLIGOMERIC MATRIX PROTEIN PRECURSOR
(COMP) (THROMBOSPONDIN5).
MMP19: (MMP19 OR MMP18 OR RASI) MATRIX METALLOPROTEINASE-19
PRECURSOR (EC 3.4.24.—) (MMP-19) (MATRIX METALLOPROTEINASE RASI)
(MMP-18).
GPC4: (GPC4) GLYPICAN-4 PRECURSOR (K-GLYPICAN).
PTN: (PTN OR NEGF1 OR HBNF1) PLEIOTROPHIN PRECURSOR (PTN)
(HEPARIN-BINDING GROWTH-ASSOCIATED MOLECULE) (HB-GAM)
(HEPARIN-BINDING GROWTH FACTOR 8) (HBGF-8) (OSTEOBLAST SPECIFIC
FACTOR 1) (OSF-1) (HEPARIN-BINDING NEURITE OUTGROWTH PROMOTING
FACTOR 1) (HBNF-1).
ALPL: (ALPL) ALKALINE PHOSPHATASE, TISSUE-NONSPECIFIC ISOZYME
PRECURSOR (EC 3.1.3.1) (AP-TNAP) (LIVER/BONE/KIDNEY ISOZYME) (TNSALP)
(AKP2 OR AKP-2).
RELN: (RELN OR RL) REELIN PRECURSOR (EC 3.4.21.—) (REELER PROTEIN).
TUBB4_HUMAN: (TUBB4) TUBULIN BETA-4 CHAIN (TUBULIN BETA-III).
SNAI2: (SNAI2 OR SLUG OR SLUGH) ZINC FINGER PROTEIN SLUG (NEURAL
CREST TRANSCRIPTION FACTOR SLUG) (SNAIL HOMOLOG 2).
IL6: (IL6 OR IFNB2 OR IL-6) INTERLEUKIN-6 PRECURSOR (IL-6) (B-CELL
STIMULATORY FACTOR 2) (BSF-2) (INTERFERON BETA-2) (HYBRIDOMA
ACTC: (ACTC OR ACTC1) ACTIN, ALPHA CARDIAC.
PRDC: (PRDC) PRDC (FLJ21195).
SDF2: (SDF2) STROMAL CELL-DERIVED FACTOR 2 PRECURSOR (SDF-2).
BMP11: (GDF11 OR BMP11) GROWTH/DIFFERENTIATION FACTOR 11
PRECURSOR (BONE MORPHOGENETIC PROTEIN 11).
FN1_REPEAT-A: (FN1 OR FN) FIBRONECTIN PRECURSOR (FN) (COLD-
INSOLUBLE GLOBULIN) (CIG).
FN1_REPEAT-B: (FN1 OR FN) FIBRONECTIN PRECURSOR (FN) (COLD-
ENG: (ENG OR END) ENDOGLIN PRECURSOR (CD105 ANTIGEN) (CELL
SURFACE MJ7/18 ANTIGEN).
CTNNA2: (CTNNA2 OR CAPR) ALPHA-2 CATENIN (ALPHA-CATENIN RELATED
PROTEIN) (ALPHA N-CATENIN).
HSCDGF: (HSCDGF OR PDGFC) SECRETORY GROWTH FACTOR-LIKE PROTEIN
FALLOTEIN (SPINAL CORD-DERIVED GROWTH FACTOR) (PLATELET-
DERIVED GROWTH FACTOR C).
VCAM1: (VCAM1 OR L1CAM OR VCAM-1) VASCULAR CELL ADHESION
PROTEIN 1 PRECURSOR (V-CAM 1) (CD106 ANTIGEN) (INCAM-100).
SNAI1: (SNAI1 OR SNAH) ZINC FINGER PROTEIN SNAI1 (SNAIL PROTEIN
HOMOLOG) (SNA PROTEIN).
IL6ST: (IL6ST) INTERLEUKIN-6 RECEPTOR BETA CHAIN PRECURSOR (IL-6R-
BETA) (INTERLEUKIN 6 SIGNAL TRANSDUCER) (MEMBRANE GLYCOPROTEIN
130) (GP130) (ONCOSTATIN M RECEPTOR) (CDW130) (CD130 ANTIGEN).
PDGFRA: (PDGFRA) ALPHA PLATELET-DERIVED GROWTH FACTOR
RECEPTOR PRECURSOR (EC 2.7.1.112) (PDGF-R-ALPHA) (CD 140A ANTIGEN).
CXCL12: (CXCL12 OR SDF1) STROMAL CELL-DERIVED FACTOR 1 PRECURSOR
(SDF-1) (CXCL12) (PRE-B CELL GROWTH STIMULATING FACTOR) (PBSF) (12-
O-TETRADECANOYLPHORBOL 13-ACETATE REPRESSED PROTEIN 1) (TPAR1)
(THYMIC LYMPHOMA CELL STIMULATING FACTOR) (TLSF).
BFGFR_1_HUMAN: (FGFR1 OR FLG OR FGFBR OR FLT2) BASIC FIBROBLAST
GROWTH FACTOR RECEPTOR 1 PRECURSOR (BFGF-R) EC 2.7.1.112) (FMS-LIKE
TYROSINE KINASE-2) (C-FGR).
EGFR-LONG: (EGFR OR ERBB1) EPIDERMAL GROWTH FACTOR RECEPTOR
PRECURSOR (EC 2.7.1.112) (RECEPTOR PROTEIN-TYROSINE KINASE ERBB-1).
FN1_EIIIA: (FN1 OR FN) FIBRONECTIN PRECURSOR (FIBRONECTIN EIILA
EDN1: (EDN1) ENDOTHELIN-1 PRECURSOR (ET-1).
ACTA2: (ACTA2 OR ACTSA OR ACTVS) AORTIC SMOOTH MUSCLE (ALPHA-
ACTIN 2).
EDN2: (EDN2) ENDOTHELIN-2 PRECURSOR (ET-2) (VASOACTIVE INTESTINAL
CONTRACTOR) (VIC).
GFAP_1_HUMAN: (GFAP) GLIAL FIBRILLARY ACIDIC PROTEIN, ASTROCYTE
(GFAP).
SERPINH1-SERPINH2: (SERPINH1 OR CBP1 OR HSP47) HEAT SHOCK PROTEIN
47 COLLAGEN BINDING PROTEIN 1 (CBP1) (COLLIGIN 1) (SERPINH2 OR CBP2)
(COLLAGEN-BINDING PROTEIN 2 PRECURSOR) (COLLIGIN 2) (RHEUMATOID
ARTHRITIS RELATED ANTIGEN RA-A47).
IGF1R: (IGF1R) INSULIN-LIKE GROWTH FACTOR I RECEPTOR PRECURSOR (EC
2.7.1.112) (CD221 ANTIGEN).
M6PR_HUMAN: (IGF2R OR MPRI) CATION-INDEPENDENT MANNOSE-6-
PHOSPHATE RECEPTOR PRECURSOR (CI MAN-6-P RECEPTOR) (CI-MPR)
(INSULIN-LIKE GROWTH FACTOR II RECEPTOR) (300 KDA MANNOSE 6-
PHOSPHATE RECEPTOR) (MPR 300) (MPR300) (CD222 ANTIGEN).
SMAD3_HUMAN: (MADH3 OR SMAD3 OR MAD33) MOTHERS AGAINST
DECAPENTAPLEGIC HOMOLOG 3 (SMAD 3) (MOTHERS AGAINST DPP
HOMOLOG 3) (MAD3) (HMAD-3) (MMAD3) (JV15-2).
ITGB1: (ITGB1 OR FNRB) INTEGRIN BETA-1 PRECURSOR (FIBRONECTIN
RECEPTOR BETA SUBUNIT) (CD29 ANTIGEN) (INTEGRIN VLA-4 BETA
SUBUNIT).
NRG1: (NRG1 OR HGL OR NDF OR HRGA OR GGF OR SMDF) PRO-
NEUREGULIN-1 PRECURSOR (PRO-NRG1) [CONTAINS: NEUREGULIN-1 (NEU
DIFFERENTIATION FACTOR) (HEREGULIN) (HRG) (BREAST CANCER CELL
DIFFERENTIATION FACTOR P45) (ACETYLCHOLINE RECEPTOR INDUCING
ACTIVITY) (ARIA) (SENSORY AND MOTOR NEURON-DERIVED FACTOR)
(GLIAL GROWTH FACTOR)].
IGF1: (IGF-I OR IGF1) INSULIN-LIKE GROWTH FACTOR I PRECURSOR
(SOMATOMEDIN). (IGF1 OR IBP1) INSULIN-LIKE GROWTH FACTOR IA
PRECURSOR (IGF-IA) (SOMATOMEDIN C). INSULIN-LIKE GROWTH FACTOR IB
PRECURSOR (IGF-IB) (SOMATOMEDIN C).
CDKN2A_1_HUMAN: (CDKN2A OR CDKN2) CYCLIN-DEPENDENT KINASE 4
INHIBITOR A (CDK4I) (P16-INK4) (P16-INK4A) (MULTIPLE TUMOR
SUPPRESSOR 1) (MTS1) (P14ARF OR ARF) (CELL CYCLE REGULATOR).
FGF1: (FGF1 OR FGFA) HEPARIN-BINDING GROWTH FACTOR 1 PRECURSOR
(HBGF-1) (ACIDIC FIBROBLAST GROWTH FACTOR) (AFGF) (BETA-
ENDOTHELIAL CELL GROWTH FACTOR) (ECGF-BETA).
FGF5: (FGF5) FIBROBLAST GROWTH FACTOR-5 PRECURSOR (FGF-5) (HBGF-5).
FGF6: (FGF6 OR HST2) FIBROBLAST GROWTH FACTOR-6 PRECURSOR (FGF-6)
(HBGF-6) (HST-2).
FGF7: (FGF7 OR KGF) KERATINOCYTE GROWTH FACTOR PRECURSOR (KGF)
(FIBROBLAST GROWTH FACTOR-7) (FGF-7) (HBGF-7).
IGF2_1: (IGF2) INSULIN-LIKE GROWTH FACTOR II PRECURSOR (IGF-II)
(SOMATOMEDIN A).
LEPR: (OBR OR LEPR OR DB OR FA) LEPTIN RECEPTOR PRECURSOR (LEP-R)
(OB RECEPTOR) (OB-R) (B219RECEPTOR).
NGFB: (NGFB) BETA-NERVE GROWTH FACTOR PRECURSOR (BETA-NGF).
VGR3: (FLT4) VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3
PRECURSOR (EC 2.7.1.112) (VEGFR-3) (TYROSINE-PROTEIN KINASE RECEPTOR
FLT4).
CDH1: (CDH1 OR UVO OR CDHE) EPITHELIAL-CADHERIN PRECURSOR (E-
CADHERIN) (UVOMORULIN) (CAM 120/80).
EDN3: (EDN3) ENDOTHELIN-3 PRECURSOR (ET-3).
FGF2_1: (FGF2 OR FGFB) HEPARIN-BINDING GROWTH FACTOR 2 PRECURSOR
(HBGF-2) (BASIC FIBROBLAST GROWTH FACTOR) (BFGF) (PROSTATROPIN).
ITGA1_2: (ITGA1) INTEGRIN ALPHA-1 (LAMININ AND COLLAGEN RECEPTOR)
(VLA-1) (CD49A).
VLDLR: (VLDLR OR LDVR) VERY LOW-DENSITY LIPOPROTEIN RECEPTOR
PRECURSOR (VLDL RECEPTOR).
ITGA5: (ITGA5 OR FNRA) INTEGRIN ALPHA-5 PRECURSOR (FIBRONECTIN
RECEPTOR ALPHA SUBUNIT) (INTEGRIN ALPHA-F) (VLA-5) (CD49E).
PCK2: (PCK2 OR PEPCK2) PHOSPHOENOLPYRUVATE CARBOXYKINASE,
MITOCHONDRIAL PRECURSOR [GTP] (EC 4.1.1.32) (PHOSPHOENOLPYRUVATE
CARBOXYLASE) (PEPCK-M).
AMBP: (AMBP OR ITIL OR HCP) AMBP PROTEIN PRECURSOR [CONTAINS:
ALPHA-1-MICROGLOBULIN (PROTEIN HC) (COMPLEX-FORMING
GLYCOPROTEIN HETEROGENEOUS IN CHARGE); INTER-ALPHA-TRYPSIN
INHIBITOR LIGHT CHAIN (ITI-LC) (BIKUNIN) (HI-30)].
TTR: (TTR OR PALB) TRANSTHYRETIN PRECURSOR (PREALBUMIN) (TBPA)
(TTR) (ATTR).
KRT14: (KRT14) KERATIN, TYPE I CYTOSKELETAL 14 (CYTOKERATIN 14)
(K14) (CK 14).
KRT17: (KRT17) KERATIN, TYPE I CYTOSKELETAL 17 (CYTOKERATIN 17)
(K17) (CK 17) (39.1) (VERSION 1).
NCAD: (CDH2 OR CDHN OR NCAD) NEURAL-CADHERIN PRECURSOR (N-
CADHERIN) (CADHERIN-2).
RHOA: (ARHA OR ARH12 OR RHOA OR RHO12) TRANSFORMING PROTEIN
RHOA (H12).
CTNNA1: (CTNNA1) ALPHA-1 CATENIN (CADHERIN-ASSOCIATED PROTEIN)
(ALPHA E-CATENIN)
HJ1: (JAG1) JAGGED 1 PRECURSOR (JAGGED1) (HJ1) NOTCH LIGAND JAGGED
ACTG2: (ACTG2 OR ACTA3 OR ACTSG) ACTIN, GAMMA-ENTERIC SMOOTH
MUSCLE (ALPHA-ACTIN 3).
FLN1: (FLNA OR FLN1 OR FLN) FILAMIN A (ALPHA-FILAMIN) (FILAMIN 1)
(ENDOTHELIAL ACTIN-BINDING PROTEIN) (ABP-280) (NONMUSCLE FILAMIN).
MYH11: (MYH11) MYOSIN HEAVY CHAIN, SMOOTH MUSCLE ISOFORM
(SMMHC) (FRAGMENT).
CRABP2: (CRABP2) RETINOIC ACID-BINDING PROTEIN II, CELLULAR (CRABP-
SOX9: (SOX9) TRANSCRIPTION FACTOR SOX-9.
WNT3: (WNT3 OR WNT-3 OR INT4) WNT-3 PROTO-ONCOGENE PROTEIN
WNT5A: (WNT5A OR WNT-5A) WNT-5A PROTEIN PRECURSOR.
Characteristic for the MSC population is the high enrichment of genes whose protein products are located within the extracellular matrix, e.g. keratins (KRT18, KRT8, KRT14, KRT17), collagens (COL1A1, COL11A1, COL12A1, COL14A1, COL15A1, COL16A1, COL4A1, COL6A1, COL7A1, COL8A1, COL1A2, COL5A2, COL6A2—1, and COL4A3—1), laminins (LAMA1, LAMA2, LAMA4, LAMA5, LAMB1, LAMB3, and LAMG1), integrins (ITGA7, ITGA8, ITGB5, ITGB6, ITGB8, ITGA1—2, ITGA5), matrix-metalloproteases (BMP1—1, MMP1, MMP13, MMP2, and MMP19) and their inhibitors (TIMP1, TIMP3). Furthermore, a conspicuous enrichment of growth factors (BMP15, NTF3, BDNF, PDGB, VEGF—1, VEGFD, VEGF—2, INHBA, VEGC, BMP7, BMP1—1, PTN, IL6, BMP11, HSCDGF, NRG1, IGF1, FGFI, FGF5, FGF6, FGF7, IGF2—1, NGFB, and FGF2—1) and growth factor receptors (ACVR2, BMPR1A, IL6ST, PDGFRA, BFGFR—1_HUMAN, EGFR-LONG, IGF1R, M6PR_HUMAN, LEPR, and VGR3) was observed.
The following genes were down-regulated in the MSC relative to the MLPC: ITGB2, ARHGAP9, CXCR4, INTEGRINB7, PECAM1, PRKCB—1, PRKCB—3, IL7R, AIF1, CD45_EX10-11, PLCG2, CD37, PRKCB—2, TCF2—1, RNF138, EAAT4, EPHA1, RPLP0, PTTG, SERPINA1—2, ITGAX, CD24, F11R, RPL4, ICAM1, LMO2, HMGB2, CD38, RPL7A, BMP3, PTHR2, S100B, OSF, SNCA, GRIK1, HTR4, CHRM1, CDKN2D, HNRPA1, IL6R, MUSLAMR, ICAM2, CSK, ITGA6, MMP9, DNMT1, PAK1, IKKB, TFRC_MIDDLE, CHI3L2, ITGA4, FGF20, NBR2, TNFRSF1B, CEBPA—3, CDO1, NFKB1, GATA2, PDGFRB, ICSBP1, KCNE3, TNNC1, ITGA2B, CCT8, LEFTA, TH, RPS24, HTR1F, TREM1, CCNB2, SELL, CD34, HMGIY, COX7A2, SELE, TNNT2, SEM2, CHEK1, CLCN5, F5, PRKCQ, ITGAL, NCAM2, ZNF257-MGC12518-ZNF92-ZNF43-ZNF273-FLJ90430, CDK1, RPL6, RPL24, IGHA1-IGHA2_M, PUM2, GJA7, HTR7, PTHR1, MAPK14, MSI2—1, KCNJ3, CD133, SYP, TFRC—5PRIME, TDGF1-TDGF3—2, FLT3, HPRT, SEMA4D, ITGAM, KIAA0152—3, ZFP42, SOX20, FLJ21190, CPN2, POU2F2, CASP8—1, CLDN10, TREM2, TERT, OLIG1, EGR2, CD44_EX3-5, CD33, CNTFR, OPN, COL9A1—2, ROBO4, HTR1D—1, IKKA, KIT, NPPA, PRKCH, FGF4, CD68, NUMB, NRG3, SALL2, NOP5, HNF4G, FIBROMODULIN, CD58, CALB1, GJB5, GJA5, POU5F—1, GDF5, POU6F1, CD44_EX16-20, BCAN, PTEN1-PTEN2, AGRIN, ALB, KCNQ4, DPPA5, EPHB2, TGFBR2, and ITGA3. Table 9 lists the genes identified as belonging to a particular pathway or family. In Table 9, “A” refers to CD as determined by ME, “B” refers to ribonucleoprotein complex as determined by GO, “C” refers to ligand/surface marker as determined by ME “D” refers to ribosome as determined by ME/GO/KE, “J” refers to external side of plasma membrane as determined by GO, and “K” refers to calcium signaling pathway as determined by KE.
Pathway analysis for genes down-regulated in MSC
TERT: (TERT OR TRT OR EST2 OR TCS1) TELOMERASE REVERSE
TRANSCRIPTASE (EC 2.7.7.—) (TELOMERASE CATALYTIC SUBUNIT) (HEST2).
CD44_EX16-20_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR
(PHAGOCYTIC GLYCOPROTEIN I) (PGP-1) (HUTCH-I) (EXTRACELLULAR MATRIX
RECEPTOR-III) (ECMR-III) (GP90 LYMPHOCYTE HOMING/ADHESION RECEPTOR)
(HERMES ANTIGEN) (HYALURONATE RECEPTOR) (HEPARAN SULFATE
PROTEOGLYCAN) (EPICAN) (CDW44).
CD44_EX3-5_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
EPHB2: (EPHB2 OR EPTH3 OR ERK OR DRT OR HEK5) EPHRIN TYPE-B RECEPTOR
2 PRECURSOR (EC 2.7.1.112) (TYROSINE-PROTEIN KINASE RECEPTOR EPH-3)
(DRT) (RECEPTOR PROTEIN-TYROSINE KINASE HEK5) (ERK).
SEMA4D: (SEMA4D OR CD100) SEMAPHORIN 4D PRECURSOR (LEUKOCYTE
ACTIVATION ANTIGEN CD100) (BB18) (A8) (GR3). (SEMA4D OR SEMAJ OR
SEMACL2) SEMAPHORIN 4D PRECURSOR (SEMAPHORIN J) (SEMA J)
(SEMAPHORIN C-LIKE 2) (M-SEMA G).
CD45_EX10-11: (PTPRC OR CD45) LEUKOCYTE COMMON ANTIGEN PRECURSOR
(EC 3.1.3.48) (L-CA) (CD45 ANTIGEN) (T200).
PRKCB_3: (PRKCB1 OR PRKCB OR PKCB) PROTEIN KINASE C, BETA TYPE (EC
2.7.1.37) (PKC-BETA) (PKC-B).
PRKCB_1: (PRKCB1 OR PRKCB OR PKCB) PROTEIN KINASE C, BETA TYPE (EC
PRKCB_2: (PRKCB1 OR PRKCB OR PKCB) PROTEIN KINASE C, BETA TYPE (EC
PRKCH: (PRKCH OR PKCL) PROTEIN KINASE C, ETA TYPE (EC 2.7.1.—) (NPKC-ETA)
(PKC-L).
TNNC1: (TNNC1 OR TNNC) TROPONIN C, SLOW SKELETAL AND CARDIAC
MUSCLES (TN-C).
RPLP0: (RPLP0) 60S ACIDIC RIBOSOMAL PROTEIN P0 (L10E).
SEM2: (SEM2) SEMAPHORIN SEM2.
CASP8_1: (MCH5 OR CASP8) CASPASE 8 PRECURSOR (EC 3.4.22.—) (ICE-LIKE
APOPTOTIC PROTEASE 5) (MORT1-ASSOCIATED CED-3 HOMOLOG) (MACH)
(FADD HOMOLOGOUS ICE/CED-3-LIKE PROTEASE) (FLICE) (APOPTOTIC
CYSTEINE PROTEASE) (APOPTOTIC PROTEASE MCH-5) (CAP4).
(HELIX-DESTABILIZING PROTEIN) (SINGLE-STRAND BINDING PROTEIN) (HNRNP
CORE PROTEIN A1).
RPL6: (RPL6) 60S RIBOSOMAL PROTEIN L6 (TAX-RESPONSIVE ENHANCER
ELEMENT BINDING PROTEIN 107) (TAXREB107) (NEOPLASM-RELATED PROTEIN
C140).
TNFRSF1B: (TNFRSF1B OR TNFR2 OR TNFBR OR TNFR-2) TUMOR NECROSIS
FACTOR RECEPTOR SUPERFAMILY MEMBER 1B PRECURSOR (TUMOR
NECROSIS FACTOR RECEPTOR 2) (TUMOR NECROSIS FACTOR BINDING
PROTEIN 2) (TBPII) (P80) (TNF-R2) (P75) (CD120B) (ETANERCEPT).
RPS24: (RPS24 OR RPS19) 40S RIBOSOMAL PROTEIN S24 (S19).
NPPA: (NPPA OR PND) ATRIAL NATRIURETIC FACTOR PRECURSOR (ANF)
(ATRIAL NATRIURETIC PEPTIDE) (ANP) (PREPRONATRIODILATIN).
RPL24: (RPL24) 60S RIBOSOMAL PROTEIN L24 (L30).
NCAM2: (NCAM2 OR NCAM21) NEURAL CELL ADHESION MOLECULE 2
PRECURSOR (N-CAM 2).
IGHA1-IGHA2_M_HUMAN: (IGHA1) IG ALPHA-1 CHAIN C REGION (IGHA2) (IG
ALPHA-2 CHAIN C REGION).
CD133: (PROM1 OR PROML1 OR PROM OR CD133 OR AC133) PROMININ 1
PRECURSOR (PROMININ-LIKE PROTEIN 1) (ANTIGEN AC133) (CD133 ANTIGEN).
ICAM2: (ICAM2 OR ICAM-2) INTERCELLULAR ADHESION MOLECULE-2
PRECURSOR (ICAM-2) (CD102) (LYMPHOCYTE FUNCTION-ASSOCIATED AG-1
COUNTER-RECEPTOR).
ITGAX: (ITGAX OR CD11C) INTEGRIN ALPHA-X PRECURSOR (LEUKOCYTE
ADHESION GLYCOPROTEIN P150,95 ALPHA CHAIN) (LEUKOCYTE ADHESION
RECEPTOR P150,95) (CD11C) (LEU M5).
F11R: (F11R OR JAM1 OR JCAM) JUNCTIONAL ADHESION MOLECULE 1
PRECURSOR (JAM) (PLATELET ADHESION MOLECULE 1) (PAM-1) (PLATELET F11
RECEPTOR) (UNQ264/PRO301).
NOP5: (NOP5) NUCLEOLAR PROTEIN NOP5 (NUCLEOLAR PROTEIN 5) (NOP58)
(HSPC120) (NOL5) (SIK SIMILAR PROTEIN).
TFRC_5PRIME: (TFRC) TRANSFERRIN RECEPTOR PROTEIN (TFR1) (TR) (TFR)
(TRFR) (CD71 ANTIGEN) (T9) (P90).
KIT: (KIT OR SL) MAST/STEM CELL GROWTH FACTOR RECEPTOR PRECURSOR
(EC 2.7.1.112) (SCFR) (PROTO-ONCOGENE TYROSINE-PROTEIN KINASE KIT) (C-
KIT) (CD117 ANTIGEN) (C-KIT RECEPTOR TYROSINE KINASE).
RPL4: (RPL4 OR RPL1) 60S RIBOSOMAL PROTEIN L4 (L1).
IL6R: (L6RA OR IL6R) INTERLEUKIN-6 RECEPTOR ALPHA CHAIN PRECURSOR
(IL-6R-ALPHA) (CD126 ANTIGEN) (IL-6R 1).
IL7R: (IL7R) INTERLEUKIN-7 RECEPTOR ALPHA CHAIN PRECURSOR (IL-7R-
ALPHA) (CDW127) (CD127 ANTIGEN).
ITGAM: (ITGAM OR CR3A OR CD11B) INTEGRIN ALPHA-M PRECURSOR (CELL
SURFACE GLYCOPROTEIN MAC-1 ALPHA SUBUNIT) (CR-3 ALPHA CHAIN)
(CD11B) (LEUKOCYTE ADHESION RECEPTOR MO1) (INTEGRIN ALPHA-M)
(NEUTROPHIL ADHERENCE RECEPTOR).
FLT3: (FLT3 OR STK1 OR FLT-3 OR FLK-2) FL CYTOKINE RECEPTOR PRECURSOR
(EC 2.7.1.112) (TYROSINE-PROTEIN KINASE RECEPTOR FLT3) (STEM CELL
TYROSINE KINASE 1) (STK-1) (CD135 ANTIGEN) (TYROSINE-PROTEIN KINASE
RECEPTOR FLK-2) (FETAL LIVER KINASE 2).
RPSA: (RPSA OR LAMR1 OR LAMBR OR P40-8) 40S RIBOSOMAL PROTEIN SA (P40)
(34/67 KDA LAMININ RECEPTOR) (COLON CARCINOMA LAMININ-BINDING
PROTEIN) (NEM/1CHD4) (MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN
MGR1-AG) (MUSLAMR).
CD58_HUMAN: (CD58 OR LFA3) LYMPHOCYTE FUNCTION-ASSOCIATED
ANTIGEN 3 PRECURSOR (AG3) (ANTIGEN CD58) (SURFACE GLYCOPROTEIN LFA-
SELE: (SELE OR ELAM1 OR ELAM-1) E-SELECTIN PRECURSOR (ENDOTHELIAL
LEUKOCYTE ADHESION MOLECULE 1) (ELAM-1) (LEUKOCYTE-ENDOTHELIAL
CELL ADHESION MOLECULE 2) (LECAM2) (CD62E).
SELL: (SELL OR LYAM1 OR LNHR OR LY-22) L-SELECTIN PRECURSOR (LYMPH
NODE HOMING RECEPTOR) (LEUKOCYTE ADHESION MOLECULE-1) (LAM-1)
(LEUKOCYTE SURFACE ANTIGEN LEU-8) (TQ1) (GP90-MEL) (LEUKOCYTE-
ENDOTHELIAL CELL ADHESION MOLECULE 1) (LECAM1) (CD62L) (LY-22)
(LYMPHOCYTE SURFACE MEL-14 ANTIGEN).
NFKB1: (NFKB1) NUCLEAR FACTOR NF-KAPPA-B P105 SUBUNIT (DNA-BINDING
FACTOR KBF1) (EBP-1) [CONTAINS: NUCLEAR FACTOR NF-KAPPA-B P50
SUBUNIT].
CD68: (CD68) MACROSIALIN PRECURSOR (CD68 ANTIGEN) (GP110).
TFRC_MIDDLE: (TFRC) TRANSFERRIN RECEPTOR PROTEIN (TFR1) (TR) (TFR)
ITGAL: (ITGAL OR CD11A OR LFA-1) INTEGRIN ALPHA-L PRECURSOR
(LEUKOCYTE ADHESION GLYCOPROTEIN LFA-1 ALPHA CHAIN) (LEUKOCYTE
FUNCTION ASSOCIATED MOLECULE 1, ALPHA CHAIN) (CD11A) (INTEGRIN
ALPHA-L).
ITGB2: (ITGB2 OR CD18) INTEGRIN BETA-2 PRECURSOR (CELL SURFACE
ADHESION GLYCOPROTEINS LFA-1/CR3/P150, 95 BETA-SUBUNIT) (CD18)
(COMPLEMENT RECEPTOR C3 BETA-SUBUNIT).
CD24: (CD24 OR CD24A) SIGNAL TRANSDUCER CD24 PRECURSOR (M1/69-J11D
HEAT STABLE ANTIGEN) (HSA) (NECTADRIN) (LY-52) (X62 HEAT STABLE
ANTIGEN) (R13-AG).
NRG3: (NRG3) PRO-NEUREGULIN-3 PRECURSOR (PRO-NRG3) [CONTAINS:
NEUREGULIN-3 (NRG-3)].
PECAM1: (PECAM1 OR PECAM-1 OR PECAM) PLATELET ENDOTHELIAL CELL
ADHESION MOLECULE PRECURSOR (PECAM-1) (CD31 ANTIGEN) (ENDOCAM)
(GPIIA′).
CD33: (CD33) MYELOID CELL SURFACE ANTIGEN CD33 PRECURSOR (GP67)
(SIGLEC-3).
CD34: (CD34) HEMATOPOIETIC PROGENITOR CELL ANTIGEN CD34 PRECURSOR.
CD37: (CD37) LEUKOCYTE ANTIGEN CD37.
CDK1: (CDC2) CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2.7.1.—) (P34
PROTEIN KINASE) (CYCLIN-DEPENDENT KINASE 1) (CDK1).
CD38: (CD38) ADP-RIBOSYL CYCLASE 1 (EC 3.2.2.5) (CYCLIC ADP-RIBOSE
HYDROLASE 1) (CADPR HYDROLASE 1) (LYMPHOCYTE DIFFERENTIATION
ANTIGEN CD38) (T10) (ACUTE LYMPHOBLASTIC LEUKEMIA CELLS ANTIGEN
CD38) (NIM-R5 ANTIGEN) (I-19) (CD38 HOMOLOG) (CD38H).
ITGA2B: (ITGA2B OR ITGAB OR GP2B) PLATELET MEMBRANE GLYCOPROTEIN
IIB PRECURSOR (GPIIB) (GPALPHA IIB) (INTEGRIN ALPHA-IIB) (CD41).
ITGA3: (ITGA3) INTEGRIN ALPHA-3 PRECURSOR (GALACTOPROTEIN B3)
(GAPB3) (VLA-3 ALPHA CHAIN) (CD49C).
ITGA4: (ITGA4 OR VLA-4) INTEGRIN ALPHA-4 PRECURSOR (INTEGRIN ALPHA-
IV) (VLA-4) (CD49D) (LYMPHOCYTE-PEYER'S PATCH ADHESION MOLECULES
ALPHA SUBUNIT) (LPAM ALPHA SUBUNIT).
ITGA6: (ITGA6) INTEGRIN ALPHA-6 PRECURSOR (VLA-6) (CD49F) (INTA6)
(INTEGRIN ALPHA 6 SUBCHAIN).
ICAM1: (ICAM1 OR ICAM-1) INTERCELLULAR ADHESION MOLECULE 1
PRECURSOR (ICAM-1) (MAJOR GROUP RHINOVIRUS RECEPTOR) (CD54) (MALA-
HTR4: (HTR4) 5-HYDROXYTRYPTAMINE 4 RECEPTOR (5-HT-4) (SEROTONIN
RECEPTOR) (5-HT4) (FRAGMENT).
CHRM1: (CHRM1) MUSCARINIC ACETYLCHOLINE RECEPTOR M1.
RPL7A: (RPL7A OR SURF3 OR SURF-3) 60S RIBOSOMAL PROTEIN L7A (SURFEIT
LOCUS PROTEIN 3) (PLA-X POLYPEPTIDE).
A significant portion of the genes (33 out of 82) down-regulated in MSC belongs to the group of CD surface markers (CD44_EX16-20_HUMAN, CD44_EX3-5_HUMAN, SEMA4D, CD45_EX10-11, TNFRSF1B, CD133, ICAM2, ITGAX, TFRC—5PRIME, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, CD58_HUMAN, SELE, SELL, CD68, TFRC_MIDDLE, ITGAL, ITGB2, CD24, PECAM1, CD33, CD34, CD37, CD38, ITGA2B, ITGA3, ITGA4, ITGA6, and ICAM1). Nearly the same set of ribosomal proteins up-regulated in the PrepaCyte cells is down-regulated in the MSC (RPLP0, RPL6, RPS24, RPL24, RPL4, RPSA, and RPL7A).
Based on the profile of up- and down-regulated genes relative to MLPC, MSC are more committed towards the mesenchymal pathways, particularly bone and cartilage as shown by strong up-regulation of keratins, collagens, integrins, matrix-metalloproteases, growth factors, and receptors. Markers of particular interest that are down-regulated in the MSC relative to MLPC are CXCR4, FLT3, TERT, hematopoietic CD markers such as CD133 and CD34, KIT, and embryonic stem cell marker POU5F (October 4). See also FIGS. 4-21, which illustrate the differences in expression between MLPC and MSC of different groups of genes (e.g., adhesion molecules; growth factors and receptors; genes involved in cell cycle, proliferation, and anti-apoptosis; transcription factors; translation regulators; connective tissue, cartilage, and bone; extracellular matrix; endothelium; hematopoiesis and the immune response; neural genes; hepatic genes; muscle, smooth muscle, and cardiac genes; genes involved in cell-cell communication; stem cell markers (hematopoietic and mesenchymal); epidermal genes; adipocytic genes; pancreatic genes; and genes involved in development/morphogenesis).
MLPC vs. LinNeg cells
The following genes were up-regulated in LinNeg cells relative to MLPC: OPN, IL3RA, CD68, CTNNB1, CALB1, PPARG, VGR1, GJB5, HB-EGF, ITGA4, UPA, HDC, GCK, FABP4, APOE, PAX5, TRK-A, THBD, TH, TREM2, KAI1, LIFRA, HTR4, KCNA7, IDH1, KITLG, CD58, MMP6, MMP12, COL18A1—2, GDNF, MAPK13, GAD1—1, LAMA3, MMP9, CDH4, F11R, ID2, TACE, HTR1D—1, CACNA1B, KCNA4, PTHR2, S100B, CD47, CD44_EX13-15, GJA5, ANPEP, CD44_EX16-20, CD44_EX11-13, EIF4A1, KCNJ6, MYH7, TIMP2, NEUROD1, KIR2.4, HNF3A, MAPK14, HTR1F, CD24, GATA2, COX7A2, CD44_EX10-12, CYPA, KCNK5, CLCN7, MTHFD2, CD34, CD44_EX3-5, and CRABP1. Table 10 lists the genes identified as belonging to a particular pathway or family. In Table 10, “A” refers to CD (cluster of differentiation) as determined by ME, “B” refers to external side of plasma membrane as determined by GO, and “C” refers to tube morphogenesis as determined by GO.
Pathway analysis for genes up-regulated in Lineage Negative Cells
CD44_EX16-20_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
CD44_EX11-13_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
VGR1: (FLT1 OR FLT OR FRT) VASCULAR ENDOTHELIAL GROWTH FACTOR
RECEPTOR 1 PRECURSOR (EC 2.7.1.112) (VEGFR-1) (TYROSINE-PROTEIN KINASE
RECEPTOR FLT) (FLT-1) (TYROSINE-PROTEIN KINASE FRT).
TACE: (ADAM17 OR TACE OR CSVP) ADAM 17 PRECURSOR (EC 3.4.24.—) (A
DISINTEGRIN AND METALLOPROTEINASE DOMAIN 17) (TNF-ALPHA CONVERTING
ENZYME) (TNF-ALPHA CONVERTASE) (SNAKE VENOM-LIKE PROTEASE) (CD156B
IL3RA: ((IL3RAX OR IL3RA OR IL3R OR IL3RX) AND (IL3RAY OR IL3RA OR IL3R OR
IL3RY)) INTERLEUKIN-3 RECEPTOR ALPHA CHAIN PRECURSOR (IL-3R-ALPHA)
(CD123 ANTIGEN).
THBD: (THBD OR THRM) THROMBOMODULIN PRECURSOR (FETOMODULIN) (TM)
(CD141 ANTIGEN).
CD58_HUMAN: (CD58 OR LFA3) LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN 3
PRECURSOR (AG3) (ANTIGEN CD58) (SURFACE GLYCOPROTEIN LFA-3).
KAI1: (KAI1 OR CD82 OR SAR2) CD82 ANTIGEN (INDUCIBLE MEMBRANE PROTEIN
R2) (C33 ANTIGEN) (IA4) (METASTASIS SUPPRESSOR KANGAI 1) (SUPPRESSOR OF
TUMORIGENICITY-6).
ANPEP: (ANPEP OR PEPN OR APN OR CD13 OR LAP1 OR LAP-1) AMINOPEPTIDASE N
(EC 3.4.11.2) (MICROSOMAL AMINOPEPTIDASE) (GP150) (MYELOID PLASMA
MEMBRANE GLYCOPROTEIN CD13) (P161 MEMBRANE PROTEIN) (MAPN) (RAPN)
(ALANYL AMINOPEPTIDASE) (AMINOPEPTIDASE M) (APM) (KIDNEY ZN
PEPTIDASE) (KZP).
CTNNB1: (CTNNB1 OR CTNNB) BETA-CATENIN.
CD44_EX10-12_HUMAN: (CD44 OR LHR) CD44 ANTIGEN PRECURSOR (PHAGOCYTIC
(EPICAN) (CDW44) (LY-24).
CD47: (CD47 OR IAP) LEUKOCYTE SURFACE ANTIGEN CD47 PRECURSOR
(ANTIGENIC SURFACE DETERMINANT PROTEIN OA3) (INTEGRIN ASSOCIATED
PROTEIN) (IAP) (MER6) (ITGP) (INTEGRIN-ASSOCIATED PROTEIN PRECURSOR).
ITGA4: (ITGA4 OR VLA-4) INTEGRIN ALPHA-4 PRECURSOR (INTEGRIN ALPHA-IV)
(VLA-4) (CD49D) (LYMPHOCYTE-PEYER'S PATCH ADHESION MOLECULES ALPHA
SUBUNIT) (LPAM ALPHA SUBUNIT).
Among the 70 genes up-regulated in the Lineage Negative cells are twelve different CD (cluster of differentiation) surface markers, including CD44 represented by five non-overlapping PIQOR probes, each specific for different adjacent exons and thus detecting various transcript variants of the CD44 gene. Most of the surface markers (CD13, CD34, CD44, CD47, CD49D, CD58, and CD141), are known to be expressed predominantly on epithelial and endothelial cells.
The following genes were down-regulated in the Lineage Negative cells relative to MLPC: RXRG, CCNE2, CDC25B, MEF-2C, ERBB2, CLCN3, EBCTF, SOX2, CRIP1, HERMES, TAL1, NFKB3, TPM1, NEUROG1, MAD1, FZD4, TNFSF11, E2IG3, RARA1, GABRA1, SMAD3, HDAC2, FN1_EIIIA, IL1R2, HGF, RARB2—1, ELAVL2, BMP4, SERPINF1, EDN3, TCF4, ACTG2, GATA4, ACTA2, CDKN1B, THROMBOSPONDIN2, CNP, LAMA1, MAP3K3, BFGFR—1, EPHB4, NCAD, FZD1—2, NRG1, EGFRLONG, ACRP, ASPIC1, SNAI1, DJ924G13.1, TFP1, HIF1A, JUNB, RPL13A, VEGF—2, TBX3, SLC16A1, HESR1, ITGB8, INTEGRINA7, ATM, P53, EN1, TRK-B, SMAD1, FLJ10884, TNC, PGH2, HSCDGF, JUN, FN1_REPEAT-A, NKX2-2, GATA5, CXCL12, ALPL, PMX2B, TIMP3, TENASCINX, PBXIP1, MMP21-22-23, and DCN—1. Table 11 lists the genes identified as belonging to a particular pathway or family. In Table 11, “A” refers to transcription factor activity as determined by GO, “B” refers to transcription factor as determined by ME, and “C” refers to tube nucleic acid binding as determined by GO.
Pathway analysis for genes down-regulated in Lineage Negative Cells
CRIP1: (CRIP1 OR CRIP) CYSTEINE-RICH PROTEIN 1 (CYSTEINE-RICH INTESTINAL
PROTEIN) (CRIP) (CYSTEINE-RICH HEART PROTEIN) (HCRHP).
ELAVL2: (ELAVL2 OR HUB) ELAV-LIKE PROTEIN 2 (HU-ANTIGEN B) (HUB) (ELAV-
LIKE NEURONAL PROTEIN 1) (NERVOUS SYSTEM-SPECIFIC RNA BINDING
PROTEIN HEL-N1).
PMX2B: (PMX2B) PAIRED MESODERM HOMEOBOX PROTEIN 2B (PAIRED-LIKE
HOMEOBOX 2B) (PHOX2B HOMEODOMAIN PROTEIN) (NEUROBLASTOMA PHOX)
(NBPHOX).
EN1: (EN1) HOMEOBOX PROTEIN ENGRAILED-1 (HU-EN-1).
NKX2-2: (NKX2-2 OR NKX2B OR NKX2.2) HOMEOBOX PROTEIN NKX-2.2
(HOMEOBOX PROTEIN NK-2 HOMOLOG B).
HDAC2: (HDAC2) HISTONE DEACETYLASE 2 (HD2).
SOX2: (SOX2) TRANSCRIPTION FACTOR SOX-2.
RARA1_HUMAN: (RARA OR NR1B1) RETINOIC ACID RECEPTOR ALPHA (RAR-
GATA5: (GATA5) TRANSCRIPTION FACTOR GATA-5 (GATA BINDING FACTOR-5).
DJ924G13.1: (DJ924G13.1 OR KIAA1221) DJ924G13.1 (KIAA1221) (PUTATIVE ZINC
FINGER PROTEIN) (BM-005) (FLJ10725) (FLJ13534) (FLJ13964) (D5ERTD689E OR
MKIAA1221) (1110068L01RIK).
JUN: (JUN) TRANSCRIPTION FACTOR AP-1 (ACTIVATOR PROTEIN 1) (AP1) (PROTO-
ONCOGENE C-JUN) (V-JUN AVIAN SARCOMA VIRUS 17 ONCOGENE HOMOLOG)
(P39).
JUNB: (JUNB) TRANSCRIPTION FACTOR JUN-B (G0S3).
NFKB3: (RELA OR NFKB3) TRANSCRIPTION FACTOR P65 (NUCLEAR FACTOR NF-
KAPPA-B P65 SUBUNIT).
HIF1A: (HIF1A) HYPOXIA-INDUCIBLE FACTOR 1 ALPHA (HIF-1 ALPHA) (ARNT
INTERACTING PROTEIN) (MEMBER OF PAS PROTEIN 1) (MOP1) (HIF1 ALPHA).
NEUROG1: (NEUROG1 OR NGN1 OR NGN OR NEUROD3 OR ATH4C) NEUROGENIN 1
(NEUROGENIC DIFFERENTIATION FACTOR 3) (NEUROD3) (NEUROGENIC BASIC-
HELIX-LOOP-HELIX PROTEIN).
TAL1: (TAL1 OR SCL OR TCL5) T-CELL ACUTE LYMPHOCYTIC LEUKEMIA-1
PROTEIN (TAL-1 PROTEIN) (STEM CELL PROTEIN) (T-CELL
LEUKEMIA/LYMPHOMA-5 PROTEIN).
TCF4: (TCF4 OR ITF2 OR SEF2) TRANSCRIPTION FACTOR 4 (IMMUNOGLOBULIN
TRANSCRIPTION FACTOR 2) (RITF-2) (ITF-2) (SL3-3 ENHANCER FACTOR 2) (SEF-2)
(CLASS A HELIX-LOOP-HELIX TRANSCRIPTION FACTOR ME2).
EBCTF: (EBF) EARLY B-CELL TRANSCRIPTION FACTOR (FRAGMENT). (COE1 OR
OLF1) TRANSCRIPTION FACTOR COE1 (OE-1) (O/E-1) (OLFACTORY NEURONAL
TRANSCRIPTION FACTOR) (OLF-1).
HESR1: (HESR-1 OR CHF2 OR HEY1) HAIRY AND ENHANCER OF SPLIT RELATED-1
(HEY1 PROTEIN).
MEF-2C: (MEF2C) MYOCYTE-SPECIFIC ENHANCER FACTOR 2C.
TBX3: (TBX3) T-BOX TRANSCRIPTION FACTOR TBX3 (T-BOX PROTEIN 3).
RARB2_1: (RARB OR NR1B2 OR HAP) RETINOIC ACID RECEPTOR BETA-2 (RAR-
BETA-2) (RAR-EPSILON).
DECAPENTAPLEGIC HOMOLOG 3 (SMAD 3) (MOTHERS AGAINST DPP HOMOLOG
3) (MAD3) (HMAD-3) (MMAD3) (JV15-2).
RXRG: (RXRG OR NR2B3) RETINOIC ACID RECEPTOR RXR-GAMMA.
SMAD1: (MADH1 OR SMAD1 OR MADR1 OR BSP1) MOTHERS AGAINST
DECAPENTAPLEGIC HOMOLOG 1 (SMAD 1) (MOTHERS AGAINST DPP HOMOLOG
1) (MAD-RELATED PROTEIN 1) (TRANSFORMING GROWTH FACTOR-BETA
SIGNALING PROTEIN-1) (BSP-1) (HSMAD1) (JV4-1).
ATM: (ATM) SERINE-PROTEIN KINASE ATM (EC 2.7.1.37) (ATAXIA
TELANGIECTASIA MUTATED) (A-T, MUTATED) (ATDC).
P53: (TP53 OR P53) CELLULAR TUMOR ANTIGEN P53 (TUMOR SUPPRESSOR P53)
(PHOSPHOPROTEIN P53).
HERMES: (HERMES OR RBPMS) RNA-BINDING PROTEIN WITH MULTIPLE
SPLICING (RBP-MS).
Surprisingly, among the 80 genes down-regulated, 30 are involved in nucleic acid binding (GO:0003676) and/or exhibit transcription factor activity (GO:0003700, ME: transcription factor) (GATA4_HUMAN, CRIP1, ELAVL2, PMX2B, EN1, NKX2-2, HDAC2, SOX2, RARA1_HUMAN, GATA5, DJ924G13.1, SNAI1, JUN, JUNB, NFKB3, HIFLA, NEUROG1, TAL1, TCF4, EBCTF, HESR1, MEF-2C, TBX3, RARB2—1, SMAD3_HUMAN, RXRG, SMAD1, ATM, P53, and HERMES). Increased expression of TBX3 and SOX2 in MLPC relative to the LinNeg cells is of particular interest.
Surface Marker Pattern
Table 12 provides the expression pattern of defined surface markers of each of the cell populations relative to MLPC. Shaded cells refer to a decrease in expression of the particular marker relative to MLPC whereas an up arrow indicates that expression was increased in that cell population relative to the MLPC.
Expression pattern of cell surface markers
CD44 ex10-12
CD44 ex11-13
CD44 ex13-15
CD44 ex16-20
CD44 ex3-5
CD44 ex7-9
CD44 ex8-10
CD45 ex10-11
Overall, MLPCs demonstrate a high degree of sternness and quiescence: down regulation of 65 genes associated with active protein synthesis (i.e.—ribosomal subunits), 18 genes linked with phosphate metabolism (e.g., kinases and phosphatases), 123 genes regulating proliferation and cell cycling (e.g., cyclins, cyclin-dependent kinases, and checkpoint proteins). MLPCs exhibited a very high degree of differentiation multipotentiality as seen in the down-regulation of 12 different clusters of genes associated with differentiation surface marker genes (e.g., epithelium and endothelium) and up-regulation of 80 genes involved in nucleic acid binding and transcription factors that regulate differentiation of tissues from all three germinal layers. Additionally 10 genes associated with maintenance of sternness in embryonic stem cells were overexpressed in MLPC compared to the other cell groups. In particular, MSCs were shown to over-express genes associated with connective and stromal tissue and were strongly committed to that lineage by array analysis.
Karyotypic Analysis of MLPC
The karyotype of MLPC (clone UM081704-1 E8, Example 5) was assessed at 20 doublings and >80 doublings and compared with the karyotype of MSC (Cambrex), normal MNC, and the KG-1 cell line, a myeloid leukemia line. MNCs were the reference cell for the other karyotypes. MLPC have a normal karyotype with complete stability through >80 doublings. No deletions, translocations, or suspicious single nucleotide polymorphisms were observed. MSC had deletions and potential epigenic changes after 20 doublings. KG-1 cells, as expected in their role as a positive neoplastic control, had major deletions and substitutions.
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