# Patent

## Title
Treatment of pluripotent cells

## Abstract

The present invention is provides a method for treating human pluripotent cells. In particular, the methods of the invention are directed to the treatment of human pluripotent cells, whereby the human pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity.

## Background
This application is a divisional of U.S. application Ser. No. 12/108,852 filed Apr. 24, 2008. The complete disclosures of the aforementioned related patent applications are hereby incorporated herein by reference for all purposes.


FIELD OF THE INVENTION
The present invention is directed to methods to treat pluripotent cells, whereby the pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.
BACKGROUND
Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent cells, such as, for example, embryonic stem cells.
In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF-3 beta, GATA-4, Mix11, CXCR4 and SOX-17.
Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, PDX-1. In the absence of PDX-1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX-1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.
The generation of a sufficient amount of cellular material for transplantation requires a source of the cellular material that can be efficiently expanded in culture, and efficiently differentiated into the tissue of interest, for example, functional β cells.
Current methods to culture human embryonic stem cells are complex; they require the use of exogenous factors, or chemically defined media in order for the cells to proliferate without loosing their pluripotency. Furthermore differentiation of embryonic stem cells often results in a decrease in the cells to expand in culture.
In one example, Cheon et al (BioReprod DOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose a feeder-free, serum-free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.
In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGFβ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
Inhibitors of glycogen synthase kinase-3 (GSK-3) are known to promote proliferation and expansion of adult stem cells. In one example, Tateishi et al. (Biochemical and Biophysical Research Communications (2007) 352: 635) show that inhibition of GSK-3 enhances growth and survival of human cardiac stem cells (hCSCs) recovered from the neonatal or adult human heart and having mesenchymal features.
For example, Rulifson et al (PNAS 144, 6247-6252, (2007)) states “Wnt signaling stimulates islet β cell proliferation.
In another example, WO2007016485 reports that addition of GSK-3 inhibitors to the culture of non-embryonic stem cells, including multipotent adult progenitor cells, leads to the maintenance of a pluripotent phenotype during expansion and results in a more robust differentiation response.
In another example, US2006030042 uses a method of inhibiting GSK-3, either by addition of Wnt or a small molecule inhibitor of GSK-3 enzyme activity, to maintain embryonic stem cells without the use of a feeder cell layer.
In another example, WO2006026473 reports the addition of a GSK-3B inhibitor, to stabilize pluripotent cells through transcriptional activation of c-myc and stabilization of c-myc protein.
In another example, WO2006100490 reports the use of a stem cell culture medium containing a GSK-3 inhibitor and a gp130 agonist to maintain a self-renewing population of pluripotent stem cells, including mouse or human embryonic stem cells.
In another example, Sato et al. (Nature Medicine (2004) 10:55-63) show that inhibition of GSK-3 with a specific pharmacological compound can maintain the undifferentiated phenotype of embryonic stem cells and sustain expression of pluripotent state-specific transcription factors such as Oct-3/4, Rex-1, and Nanog.
In another example, Maurer et al (Journal of Proteome Research (2007) 6:1198-1208) show that adult, neuronal stem cells treated with a GSK-3 inhibitor show enhanced neuronal differentiation, specifically by promoting transcription of β-catenin target genes and decreasing apoptosis.
In another example, Gregory et al (Annals of the New York Academy of Sciences (2005) 1049:97-106) report that inhibitors of GSK-3B enhance in vitro osteogenesis.
In another example, Feng et al (Biochemical and Biophysical Research Communications (2004) 324:1333-1339) show that hematopoietic differentiation from embryonic stem cells is associated with down-regulation of the Wnt/β-catenin pathway, where Wnt is a natural inhibitor of GSK3.
Therefore, there still remains a significant need to develop methods for treating pluripotent stem cell such that they can be expanded to address the current clinical needs, while retaining the potential to differentiate into pancreatic endocrine cells, pancreatic hormone expressing cells, or pancreatic hormone secreting cells.
SUMMARY
The present invention provides a method to expand and differentiate pluripotent cells by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.
In one embodiment, the present invention provides a method to expand and differentiate pluripotent cells, comprising the steps of:

    
    
        a. Culturing pluripotent cells, and
        b. Treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.
    
    


In one embodiment, the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.
The pluripotent cells may be human embryonic stem cells, or they may be cells expressing pluripotency markers derived from human embryonic stem cells, according to the methods disclosed in 60/913,475.
In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (I):




In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (II):




In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (III):







BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the effect of a range of concentrations of the compound JNJ 17189731 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 2 shows the effect of a range of concentrations of the compound JNJ 17163796 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 3 shows the effect of a range of concentrations of the compound JNJ 17223375 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 4 shows the effect of a range of concentrations of the compound JNJ 18157698 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 5 shows the effect of a range of concentrations of the compound JNJ 26158015 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 6 shows the effect of a range of concentrations of the compound JNJ 26483197 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 7 shows the effect of a range of concentrations of the compound JNJ 26483249 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 8 shows the effect of a range of concentrations of the compound JNJ 10220067 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).
FIG. 9 shows the expression of CXCR4 on the surface of cells, as determined by immunofluorescent staining and flow cytometric analysis, on cells treated with the compounds shown, according to the methods described in Example 8.
FIG. 10 shows the expression of CXCR4 (Panel A), HNF-3 beta (Panel B), and Sox-17 (Panel C), as determined by real-time PCR, in cells treated with the compounds shown, according to the methods described in Example 8.
FIG. 11 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and Pdx-1 expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare). Cells were treated according to the methods described in Example 9.
FIG. 12 shows the effect of a range of concentrations of the compounds shown on Pdx-1 expression (white bars) and HNF-6 (black bars), as determined by real-time PCR. Cells were treated according to the methods described in Example 9.
FIG. 13 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and insulin expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare). Cells were treated according to the methods described in Example 10.
FIG. 14 shows effect of a range of concentrations of the compounds shown on Pdx-1 expression (white bars) and insulin (black bars), as determined by real-time PCR. Cells were treated according to the methods described in Example 10.
FIG. 15 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and insulin expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare). Cells were treated according to the methods described in Example 11.



DETAILED DESCRIPTION
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments, or applications of the present invention.
Definitions
Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
“β-cell lineage” refer to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN-3, NRx2.2, NRx6.1, NeuroD, Is1-1, HNF-3 beta, MAFA, Pax4, and Pax6. Cells expressing markers characteristic of the β cell lineage include β cells.
“Cells expressing markers characteristic of the definitive endoderm lineage” as used herein refer to cells expressing at least one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cer1, Noda1, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
“Cells expressing markers characteristic of the pancreatic endoderm lineage” as used herein refer to cells expressing at least one of the following markers: PDX-1, HNF-1beta, PTF-1 alpha, HNF-6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.
“Cells expressing markers characteristic of the pancreatic endocrine lineage” as used herein refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, Ngn-3, or PTF-1 alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.
“Definitive endoderm” as used herein refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF-3 beta, GATA-4, SOX-17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and Mix11.
“Extraembryonic endoderm” as used herein refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, and SPARC.
“Markers” as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
“Mesendoderm cell” as used herein refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta, GSC, FGF17, GATA-6.
“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
“Pancreatic hormone secreting cell” as used herein refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
“Pre-primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Noda1, or FGF8
“Primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
In one embodiment, the present invention provides a method for the expansion and differentiation of pluripotent cells comprising treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.
In one embodiment, the present invention provides a method to expand and differentiate pluripotent cells, comprising the steps of:

    
    
        c. Culturing pluripotent cells, and
        d. Treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.
    
    


In one embodiment, the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.
Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, Hnf-3beta, GSC, Cer1, Noda1, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Contemplated in the present invention is a cell, derived from a pluripotent cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
The pluripotent cells may be treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours. Alternatively, the pluripotent cells may be treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours. Alternatively, the pluripotent cells may be treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.
In one embodiment, the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 100 μM. Alternatively, the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μM to about 10 μM. Alternatively, the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μM.
Compounds Suitable for Use in the Methods of the Present Invention
In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (I):




wherein:
R1 is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl and nitrile, or pyrimidinyl;
R2 is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl and nitrile, or pyrimidinyl which is optionally C1-4alkyl substituted, and at least one of R1 and R2 is pyrimidinyl;
R3 is hydrogen, 2-(trimethylsilyl)ethoxymethyl, C1-5alkoxycarbonyl, aryloxycarbonyl, arylC1-5alkyloxycarbonyl, arylC1-5alkyl, substituted arylC1-5alkyl wherein the one or more aryl substituents are independently selected from the group consisting of C1-5alkyl, C1-5alkoxy, halogen, amino, C1-5 alkylamino, and diC1-5 alkylamino, phthalimidoC1-5alkyl, aminoC1-5 alkyl, diaminoC1-5 alkyl, succinimidoC1-5 alkyl, C1-5 alkylcarbonyl, arylcarbonyl, C1-5 alkylcarbonylC1-5 alkyl and aryloxycarbonylC1-5 alkyl;
R4 is -(A)-(CH2)q—X;
A is vinylene, ethynylene or




R5 is selected from the group consisting of hydrogen, C1-5alkyl, phenyl and phenylC1-5alkyl;

q is 0-9;
X is selected from the group consisting of hydrogen, hydroxy, vinyl, substituted vinyl wherein one or more vinyl substituents are each selected from the group consisting of fluorine, bromine, chlorine and iodine, ethynyl, substituted ethynyl wherein the ethynyl substituents are selected from the group consisting of fluorine, bromine chlorine and iodine, C1-5alkyl, substituted C1-5alkyl wherein the one or more alkyl substituents are each selected from the group consisting of C1-5alkoxy, trihaloalkyl, phthalimido and amino, C3-7cycloalkyl, C1-5alkoxy, substituted C1-5alkoxy wherein the alkyl substituents are selected from the group consisting of phthalimido and amino, phthalimidooxy, phenoxy, substituted phenoxy wherein the one or more phenyl substituents are each selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy, phenyl, substituted phenyl wherein the one or more phenyl substituents are each selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy, arylC1-5alkyl, substituted arylC1-5alkyl wherein the one or more aryl substituents are each selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy, aryloxyC1-5alkylamino, C1-5 alkylamino, diC1-5 alkylamino, nitrile, oxime, benxyloxyimino, C1-5 alkyloxyimino, phthalimido, succinimido, C1-5 alkylcarbonyloxy, phenylcarbonyloxy, substituted phenylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy, phenylC1-5alkylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy, aminocarbonyloxy, C1-5alkylaminocarbonyloxy, diC1-5 alkylaminocarbonyloxy, C1-5 alkoxycarbonyloxy, substituted C1-5alkoxycarbonyloxy wherein the one or more alkyl substituents are each selected from the group consisting of methyl, ethyl, isopropyl and hexyl, phenoxycarbonyloxy, substituted phenoxycarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C1-5alkyl, C1-5 alkoxy and halogen, C1-5 alkylthio, substituted C1-5alkylthio wherein the alkyl substituents are selected from the group consisting of hydroxy and phthalimido, C1-5alkylsulfonyl, phenylsulfonyl, substituted phenylsulfonyl wherein the one or more phenyl substituents are each selected from the group consisting of bromine, fluorine, chloride, C1-5alkoxy and trifluoromethyl; with the proviso that if A is




q is 0 and X is H, then R3 may not be 2-(trimethylsilyl)ethoxymethyl; and pharmaceutically acceptable salts thereof.

An example of the invention includes a compound of Formula (I) wherein R1 is substituted phenyl and R2 is pyrimidin-3-yl.
An example of the invention includes a compound of Formula (I) wherein R1 is 4-fluorophenyl.
An example of the invention includes a compound of Formula (I) wherein R3 is hydrogen, arylC1-5alkyl, or substituted arylC1-5alkyl.
An example of the invention includes a compound of Formula (I) wherein R3 is hydrogen or phenylC1-5alkyl.
An example of the invention includes a compound of Formula (I) wherein A is ethynylene and q is 0-5.
An example of the invention includes a compound of Formula (I) wherein X is succinimido, hydroxy, methyl, phenyl, C1-5alkylsulfonyl, C3-6cycloalkyl, C1-5alkylcarbonyloxy, C1-5alkoxy, phenylcarbonyloxy, C1-5alkylamino, diC1-5alkylamino or nitrile.
Compounds of Formula (I) are disclosed in commonly assigned U.S. Pat. No. 6,214,830, the complete disclosure of which is herein incorporated by reference.
An example of the invention includes a compound of Formula (I) wherein the compound is selected from the group consisting of:











Compound
Name
















1
5(4)-(4-fluorophenyl)-4(5)-(4-pyridyl)imidazole,


2
4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridyl)imidazole,


3
5-(4-fluorophenyl)-1-(3-phenylpropyl)-4-(4-pyridyl)imidazole,


4
4-(4-fluorophenyl)-2-iodo-1-(3-phenylpropyl)-5-(4-



pyridyl)imidazole,


5
4-(4-fluorophenyl)-2-(4-hydroxybutyn-1-yl)-1-(3-phenylpropyl)-



5-(4-pyridyl)imidazole,


6
4-(4-fluorophenyl)-5-(4-pyridyl)-1-[2-



(trimethylsilyl)ethoxymethyl]-imidazole,


7
5-(4-fluorophenyl)-4-(4-pyridyl)-1-[2-



(trimethylsilyl)ethoxymethyl]-imidazole,


8
5-(4-fluorophenyl)-2-iodo-4-(4-pyridyl)-1-[2-



(trimethylsilyl)ethoxymethyl]-imidazole,


9
5-(4-fluorophenyl)-4-(4-pyridyl)-2-(trimethylsilyl)ethinyl-1-[2-



(trimethylsilyl)ethoxymethyl]-imidazole,


10
2-(2-chlorovinyl)-5-(4-fluorophenyl)-4-(4-pyridyl)-imidazole,


11
5-(4-fluorophenyl)-4-(4-pyridyl)-1-[2-



(trimethylsilyl)ethoxymethyl]-imidazole-2-carboxaldehyde,


12
2-[2,2-dibromoethylene-1-yl]-5-(4-fluorophenyl)-4-(4-pyridyl)-1-



[2-(trimethylsilyl)ethoxymethyl]-imidazole-2-carboxaldehyde,


13
5(4)-(4-fluorophenyl)-2-(3-hydroxy-3-phenyl-propyn-1-yl)-4(5)-



(4-pyridyl)imidazole,


14
5-(4-fluorophenyl)-4-(4-pyridyl)-1-[2-



(trimethylsilyl)ethoxymethyl]-2-oximinoimidazole,


15
5-(4-fluorophenyl)-4-(4-pyridyl)-2-imidazole oxime,


16
2-(5-chloropentyn-1-yl)-4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-



(4-pyridyl)imidazole,


17
4-(4-fluorophenyl)-2-(4-N-phenylcarbamoyloxybutyn-1-yl)1-(3-



phenylpropyl)-5-(4-pyridyl)imidazole,


17
2-(4-chlorobutyn-1-yl)-4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-



(4-pyridyl)imidazole, and


18
2-(4-dimethylaminobutyn-1-yl)-4-(4-fluorophenyl)-1-(3-



phenylpropyl)-5-(4-pyridyl)imidazole.









An example of the invention includes a compound of Formula (I) wherein the compound is Compound 5 of the formula:




In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (II):




Wherein:
R is selected from the group consisting of Ra, —C1-8alkyl-Ra, —C2-8alkenyl-Ra, —C2-8alkynyl-Ra and cyano;
Ra is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl;
R1 is selected from the group consisting of hydrogen, —C1-8alkyl-R5, —C2-8alkenyl-R5, —C2-8alkynyl-R5, —C(O)—(C1-8)alkyl-R9, —C(O)-aryl-R8, —C(O)—O—(C1-8)alkyl-R9, —C(O)—O-aryl-R8, —C(O)—NH(C1-8alkyl-R9), —C(O)—NH(aryl-R8), —C(O)—N(C1-8alkyl-R9)2, —SO2—(C1-8)alkyl-R9, —SO2-aryl-R8, -cycloalkyl-R6, -heterocyclyl-R6, -aryl-R6 and -heteroaryl-R6; wherein heterocyclyl and heteroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or heteroaryl ring carbon atom;
R5 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —O—(C1-8)alkyl, —O—(C1-8)alkyl-OH, —O—(C1-8)alkyl-O—(C1-8)alkyl, —O—(C1-8)alkyl-NH2, —O—(C1-8)alkyl-NH(C1-8alkyl), —O—(C1-8)alkyl-N(C1-8alkyl)2, —O—(C1-8)alkyl-S—(C1-8)alkyl, —O—(C1-8)alkyl-SO2—(C1-8)alkyl, —O—(C1-8)alkyl-SO2—NH2, —O—(C1-8)alkyl-SO2—NH(C1-8alkyl), —O—(C1-8)alkyl-SO2—N(C1-8alkyl)2, —O—C(O)H, —O—C(O)—(C1-8)alkyl, —O—C(O)—NH2, —O—C(O)—NH(C1-8alkyl), —O—C(O)—N(C1-8alkyl)2, —O—(C1-8)alkyl-C(O)H, —O—(C1-8)alkyl-C(O)—(C1-8)alkyl, —O—(C1-8)alkyl-CO2H, —O—(C1-8)alkyl-C(O)—O—(C1-8)alkyl, —O—(C1-8)alkyl-C(O)—NH2, —O—(C1-8)alkyl-C(O)—NH(C1-8alkyl), —O—(C1-8)alkyl-C(O)—N(C1-8alkyl)2, —C(O)H, —C(O)—(C1-8)alkyl, —CO2H, —C(O)—O—(C1-8)alkyl, —C(O)—NH2, —C(NH)—NH2, —C(O)—NH(C1-8alkyl), —C(O)—N(C1-8alkyl)2, —SH, —S—(C1-8)alkyl, —S—(C1-8)alkyl-S—(C1-8)alkyl, —S—(C1-8)alkyl-O—(C1-8)alkyl, —S—(C1-8)alkyl-O—(C1-8)alkyl-OH, —S—(C1-8)alkyl-O—(C1-8)alkyl-NH2, —S—(C1-8)alkyl-O—(C1-8)alkyl-NH(C1-8alkyl), —S—(C1-8)alkyl-O—(C1-8)alkyl-N(C1-8alkyl)2, —S—(C1-8)alkyl-NH(C1-8alkyl), —SO2—(C1-8)alkyl, —SO2—NH2, —SO2—NH(C1-8alkyl), —SO2—N(C1-8alkyl)2, —N—R7, cyano, (halo)1-3, hydroxy, nitro, oxo, -cycloalkyl-R6, -heterocyclyl-R6, -aryl-R6 and -heteroaryl-R6;
R6 is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —C1-8alkyl, —C2-8alkenyl, —C2-8alkynyl, —C(O)H, —C(O)—(C1-8)alkyl, —CO2H, —C(O)—O—(C1-8)alkyl, —C(O)—NH2, —C(NH)—NH2, —C(O)—NH(C1-8alkyl), —C(O)—N(C1-8)alkyl)2, —SO2—(C1-8)alkyl, —SO2—NH2, —SO2—NH(C1-8alkyl), —SO2—N(C1-8alkyl)2, —(C1-8)alkyl-N—R7, —(C1-8)alkyl-(halo)1-3, —(C1-8)alkyl-OH, -aryl-R8, —(C1-8)alkyl-aryl-R8 and —(C1-8)alkyl-heteroaryl-R8; with the proviso that, when R6 is attached to a carbon atom, R6 is further selected from the group consisting of —C1-8alkoxy, —(C1-8)alkoxy-(halo)1-3, —SH, —S—(C1-8)alkyl, —N—R7, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R8;
R7 is 2 substituents independently selected from the group consisting of hydrogen, —C1-8alkyl, —C2-8alkenyl, —C2-8alkynyl, —(C1-8)alkyl-OH, —(C1-8)alkyl-O—(C1-8)alkyl, —(C1-8)alkyl-NH2, —(C1-8)alkyl-NH(C1-8alkyl), —(C1-8)alkyl-N(C1-8alkyl)2, —(C1-8)alkyl-S—(C1-8)alkyl, —C(O)H, —C(O)—(C1-8)alkyl, —C(O)—O—(C1-8)alkyl, —C(O)—NH2, —C(O)—NH(C1-8alkyl), —C(O)—N(C1-8alkyl)2, —SO2—(C1-8)alkyl, —SO2—NH2, —SO2—NH(C1-8alkyl), —SO2—N(C1-8alkyl)2, —C(N)—NH2, -cycloalkyl-R8, —(C1-8)alkyl-heterocyclyl-R8, -aryl-R8, —(C1-8)alkyl-aryl-R8 and —(C1-8)alkyl-heteroaryl-R8;
R8 is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —C1-8alkyl, —(C1-8)alkyl-(halo)1-3 and —(C1-8)alkyl-OH; with the proviso that, when R8 is attached to a carbon atom, R8 is further selected from the group consisting of —C1-8alkoxy, —NH2, —NH(C1-8alkyl), —N(C1-8alkyl)2, cyano, halo, —(C1-8)alkoxy-(halo)1-3, hydroxy and nitro;
R9 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —C1-8alkoxy, —NH2, —NH(C1-8alkyl), —N(C1-8alkyl)2, cyano, (halo)1-3, hydroxy and nitro;
R2 is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —C1-8alkyl-R5, —C2-8alkenyl-R5, —C2-8alkynyl-R5, —C(O)H, —C(O)—(C1-8)alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-8alkyl-R9), —C(O)—N(C1-8alkyl-R9)2, —C(O)—NH(aryl-R8), —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —CO2H, —C(O)—O—(C1-8)alkyl-R9, —C(O)—O-aryl-R8, —SO2—(C1-8)alkyl-R9, —SO2-aryl-R8, -cycloalkyl-R6, -aryl-R6 and —(C1-8)alkyl-N—R7; with the proviso that, when R2 is attached to a carbon atom, R2 is further selected from the group consisting of —C1-8alkoxy-R5, —N—R7, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R6 and -heteroaryl-R6;
R3 is 1 to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —C1-8alkyl-R10, —C2-8alkenyl-R10, —C2-8alkynyl-R10, —C1-8alkoxy-R10, —C(O)H, —C(O)—(C1-8)alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-8alkyl-R9), —C(O)—N(C1-8alkyl-R9)2, —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —C(NH)—NH2, —CO2H, —C(O)—O—(C1-8)alkyl-R9, —C(O)—O-aryl-R8, —SO2—(C1-8)alkyl-R9, —SO2-aryl-R8, —N—R7, cyano, halogen, hydroxy, nitro, -cycloalkyl-R8, -heterocyclyl-R8, -aryl-R8 and -heteroaryl-R8;
R4 is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —C1-8alkyl-R10, —C2-8alkenyl-R10, —C2-8alkynyl-R10, —C1-8alkoxy-R10, —C(O)H, —C(O)—(C1-8)alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-8alkyl-R9), —C(O)—N(C1-8alkyl-R9)2, —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —C(NH)—NH2, —CO2H, —C(O)—O—(C1-8)alkyl-R9, —C(O)—O-aryl-R8, —SH, —S—(C1-8)alkyl-R10, —SO2—(C1-8)alkyl-R9, —SO2-aryl-R8, —SO2—NH2, —SO2—NH(C1-8alkyl-R9), —SO2—N(C1-8alkyl-R9)2, —N—R7, cyano, halogen, hydroxy, nitro, -cycloalkyl-R8, -heterocyclyl-R8, -aryl-R8 and -heteroaryl-R8;
R10 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —NH2, —NH(C1-8alkyl), —N(C1-8alkyl)2, cyano, (halo)1-3, hydroxy, nitro and oxo; and,
Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H); with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H); and pharmaceutically acceptable salts thereof.
Embodiments of the present invention include compounds of Formula (II) wherein, R is selected from the group consisting of Ra, —C1-4alkyl-Ra, —C2-4alkenyl-Ra, —C2-4alkynyl-Ra and cyano.
Embodiments of the present invention include compounds of Formula (II) wherein, Ra is selected from the group consisting of heterocyclyl, aryl and heteroaryl.
In one embodiment, Ra is selected from the group consisting of dihydro-pyranyl, phenyl, naphthyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, azaindolyl, indazolyl, benzofuryl, benzothienyl, dibenzofuryl and dibenzothienyl.
Embodiments of the present invention include compounds of Formula (II) wherein, R1 is selected from the group consisting of hydrogen, —C1-4alkyl-R5, —C2-4alkenyl-R5, —C2-4alkynyl-R5, —C(O)—(C1-4)alkyl-R9, —C(O)-aryl-R8, —C(O)—O—(C1-4alkyl-R9, —C(O)—O-aryl-R8, —C(O)—NH(C1-4alkyl-R9), —C(O)—NH(aryl-R8), —C(O)—N(C1-4alkyl-R9)2, —SO2—(C1-4alkyl-R9, —SO2-aryl-R8, -cycloalkyl-R6, -heterocyclyl-R6, -aryl-R6 and -heteroaryl-R6; wherein heterocyclyl and heteroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or heteroaryl ring carbon atom.
In one embodiment, R1 is selected from the group consisting of hydrogen, —C1-4alkyl-R5, -aryl-R6 and -heteroaryl-R6; wherein heteroaryl is attached to the azaindole nitrogen atom in the one position via a heteroaryl ring carbon atom.
In one embodiment, R1 is selected from the group consisting of hydrogen, —C1-4alkyl-R5 and -naphthyl-R6.
Embodiments of the present invention include compounds of Formula (II) wherein, R5 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —O—(C1-4alkyl, —O—(C1-4alkyl-OH, —O—(C1-4alkyl-O—(C1-4alkyl, —O—(C1-4alkyl-NH2, —O—(C1-4alkyl-NH(C1-4alkyl), —O—(C1-4alkyl-N(C1-4alkyl)2, —O—(C1-4alkyl-S—(C1-4)alkyl, —O—(C1-4)alkyl-SO2—(C1-4)alkyl, —O—(C1-4alkyl-SO2—NH2, —O—(C1-4alkyl-SO2—NH(C1-4alkyl), —O—(C1-4alkyl-SO2—N(C1-4alkyl)2, —O—C(O)H, —O—C(O)—(C1-4alkyl, —O—C(O)—NH2, —O—C(O)—NH(C1-4alkyl), —O—C(O)—N(C1-4alkyl)2, —O—(C1-4alkyl-C(O)H, —O—(C1-4alkyl-C(O)—(C1-4)alkyl, —O—(C1-4alkyl-CO2H, —O—(C1-4alkyl-C(O)—O—(C1-4alkyl, —O—(C1-4)alkyl-C(O)—NH2, —O—(C1-4alkyl-C(O)—NH(C1-4alkyl), —O—(C1-4alkyl-C(O)—N(C1-4alkyl)2, —C(O)H, —C(O)—(C1-4alkyl, —CO2H, —C(O)—O—(C1-4alkyl, —C(O)—NH2, —C(NH)—NH2, —C(O)—NH(C1-4alkyl), —C(O)—N(C1-4alkyl)2, —SH, —S—(C1-4alkyl, —S—(C1-4alkyl-S—(C1-4)alkyl, —S—(C1-4alkyl-O—(C1-4)alkyl, —S—(C1-4alkyl-O—(C1-4)alkyl-OH, —S—(C1-4alkyl-O—(C1-4)alkyl-NH2, —S—(C1-4alkyl-O—(C1-4)alkyl-NH(C1-4alkyl), —S—(C1-4alkyl-O—(C1-4)alkyl-N(C1-4alkyl)2, —S—(C1-4alkyl-NH(C1-4alkyl), —SO2—(C1-4alkyl, —SO2—NH2, —SO2—NH(C1-4alkyl), —SO2—N(C1-4alkyl)2, —N—R7, cyano, (halo)1-3, hydroxy, nitro, oxo, -cycloalkyl-R6, -heterocyclyl-R6, -aryl-R6 and -heteroaryl-R6.
In one embodiment, R5 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —O—(C1-4alkyl, —N—R7, hydroxy and -heteroaryl-R6.
In one embodiment, R5 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —O—(C1-4alkyl, —N—R7, hydroxy, -imidazolyl-R6, -triazolyl-R6 and -tetrazolyl-R6.
Embodiments of the present invention include compounds of Formula (II) wherein, R6 is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —C1-4alkyl, —C2-4alkenyl, —C2-4alkynyl, —C(O)H, —C(O)—(C1-4alkyl, —CO2H, —C(O)—O—(C1-4alkyl, —C(O)—NH2, —C(NH)—NH2, —C(O)—NH(C1-4alkyl), —C(O)—N(C1-4)alkyl)2, —SO2—(C1-4alkyl, —SO2—NH2, —SO2—NH(C1-4alkyl), —SO2—N(C1-4alkyl)2, —(C1-4)alkyl-N—R7, —(C1-4)alkyl-(halo)1-3, —(C1-4alkyl-OH, -aryl-R8, —(C1-4)alkyl-aryl-R8 and —(C1-4)alkyl-heteroaryl-R8; with the proviso that, when R6 is attached to a carbon atom, R6 is further selected from the group consisting of —C1-4alkoxy, —(C1-4)alkoxy-(halo)1-3, —SH, —S—(C1-4)alkyl, —N—R7, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R8.
In one embodiment, R6 is hydrogen.
Embodiments of the present invention include compounds of Formula (II) wherein, R7 is 2 substituents independently selected from the group consisting of hydrogen, —C1-4alkyl, —C2-4alkenyl, —C2-4alkynyl, —(C1-4)alkyl-OH, —(C1-4)alkyl-O—(C1-4alkyl, —(C1-4)alkyl-NH2, —(C1-4)alkyl-NH(C1-4alkyl), —(C1-4)alkyl-N(C1-4alkyl)2, —(C1-4)alkyl-S—(C1-4alkyl, —C(O)H, —C(O)—(C1-4alkyl, —C(O)—O—(C1-4alkyl, —C(O)—NH2, —C(O)—NH(C1-4alkyl), —C(O)—N(C1-4alkyl)2, —SO2—(C1-4)alkyl, —SO2—NH2, —SO2—NH(C1-4alkyl), —SO2—N(C1-4alkyl)2, —C(N)—NH2, -cycloalkyl-R8, —(C1-4)alkyl-heterocyclyl-R8, -aryl-R8, —(C1-4)alkyl-aryl-R8 and —(C1-4)alkyl-heteroaryl-R8.
In one embodiment R2 is 2 substituents independently selected from the group consisting of hydrogen, —C1-4alkyl, —C(O)H, —C(O)—(C1-4alkyl, —C(O)—O—(C1-4)alkyl, —SO2—NH2, —SO2—NH(C1-4alkyl) and —SO2—N(C1-4alkyl)2.
Embodiments of the present invention include compounds of Formula (II) wherein, R8 is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —C1-4alkyl, —(C1-4)alkyl-(halo)1-3 and —(C1-4)alkyl-OH; with the proviso that, when R8 is attached to a carbon atom, R8 is further selected from the group consisting of —C1-4alkoxy, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, cyano, halo, —(C1-4)alkoxy-(halo)1-3, hydroxy and nitro.
In one embodiment, R8 is hydrogen.
Embodiments of the present invention include compounds of Formula (II) wherein, R9 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —C1-4alkoxy, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, cyano, (halo)1-3, hydroxy and nitro.
In one embodiment, R9 is hydrogen.
Embodiments of the present invention include compounds of Formula (II) wherein, R2 is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —C1-4alkyl-R5, —C2-4alkenyl-R5, —C2-4alkynyl-R5, —C(O)H, —C(O)—(C1-4alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-4alkyl-R9), —C(O)—N(C1-4alkyl-R9)2, —C(O)—NH(aryl-R8), —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —CO2H, —C(O)—O—(C1-4alkyl-R9, —C(O)—O-aryl-R8, —SO2—(C1-4alkyl-R9, —SO2-aryl-R8, -cycloalkyl-R6, -aryl-R6 and —(C1-4)alkyl-N—R7; with the proviso that, when R2 is attached to a carbon atom, R2 is further selected from the group consisting of —C1-4alkoxy-R5, —N—R2, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R6 and -heteroaryl-R6.
In one embodiment, R2 is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —C1-4alkyl-R5, —C2-4alkenyl-R5, —C2-4alkynyl-R5, —CO2H, —C(O)—O—(C1-4alkyl-R9, -cycloalkyl-R6, -aryl-R6 and —(C1-4)alkyl-N—R7; with the proviso that, when R2 is attached to a nitrogen atom, a quaternium salt is not formed; and, with the proviso that, when R2 is attached to a carbon atom, R2 is further selected from the group consisting of —C1-4alkoxy-R5, —N—R7, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R6 and -heteroaryl-R6.
In one embodiment, R2 is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —C1-4alkyl-R5 and -aryl-R6; with the proviso that, when R2 is attached to a nitrogen atom, a quaternium salt is not formed; and, with the proviso that when R2 is attached to a carbon atom, R2 is further selected from the group consisting of —N—R7, halogen, hydroxy and -heteroaryl-R6.
Embodiments of the present invention include compounds of Formula (II) wherein, R3 is 1 to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —C1-4alkyl-R10, —C2-4alkenyl-R10, —C2-4alkynyl-R10, —C1-4alkoxy-R10, —C(O)H, —C(O)—(C1-4alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-4alkyl-R9), —C(O)—N(C1-4alkyl-R9)2, —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —C(NH)—NH2, —CO2H, —C(O)—O—(C1-4alkyl-R9, —C(O)—O-aryl-R8, —SO2—(C1-8)alkyl-R9, —SO2-aryl-R8, —N—R7, —(C1-4)alkyl-N—R7, cyano, halogen, hydroxy, nitro, -cycloalkyl-R8, -heterocyclyl-R8, -aryl-R8 and -heteroaryl-R8.
In one embodiment, R3 is one substituent attached to a carbon atom selected from the group consisting of hydrogen, —C1-4alkyl-R10, —C2-4alkenyl-R10, —C2-4alkynyl-R10, —C1-4alkoxy-R10, —C(O)H, —CO2H, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, cyano, halogen, hydroxy and nitro.
In one embodiment, R3 is one substituent attached to a carbon atom selected from the group consisting of hydrogen, —C1-4alkyl-R10, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, halogen and hydroxy.
Embodiments of the present invention include compounds of Formula (II) wherein, R4 is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —C1-4alkyl-R10, —C2-4alkenyl-R10, —C2-4alkynyl-R10, —C1-4alkoxy-R10, —C(O)H, —C(O)—(C1-4alkyl-R9, —C(O)—NH2, —C(O)—NH(C1-4alkyl-R9), —C(O)—N(C1-4alkyl-R9)2, —C(O)-cycloalkyl-R8, —C(O)-heterocyclyl-R8, —C(O)-aryl-R8, —C(O)-heteroaryl-R8, —C(NH)—NH2, —CO2H, —C(O)—O—(C1-4alkyl-R9, —C(O)—O-aryl-R8, —SH, —S—(C1-4alkyl-R10, —SO2—(C1-4alkyl-R9, —SO2-aryl-R8, —SO2—NH2, —SO2—NH(C1-4alkyl-R9), —SO2—N(C1-4alkyl-R9)2, —N—R7, cyano, halogen, hydroxy, nitro, -cycloalkyl-R8, -heterocyclyl-R8, -aryl-R8 and -heteroaryl-R8.
In one embodiment, R4 is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —C1-4alkyl-R10, —C2-4alkenyl-R10, —C2-4alkynyl-R10, —C1-4alkoxy-R10, —C(O)H, —CO2H, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, cyano, halogen, hydroxy, nitro, -cycloalkyl, -heterocyclyl, -aryl and -heteroaryl.
In one embodiment, R4 is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, C1-4alkyl-R10, C1-4alkoxy-R10, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, halogen and hydroxy.
In one embodiment, R4 is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, C1-4alkyl-R10, C1-4alkoxy-R10, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, chlorine, fluorine and hydroxy.
Embodiments of the present invention include compounds of Formula (II) wherein, R10 is 1 to 2 substituents independently selected from the group consisting of hydrogen, —NH2, —NH(C1-4alkyl), —N(C1-4alkyl)2, cyano, (halo)1-3, hydroxy, nitro and oxo.
In one embodiment, R10 is 1 to 2 substituents independently selected from the group consisting of hydrogen and (halo)1-3.
In one embodiment, R10 is 1 to 2 substituents independently selected from the group consisting of hydrogen and (fluoro)3.
Embodiments of the present invention include compounds of Formula (II) wherein, Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H); with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H).
In one embodiment, Y and Z are independently selected from the group consisting of O and (H,H); with the proviso that one of Y and Z is O, and the other is selected from the group consisting of O and (H,H).
In one embodiment, Y and Z are independently selected from O.
Compounds of Formula (II) are disclosed in commonly assigned U.S. Pat. No. 7,125,878, the complete disclosure of which is herein incorporated by reference.
An example of the invention includes a compound of Formula (II) wherein the compound is selected from the group consisting of:











Compound
Name
















1
3-(2-chlorophenyl)-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-



b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


2
3-(2-chlorophenyl)-4-[1-[3-(dimethylamino)propyl]-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


3
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-



naphthalenyl)-1H-pyrrole-2,5-dione,


4
3-[1-[3-(dimethylamino)propyl]-1H-pyrrolo[2,3-b]pyridin-3-yl]-



4-(1-naphthalenyl)-1H-pyrrole-2,5-dione,


5
3-(5-chlorobenzo[b]thien-3-yl)-4-[1-(3-hydroxypropyl)-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


6
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1H-



indazol-3-yl)-1H-pyrrole-2,5-dione,


7
3-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-[1-(3-



hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


8
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(2-



methoxyphenyl)-1H-pyrrole-2,5-dione,


9
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(3-



methoxyphenyl)-1H-pyrrole-2,5-dione,


10
3-(2-chloro-4-fluorophenyl)-4-[1-(3-hydroxypropyl)-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


11
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-[2-



(trifluoromethyl)phenyl]-1H-pyrrole-2,5-dione,


12
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(2-



pyridinyl)-1H-pyrrole-2,5-dione,


13
3-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]-4-[1-(3-



hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


14
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(2-



thienyl)-1H-pyrrole-2,5-dione,


15
3-(2,5-dichloro-3-thienyl)-4-[1-(3-hydroxypropyl)-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


16
3-[1-(3-hydroxypropyl)-1H-pyrazol-3-yl]-4-[1-(3-



hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


17
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1H-



imidazol-2-yl)-1H-pyrrole-2,5-dione,


18
3-[1-(3-hydroxypropyl)-1H-imidazol-4-yl]-4-[1-(3-



hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


19
3-[1-(2-hydroxyethyl)-1H-imidazol-4-yl]-4-[1-(3-



hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


20
3-[1-[3-(dimethylamino)propyl]-1H-indazol-3-yl]-4-[1-(2-



naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-



dione,


21
3-[1-(3-hydroxypropyl)-1H-indazol-3-yl]-4-[1-(2-naphthalenyl)-



1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


22
3-[(E)-2-(4-fluorophenyl)ethenyl]-4-[1-(3-hydroxypropyl)-1H-



pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


23
3-(3,4-dihydro-2H-pyran-6-yl)-4-[1-(3-hydroxypropyl)-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


24
4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-[3,3′-bi-



1H-pyrrole]-2,5-dione,


25
3-(2-benzofuranyl)-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-



b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


26
3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-



methyl-1H-pyrazol-3-yl)-1H-pyrrole-2,5-dione,


27
2,5-dihydro-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-



yl]-2,5-dioxo-1H-pyrrole-3-carbonitrile,


28
3-dibenzo[b,d]thien-4-yl-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-



b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


29
3-(4-dibenzofuranyl)-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-



b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


30
3-(2-hydroxyphenyl)-4-[1-(3-methoxypropyl)-1H-pyrrolo[2,3-



b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


31
3-(3,4-dimethoxyphenyl)-4-[1-(3-methoxypropyl)-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


32
3-(3,4-dihydroxyphenyl)-4-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-



b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


33
3-(2-methoxyphenyl)-4-[1-(2-naphthalenyl)-1H-pyrrolo[2,3-



b]pyridin-3-yl]-1H-pyrrole-2,5-dione,


34
[3-[3-[2,5-dihydro-4-(2-methoxyphenyl)-2,5-dioxo-1H-pyrrol-3-



yl]-1H-pyrrolo[2,3-b]pyridin-1-yl]propyl]-carbamic acid 2-



methylpropyl ester,


35
3-[1-(3-aminopropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(2-



methoxyphenyl)-1H-pyrrole-2,5-dione,


36
N-[3-[3-[2,5-dihydro-4-(2-methoxyphenyl)-2,5-dioxo-1H-pyrrol-



3-yl]-1H-pyrrolo[2,3-b]pyridin-1-yl]propyl]-acetamide,


37
N-[3-[3-[2,5-dihydro-4-(2-methoxyphenyl)-2,5-dioxo-1H-pyrrol-



3-yl]-1H-pyrrolo[2,3-b]pyridin-1-yl]propyl]-sulfamide,


38
3-(2-methoxyphenyl)-4-[1-[3-(1H-tetrazol-1-yl)propyl]-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


39
3-(2-methoxyphenyl)-4-[1-[3-(2H-tetrazol-2-yl)propyl]-1H-



pyrrolo[2,3-b]pyridine-3-yl]-1H-pyrrole-2,5-dione,


40
3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-



pyrazin-2-yl-pyrrole-2,5-dione,


41
3-(2,4-dimethoxy-pyrimidin-5-yl)-4-[1-(3-hydroxy-propyl)-1H-



pyrrolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione,


42
4-{3-[4-(2,4-dimethoxy-pyrimidin-5-yl)-2,5-dioxo-2,5-dihydro-



1H-pyrrol-3-yl]-pyrrolo[2,3-b]pyridin-1-yl}-butyronitrile,


43
4-{3-[4-(1-methyl-1H-pyrazol-3-yl)-2,5-dioxo-2,5-dihydro-1H-



pyrrol-3-yl]-pyrrolo[2,3-b]pyridin-1-yl}-butyronitrile, and


44
3-(2,4-dimethoxy-pyrimidin-5-yl)-4-(1-phenethyl-1H-



pyrrolo[2,3-b]pyridine-3-yl)-pyrrole-2,5-dione.









An example of the invention includes a compound of Formula (II) wherein the compound is selected from the group consisting of:







In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (III):




wherein
A and E are independently selected from the group consisting of a hydrogen substituted carbon atom and a nitrogen atom; wherein




is independently selected from the group consisting of 1H-indole, 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 1H-indazole;

Z is selected from 0; alternatively, Z is selected from dihydro; wherein each hydrogen atom is attached by a single bond;
R4 and R5 are independently selected from C1-8alkyl, C2-8alkenyl and C2-8alkynyl optionally substituted with oxo;
R2 is selected from the group consisting of —C1-8alkyl-, —C2-8alkenyl-, —C2-8alkynyl-, —O—(C1-8)alkyl-O—, —O—(C2-8)alkenyl-O—, —O—(C2-8)alkynyl-O—, —C(O)—(C1-8)alkyl-C(O)— (wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, —C(O)O—(C1-8)alkyl, —C1-8alkyl-C(O)O—(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy, hydroxy(C1-8)alkyl and oxo; and, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaryl, heterocyclyl(C1-8)alkyl, aryl(C1-8)alkyl, heteroaryl(C1-8)alkyl, spirocycloalkyl and spiroheterocyclyl (wherein any of the foregoing cycloalkyl, heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein any of the foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyl, heterocyclyl, aryl, heteroaryl (wherein cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein heterocyclyl is optionally substituted with oxo), —(O—(CH2)1-6)0-5—O—, —O—(CH2)1-6—O—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—O—(CH2)1-6—O—, —(O—(CH2)1-6)0-5—NR6—, —O—(CH2)1-6—NR6—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—NR6, —(O—(CH2)1-6)0-5—S—, —O—(CH2)1-6—S—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—S—, —NR6—, —NR6—NR7—, —NR6—(CH2)1-6—NR7—, —NR6—(CH2)1-6—NR7—(CH2)1-6—NR8—, —NR6—C(O)—, —C(O)—NR6—, —C(O)—(CH2)0-6—NR6—(CH2)0-6—C(O)—, —NR6—(CH2)0-6—C(O)—(CH2)1-6—C(O)—(CH2)0-6—NR7—, —NR6—C(O)—NR7—, —NR6—C(NR7)—NR8—, —O—(CH2)1-6—NR6—(CH2)1-6—S—, —S—(CH2)1-6—NR6—(CH2)1-6—O—, —S—(CH2)1-6—NR6—(CH2)1-6—S—, —NR6—(CH2)1-6—S—(CH2)1-6—NR7— and —SO2— (wherein R6, R7 and R8 are independently selected from the group consisting of hydrogen, C1-8alkyl, C1-8alkoxy(C1-8)alkyl, carboxyl(C1-8)alkyl, amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), hydroxy(C1-8)alkyl, heterocyclyl(C1-8)alkyl, aryl(C1-8)alkyl and heteroaryl(C1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein heterocyclyl is optionally substituted with oxo)); with the proviso that, if A and E are selected from a hydrogen substituted carbon atom, then R2 is selected from the group consisting of —C2-8alkynyl-, —O—(C1-8)alkyl-O—, —O—(C2-8)alkenyl-O—, —O—(C2-8)alkynyl-O—, —C(O)—(C1-8)alkyl-C(O)— (wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, —C(O)O—(C1-8)alkyl, —C1-8alkyl-C(O)O—(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy, hydroxy(C1-8)alkyl and oxo; and, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaryl, heterocyclyl(C1-8)alkyl, aryl(C1-8)alkyl, heteroaryl(C1-8)alkyl, spirocycloalkyl and spiroheterocyclyl (wherein any of the foregoing cycloalkyl, heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein any of the foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyl (wherein cycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl), —(O—(CH2)1-6)1-5—O—, —O—(CH2)1-6—O—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—O—(CH2)1-6—O—, —(O—(CH2)1-6)1-5—NR6—, —O—(CH2)1-6—NR6—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—NR6—, —(O—(CH2)1-6)0-5—S—, —O—(CH2)1-6—S—(CH2)1-6—O—, —O—(CH2)1-6—O—(CH2)1-6—S—, —NR6—NR7—, —NR6—(CH2)1-6—NR7—, —NR6—(CH2)1-6—NR7—(CH2)1-6—NR8—, —NR9—C(O)—, —C(O)—NR9—, —C(O)—(CH2)0-6—NR6—(CH2)0-6—C(O)—, —NR6—(CH2)0-6—C(O)—(CH2)1-6—C(O)—(CH2)0-6—NR7—, —NR6—C(O)—NR7—, —NR6—C(NR7)—NR8—, —O—(CH2)1-6—NR6—(CH2)1-6—S—, —S—(CH2)1-6—NR6—(CH2)1-6—O—, —S—(CH2)1-6—NR6—(CH2)1-6—S— and —NR6—(CH2)1-6—S—(CH2)1-6—NR7— (wherein R6, R7 and R8 are independently selected from the group consisting of hydrogen, C1-8alkyl, C1-8alkoxy(C1-8)alkyl, carboxyl(C1-8)alkyl, amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), hydroxy(C1-8)alkyl, heterocyclyl(C1-8)alkyl, aryl(C1-8)alkyl and heteroaryl(C1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein heterocyclyl is optionally substituted with oxo); and, wherein R9 is selected from the group consisting of C1-8alkyl, C1-8alkoxy(C1-8)alkyl, carboxyl(C1-8)alkyl, amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), hydroxy(C1-8)alkyl, heterocyclyl(C1-8)alkyl, aryl(C1-8)alkyl and heteroaryl(C1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C1-8alkyl, C1-8alkoxy, C1-8alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl; and, wherein heterocyclyl is optionally substituted with oxo)); and,
R1 and R3 are independently selected from the group consisting of hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl (wherein alkyl, alkenyl and alkynyl are optionally substituted with a substituent selected from the group consisting of C1-8alkoxy, alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), (halo)1-3, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy, hydroxy(C1-8)alkyl and oxo), C1-8alkoxy, C1-8alkoxycarbonyl, (halo)1-3(C1-8)alkoxy, C1-8alkylthio, aryl, heteroaryl (wherein aryl and heteroaryl are optionally substituted with a substituent selected from the group consisting of C1-8alkyl, C1-8alkoxy, alkoxy(C1-8)alkyl, carboxyl, carboxyl(C1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), amino(C1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), halogen, (halo)1-3(C1-8)alkyl, (halo)1-3(C1-8)alkoxy, hydroxy and hydroxy(C1-8)alkyl), amino (substituted with a substituent independently selected from the group consisting of hydrogen and C1-4alkyl), cyano, halogen, hydroxy and nitro; and pharmaceutically acceptable salts thereof.
In one embodiment, a compound of Formula (III) is a compound selected from the group consisting of:










wherein all other variables are as previously defined; and, pharmaceutically acceptable salts thereof.
In one embodiment, a compound of Formula (III) is a compound selected from the group consisting of:




wherein all other variables are as previously defined; and, pharmaceutically acceptable salts thereof.
Compounds of Formula (III) are disclosed in commonly assigned U.S. Pat. No. 6,828,327, the complete disclosure of which is herein incorporated by reference.
An example of the invention includes a compound of Formula (III) wherein the compound is selected from the group consisting of:











Compound
Name
















1
6,7,9,10,12,13,15,16-octahydro-23H-5,26: 17,22-dimetheno-5H-



dipyrido[2,3-k: 3′,2′-q]pyrrolo[3,4-



n][1,4,7,10,19]trioxadiazacyclohenicosine-23,25(24H)-dione,


2
10,11,13,14,16,17,19,20,22,23-decahydro-9,4: 24,29-dimetheno-1H-



dipyrido[2,3-n: 3′,2′-t]pyrrolo[3,4-



q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione,


3
10,11,13,14,16,17,19,20,22,23,25,26-dodecahydro-9,4: 27,32-



dimetheno-1H-dipyrido[2,3-q: 3′,2′-w]pyrrolo[3,4-



t][1,4,7,10,13,16,25]pentaoxadiazacycloheptacosine-1,3(2H)-dione,


4
6,7,9,10,12,13-hexahydro-20H-5,23: 14,19-dimetheno-5H-



dibenzo[h,n]pyrrolo[3,4-k][1,4,7,16]dioxadiazacyclooctadecine-



20,22(21H)-dione,


5
6,7,9,10,12,13,15,16-octahydro-23H-5,26: 17,22-dimetheno-5H-



dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]trioxadiazacycloheneicosine-



23,25(24H)-dione,


6
10,11,13,14,16,17,19,20,22,23-decahydro-9,4: 24,29-dimetheno-1H-



dibenzo[n,t]pyrrolo[3,4-



q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione,


7
10,11,13,14,16,17,19,20,22,23,25,26-dodecahydro-9,4: 27,32-



dimetheno-1H-dibenzo[q,w]pyrrolo[3,4-



t][1,4,7,10,13,16,25]pentaoxadiazacycloheptacosine-1,3(2H)-dione,


8
12-hydro-6H,19H-5,22: 13,18: 7,11-trimethenopyrido[2,3-



j]pyrrolo[3,4-m][1,9]benzodiazacycloheptadecine-19,21(20H)-dione,


9
12-hydro-6H,19H-5,22: 13,18-dimetheno-7,11-nitrilopyrido[2,3-



j]pyrrolo[3,4-m][1,9]benzodiazacycloheptadecine-19,21(20H)-dione,


10
6,7,9,10,12,13-hexahydro-20H-5,23: 14,19-dimetheno-5H-



pyrido[2,3-k]pyrrolo[3,4-



n][4,7,1,10]benzodioxadiazacyclooctadecine-20,22(21H)-dione,


11
6,7,9,10,12,13,15,16-octahydro-23H-5,26: 17,22-dimetheno-5H-



pyrido[2,3-n]pyrrolo[3,4-



q][4,7,10,1,13]benzotrioxadiazacycloheneicosine-23,25(24H)-dione,


12
11-ethyl-6,7,10,11,12,13,15,16-octahydro-23H-5,26: 17,22-



dimetheno-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


13
6,7,10,11,12,13,15,16-octahydro-11-methyl-23H-5,26: 17,22-



dimetheno-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


14
6,7,10,11,12,13,15,16-octahydro-11-(1-methylethyl)-23H-



5,26: 17,22-dimetheno-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


15
7,8,9,10,11,12,13,14,15,16-decahydro-8,11,14-trimethyl-6H,23H-



5,26: 17,22-dimethenodibenzo[n,t]pyrrolo[3,4-



q][1,4,7,10,13]pentaazacycloheneicosine-23,25(24H)-dione,


16
6,7,10,11,12,13,15,16-octahydro-11-methyl-23H-5,26-metheno-



17,22-nitrilo-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


17
11-ethyl-6,7,10,11,12,13,15,16-octahydro-23H-5,26-metheno-17,22-



nitrilo-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


18
11-ethyl-6,7,10,11,12,13,15,16-octahydro-23H-5,26: 17,22-



dimetheno-5H,9H-dipyrido[2,3-k: 3′,2′-q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


19
6,7,9,10,12,13,15,16-octahydro-23H-5,26: 17,22-dimetheno-5H-



dipyrido[2,3-k: 3′,2′-q]pyrrolo[3,4-



n][1,7,4,10,19]dioxathiadiazacycloheneicosine-23,25(24H)-dione,


20
7,8,9,10,11,12,13,14,15,16-decahydro-(6H,23H-5,26: 17,22-



dimethenodipyrido[2,3-n: 3′,2′-t]pyrrolo[3,4-



q][1,7,13]triazacycloheneicosine-23,25(24H)-dione,


21
11-ethyl-7,8,9,10,11,12,13,14,15,16-decahydro-6H,23H-5,26: 17,22-



dimethenodipyrido[2,3-n: 3′,2′-t]pyrrolo[3,4-



q][1,7,13]triazacycloheneicosine-23,25(24H)-dione,


22
6,7,8,9,10,11,12,13,14,15-decahydro-22H-5,25: 16,21-dimetheno-



5H-dipyrido[2,3-m: 3′,2′-s]pyrrolo[3,4-p][1,6,12]triazacycloeicosine-



22,24(23H)-dione,


23
10-ethyl-6,7,8,9,10,11,12,13,14,15-decahydro-22H-5,25: 16,21-



dimetrieno-5H-dipyrido[2,3-m: 3′,2′-s]pyrrolo[3,4-



p][1,6,12]triazacycloeicosine-22,24(23H)-dione,


24
7,8,9,15,16,17,18-heptahydro-6H,25H-5,28: 19,24-dimetheno-10,14-



nitrilodipyrido[2,3-b: 3′,2′-h]pyrrolo[3,4-e][1,10]diazacyclotricosine-



25,27(26H)-dione,


25
7,8,9,10,11,13,14,15,16-nonahydro-6H,23H-5,26: 17,22-



dimetrienodipyrido[2,3-b: 3′,2′-h]pyrrolo[3,4-



e][1,10]diazacycloheneicosine-12,23,25(24H)-trione,


26
7,8,9,11,12,13,14-heptahydro-6H,21H-5,24: 15,20-



dimethenodipyrido[2,3-b: 3′,2′-h]pyrrolo[3,4-



e][1,10]diazacyclononadecine-10,21,23(22H)-trione,


27
6,7,8,9,10,11,12,13,14,15-decahydro-7,14-dihydroxy-(7R,14R)-22H-



5,25: 16,21-dimetheno-5H-dipyrido[2,3-b: 3′,2′-h]pyrrolo[3,4-



e][1,10]diazacycloeicosine-22,24(23H)-dione,


28
6,7,9,10,12,13-hexahydro-20H-5,23: 14,19-dimetheno-5H-



dipyrido[2,3-h: 3′,2′-n]pyrrolo[3,4-



k][1,4,7,16]dioxadiazacyclooctadecine-20,22(21H)-dione,


29
6,7,10,11,12,13,15,16-octahydro-11-(2-methoxyethyl)-23H-5,26-



metheno-17,22-nitrilo-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


30
6,7,10,11,12,13,15,16-octahydro-11-(2-hydroxyethyl)-23H-



5,26: 17,22-dimetheno-5H,9H-dibenzo[k,q]pyrrolo[3,4-



n][1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione, and


31
6,7,9,10,12,13,14,15,16,17-decahydro-14-methyl-24H-5,27: 18,23-



dimetheno-5H-dibenzo[l,r]pyrrolo[3,4-



o][1,4,7,11,20]dioxatriazacyclodocosine-24,26(25H)-dione.









An example of the invention includes a compound of Formula (III) wherein the compound is selected from the group consisting of:




Other examples of the invention include a compound selected from the group consisting of:











Compound
Name







 1a
To be provided


 2a
3-[1-[3-[(2-hydroxyethyl)methylamino]propyl]-1H-indazol-3-yl]-



4-[1-(3-pyridinyl)-1H-indol-3-yl]-1H-pyrrole-2,5-dione,


 3a
3,5-dichloro-N-[3-chloro-4-[(3,4,12,12a-tetrahydro-1H-



[1,4]thiazino[3,4-c][1,4]benzodiazepin-11(6H)-



yl)carbonyl]phenyl]-benzamide,


 4a
3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyridin-3-yl-1H-indol-



3-yl)-pyrrole-2,5-dione,


 5a
3-(2-methoxy-phenyl)-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-



2,5-dione,


 6a
6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-



pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile,


 7a
3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-imidazol-1-yl-



propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione,


 8a
3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-[1,2,3 ]triazol-1-yl-



propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione,


 9a
3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-



methyl-1H-pyrazol-3-yl)-pyrrole-2,5-dione,


10a
To be provided


11a
3-[1-(3-hydroxy-3-methyl-butyl)-1H-indazol-3-yl]-4-(1-pyridin-3-



yl-1H-indol-3-yl)-pyrrole-2,5-dione,


12a
3-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-4-(1-pyrimidin-5-yl-1H-



indol-3-yl)-pyrrole-2,5-dione,


13a
3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyrimidin-5-yl-1H-



indol-3-yl)-pyrrole-2,5-dione,


14a
(11Z)-8,9,10,13,14,15-hexahydro-2,6: 17,21-



di(metheno)pyrrolo[3,4-h][1,15,7] dioxazacyclotricosine-



22,24(1H,23H)-dione,


15a
3-(5-chloro-1-pyridin-3-yl-1H-indol-3-yl)-4-[1-(3-hydroxy-



propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione,


16a
3-(2-methoxy-phenyl)-4-[1-(3-methoxy-propyl)-1H-pyrrolo[3,2-



c]pyridin-3-yl]-pyrrole-2,5-dione,


17a
3-[1-(3-hydroxy-propyl)-1H-indazol-3-yl]-4-[1-(tetrahydro-pyran-



4-yl)-1H-indol-3-yl]-pyrrole-2,5-dione,


18a
2-{3-[4-(5-chloro-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-



1H-pyrrol-3-yl]-indazol-1-yl}-N-(2-hydroxy-ethyl)-acetamide,


19a
4-(3-chloro-phenyl)-6-(3-dimethylamino-propyl)-5,6-dihydro-4H-



2,4,6-triaza-cyclopenta[c]fluorine-1,3-dione,


20a
14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26: 17,22-



dimethenodibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione,


21a
14-benzyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26: 17,22-



di(metheno)dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,


22a
3-(1-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-yl)-4-[1-



(2-hydroxy-ethyl)-1H-indol-3-yl]-pyrrole-2,5-dione,


23a
6,7,8,9,10,11,12,13-octahydro-8,11-dimethyl-5,23: 14,19-



dimetheno-20H-dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10]tetraazacyclooctadecine-20,22(21H)-dione,


24a
7,8,9,10,12,13,16,17,18,19-decahydro-8,17-dimethyl-15H,26H-



5,29: 20,25-dimetheno-6H-dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19,22]dioxatetraazacyclotetracosine-26,28(27H)-dione,


25a
14-(2-furylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-



5,26: 17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,


26a
14-(2-thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-



5,26: 17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,


27a
14-(1-naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-



5,26: 17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,


28a
14-(pyridin-4-ylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-



5,26: 17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-



n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,


29a
3-[1-(2-{2-[2-(1,2,3,4-tetrahydro-naphthalen-1-ylamino)-ethoxy]-



ethoxy}-ethyl)-1H-indol-3-yl]-4-{1-[2-(1,2,3,4-tetrahydro-



naphthalen-1-ylamino)-ethyl]-1H-indol-3-yl}-pyrrole-2,5-dione,


30a
3-[1-(3-dimethylamino-phenyl)-1H-indol-3-yl]-4-[1-(2-hydroxy-



ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione,


31a
3-[5-chloro-1-(6-dimethylamino-pyridin-3-yl)-1H-indol-3-yl]-4-[1-



(2-hydroxy-ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione, and


32a
5-(5-chloro-3-{4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-2,5-dioxo-



2,5-dihydro-1H-pyrrol-3-yl}-indol-1-yl)-nicotinic acid methyl



ester.









Other examples of the invention include a compound selected from the group consisting of:

























Cells Suitable for Treatment According to the Methods of the Present Invention
Pluripotent cells, suitable for use in the present invention express at least one of the following pluripotency markers selected from the group consisting of: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, and Tra1-81.
In one embodiment, the pluripotent cells are embryonic stem cells. In an alternate embodiment, the pluripotent cells are cells expressing pluripotency markers derived from embryonic stem cells. In one embodiment, the embryonic stem cells are human.
Isolation, Expansion and Culture of Human Embryonic Stem Cells
Characterization of Human Embryonic Stem Cells:
Human embryonic stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of human embryonic stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1. Undifferentiated human embryonic stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
Another desirable phenotype of propagated human embryonic stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of human embryonic stem cells can be confirmed, for example, by injecting cells into SCID mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
Propagated human embryonic stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype”, which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
Sources of Human Embryonic Stem Cells:
Types of human embryonic stem cells that may be used include established lines of human embryonic cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BG01v (BresaGen, Athens, Ga.).
In one embodiment, Human embryonic stem cells are prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).
Culture of Human Embryonic Stem Cells:
In one embodiment, human embryonic stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of human embryonic stem cells without undergoing substantial differentiation. The growth of human embryonic stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type. Alternatively, the growth of human embryonic stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
In an alternate embodiment, human embryonic stem cells are initially cultured layer of feeder cells that support the human embryonic stem cells in various ways. The human embryonic are then transferred to a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of human embryonic stem cells without undergoing substantial differentiation.
Examples of conditioned media suitable for use in the present invention are disclosed in US20020072117, U.S. Pat. No. 6,642,048, WO2005014799, and Xu et al (Stem Cells 22: 972-980, 2004).
An example of a chemically defined medium suitable for use in the present invention may be found in US20070010011.
Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; (3-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
In one embodiment, the human embryonic stem cells are plated onto a suitable culture substrate that is treated prior to treatment according to the methods of the present invention. In one embodiment, the treatment is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a the suitable culture substrate is Matrigel® (Becton Dickenson). Matrigel® is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
Other extracellular matrix components and component mixtures are suitable as an alternative. This may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
The human embryonic stem cells are plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
Isolation, Expansion and Culture of Cells Expressing Pluripotency Markers that are Derived from Human Embryonic Stem Cells
In one embodiment, cells expressing pluripotency markers are derived from human embryonic stem cells by a method comprising the steps of:

    
    
        a. Culturing human embryonic stem cells,
        b. Differentiating the human embryonic stem cells into cells expressing markers characteristic of definitive endoderm cells, and
        c. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix prior to culturing the cells.
    
    


In one embodiment, cells expressing pluripotency markers are derived from human embryonic stem cells by a method comprising the steps of:

    
    
        a. Culturing human embryonic stem cells, and
        b. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.
    
    


Cell Culture Under Hypoxic Conditions on a Tissue Culture Substrate that is not Pre-Treated with a Protein or an Extracellular Matrix
In one embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 1 to about 20 days. In an alternate embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 5 to about 20 days. In an alternate embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 15 days.
In one embodiment, the hypoxic condition is about 1% O2 to about 20% O2. In an alternate embodiment, the hypoxic condition is about 2% O2 to about 10% O2. In an alternate embodiment, the hypoxic condition is about 3% O2.
The cells may be cultured, under hypoxic conditions on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum, activin A, and a Wnt ligand. Alternatively, the medium may also contain IGF-1.
The culture medium may have a serum concentration in the range of about 2% to about 5%. In an alternate embodiment, the serum concentration may be about 2%.
Activin A may be used at a concentration from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 μg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
The Wnt ligand may be used at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the Wnt ligand may be used at a concentration of about 10 ng/ml to about 100 ng/ml. In one embodiment, the concentration of the Wnt ligand is about 20 ng/ml.
IGF-1 may be used at a concentration of about 1 ng/ml to about 100 ng/ml. In an alternate embodiment, the IGF-1 may be used at a concentration of about 10 ng/ml to about 100 ng/ml. In one embodiment, the concentration of IGF-1 is about 50 ng/ml.
The cells expressing pluripotency markers derived by the methods of the present invention are capable of expansion in culture under hypoxic conditions, on tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.
The cells expressing pluripotency markers derived by the methods of the present invention express at least one of the following pluripotency markers selected from the group consisting of: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, and Tra1-81.
Further Differentiation of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art.
For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 24: 1392-1401, (2006).
Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of Pdx1, HNF-1beta, PTF1a, HNF-6, HB9 and PROX1. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
Further Differentiation of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art.
For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1, Pax-4, Ngn-3, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.
In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdx1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Is1-1, HNF-3 beta, MAFA, Pax4, and Pax6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.
Detection of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
For example, characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.
After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage.
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endoderm lineage. Pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1beta.
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage. Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN-3, NeuroD, Islet-1.
Markers characteristic of cells of the β cell lineage are well known to those skilled in the art, and additional markers characteristic of the β cell lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the 3-cell lineage. β cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, Pdx1 (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Is11, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3beta, and MafA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the β cell lineage.
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
The present invention is further illustrated, but not limited by, the following examples.
Example 1
Human Embryonic Stem Cell Culture
Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
The human embryonic stem cell lines H1, H7 and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2×104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO2/within a humidified tissue culture incubator. When confluent (approximately 5-7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surface using a 5-ml pipette. Cells were spun at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells in fresh culture medium.
In parallel, H1, H7, and H9 human embryonic stem cells were also seeded on plates coated with a 1:30 dilution of growth factor reduced MATRIGELTM (BD Biosciences) and cultured in MEF-conditioned media supplemented with 8 ng/ml bFGF. The cells cultured on MATRIGELTM were routinely passaged with collagenase IV (Invitrogen/GIBCO), Dispase (BD Biosciences) or Liberase enzyme (Source). Some of the human embryonic stem cell cultures were incubated under hypoxic conditions (approximately 3% O2).
Example 2
Derivation and Culture of Cells Expressing Pluripotency Markers, Derived from Human Embryonic Stem Cells
Cells from the human embryonic stem cell lines H1 and H9 various passages (Passage 30-54) were cultured under hypoxic conditions (approximately 3% O2) for at least three passages. The cells were cultured in MEF-CM supplemented with 8 ng/ml of bFGF and plated on MATRIGEL coated plates according to Example 1.
Cells were then treated with DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml WNT-3a (Catalog#1324-WN-002, R&D Systems, MN), and 100 ng/ml Activin-A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml Activin-A (AA) for an additional 3 to 4 days. This protocol resulted in significant upregulation of definitive endoderm markers.
The cells were then treated with TrypLETM Express solution (Invitrogen, CA) for 5 mins. Released cells were resuspended in DMEM-F12+2% FBS medium, recovered by centrifugation, and counted using a hemocytometer. The released cells were seeded at 1000-10,000 cells/cm2 on tissue culture polystyrene (TCPS) treated flasks and cultured in DMEM-F12+2% FBS+100 ng/ml activin-A+20 ng/ml WNT-3A under hypoxic conditions (approximately 3% O2) at 37° C. in standard tissue culture incubator. The TCPS flaks were not coated with MATRIGEL or other extarcellular matrix proteins. The media was changed daily. In some cultures, the media was further supplemented with 10-50 ng/ml of IGF-I (insulin growth factor-I from R&D Systems, MN) or 1×ITS (Insulin, transferrin, and selenium from Invitrogen, Ca). In some of the culture conditions the basal media (DM-F12+2% FBS) was further supplemented with 0.1 mM mercaptoethanol (Invitrogen, CA) and non-essential amino acids (1×, NEAA from Invitrogen, CA).
Following 5 to 15 days of culturing, distinct cell colonies appeared surrounded by a large number of enlarged cells that appear to be in senescence. At approximately 50 to 60% confluency, the cultures were passaged by exposure to TrypLETM Express solution for 5 mins at room temperature. The released cells were resuspended in DMEM-F12+2% FBS medium, recovered by centrifugation, and seeded at 10,000 cells/cm2 on tissue culture polystyrene (TCPS) treated flasks in DMEM-F12+2% FBS+100 ng/ml activin-A+20 ng/ml WNT-3A+/−50 ng/ml of IGF-I. This media will be further referred to as the “growth media”.
Example 3
Derivation of Cells Expressing Pluripotency Markers from a Single Cell Suspension of Human Embryonic Stem Cells
Cells from the human embryonic stem cell lines H1 P33 and H9 P45 were cultured under hypoxic conditions (approximately 3% O2) for at least three passages. The cells were cultured in MEF-CM supplemented with 8 ng/ml of bFGF and plated on MATRIGEL coated plates according to Example 1. At approximately 60% confluency, the cultures were exposed to TrypLETM Express solution (Invitrogen, CA) for 5 mins. Released cells were resuspended in DMEM-F12+2% FBS medium, recovered by centrifugation, and counted using a hemocytometer. The released cells were seeded at 1000 to 10,000 cells/cm2 on tissue culture polystyrene (TCPS) treated flasks and cultured in DM-F12+2% FBS+100 ng/ml activin-A+20 ng/ml WNT-3A+50 ng/ml of IGF-I+0.1 mM mercaptoethanol (Invitrogen, CA) and non-essential amino acids (1×, NEAA from Invitrogen, CA) under hypoxic conditions (approximately 3% O2) at 37° C. in standard tissue culture incubator. The TCPS flasks were not coated with MATRIGEL or other extarcellular matrix proteins. The media was changed daily. The first passage cells are referred to as P1.
Example 4
Various Growth Media Useful for Expansion of Cells Expressing Pluripotency Markers Derived from Human Embryonic Stem Cells
Cells expressing pluripotency markers derived from human embryonic stem cells have been successfully cultured in the following media compositions for at least 2-30 passages:

    
    
        1. DM-F12+2% FBS+100 ng/ml AA+20 ng/ml WNT-3A
        2. DM-F12+2% FBS+100 ng/ml AA+20 ng/ml WNT-3A+50 ng/ml IGF-I
        3. DM-F12+2% FBS+100 ng/ml AA+20 ng/ml WNT-3A+10 ng/ml IGF-I
        4. DM-F12+2% FBS+50 ng/ml AA+20 ng/ml WNT-3A+50 ng/ml IGF-I
        5. DM-F12+2% FBS+50 ng/ml AA+10 ng/ml WNT-3A+50 ng/ml IGF-I
        6. DM-F12+2% FBS+50 ng/ml AA+20 ng/ml WNT-3A+10 ng/ml IGF-I
        7. DM-F12+2% FBS+100 ng/ml AA+10 ng/ml WNT-3A+10 ng/ml IGF-I
        8. HEScGRO defined media (Chemicon, CA)
    
    


The basal component of the above listed media may be replaced with similar media such as, RPMI, DMEM, CRML, KnockoutTMDMEM, and F12.
Example 4
Effects of Inhibitors of GSK-3β Enzyme Activity on the Viability of Cells Expressing Pluripotency Markers
Derivation and maintenance of cells expressing pluripotency makers was conducted as has been described in Example 2. Cells were grown in DMEM:F12 supplemented with 2% FCS (Invitrogen), 100 ng/ml Activin A, 20 ng/ml Wnt-3a, and 50 ng/ml IGF (R&D Biosystems). Cells were seeded at a density of 10,000 cells/cm2 on Falcon polystyrene flasks and grown in monolayer culture at 37° C., 5% CO2, low oxygen. After reaching 60-70% confluence, cells were passed by washing the monolayer with PBS and incubating with TrypLE (Invitrogen) for 3-5 minutes to allow detachment and single cell dispersal.
Screening was conducted using test compounds from a proprietary library of small molecules selected for their ability to inhibit GSK-3B enzyme activity. Compounds from this library were made available as 1 mM stocks, in a 96-well plate format in 50 mM HEPES, 30% DMSO. For assay, cells expressing pluripotency markers were washed, counted, and plated in normal culture medium at a seeding density of 20,000 cells per well in 96-well clear-bottom, dark-well plates (Costar). This seeding density was previously determined to yield optimal monolayer formation in overnight culture. On the following day, culture medium was removed, cell monolayers were rinsed three times with PBS, and test compounds were added to the wells in 80 μl aliquots, each diluted into assay medium at a final assay concentration of 10 μM. On day 2 of the assay, medium was removed from each well and replaced with a fresh aliquot of test compounds diluted into assay medium. Assay medium on days 1 and 2 of culture consisted of DMEM:F12 supplemented with 0.5% FCS and 100 ng/ml Activin A. On days 3 and 4 of culture, medium was removed from each well and replaced with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A (no test compound). On day 4 of assay, 15 μl of MTS (Promega) was added to each well and plates were incubated at 37° C. for 1.5 to 4 hours prior to reading optical density at 490 nm on a SpectraMax (Molecular Devices) instrument. Statistical measures consisting of mean, standard deviation, and coefficient of variation were calculated for each duplicate set. Toxicity was calculated for each test well relative to a positive control (wells treated with Activin A and Wnt3a on days 1 and 2 of culture).
Table II is a compilation of all screening results. Cells expressing pluripotency markers were plated initially as a confluent monolayer in this assay; hence, the results are representative of a toxicity measure over the four-day culture period. Results are expressed as percentage viability of control, and demonstrate variable toxicity for some compounds at the 10 μM screening concentration used. A larger proportion of the compounds have minimal or no measurable toxicity in this cell-based assay.
A small panel of select compounds was repeat tested over a narrow dose titration range, again using cells expressing pluripotency markers in a similar assay as described above. Table III is a summary of these results, demonstrating variable dose titration effects for a range of toxic and non-toxic compounds.
Example 5
Effects of Inhibitors of GSK-3β Enzyme Activity on the Differentiation and Proliferation of Human Embryonic Stem Cells Determined Using a High Content Screening Assay
Maintenance of human embryonic stem cells (H9 line) was conducted as described in Example 1. Colonies of cells were maintained in an undifferentiated, pluripotent state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37° C. followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual collagenase. Cell clusters were split at a 1:3 ratio for routine maintenance culture or a 1:1 ratio for immediate assay. The human embryonic stem cell lines used were maintained at passage numbers less than passage 50 and routinely evaluated for normal karyotypic phenotype and absence of mycoplasma contamination.
Cell clusters used in the assay were evenly resuspended in normal culture medium and plated onto MATRIGEL-coated 96-well Packard VIEWPLATES (PerkinElmer) in volumes of 100 μl/well. MEF conditioned medium supplemented with 8 ng/ml bFGF was used for initial plating and recovery. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Plates were maintained at 37° C., 5% CO2 in a humidified box throughout the duration of assay.
Screening was conducted using test compounds from a proprietary library of small molecules selected for their ability to inhibit GSK-3B enzyme activity. Compounds from this library were made available as 1 mM stocks, in a 96-well plate format in 50 mM HEPES, 30% DMSO. Screening compounds were tested in triplicate or duplicate sets. Primary screening assays were initiated by aspirating culture medium from each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 80 to 100 μl per well were added back containing DMEM:F12 base medium (Invitrogen) supplemented with 0.5% FCS (HyClone) and 100 ng/ml activin A (R&D Biosystems) plus 101.1M test compound. Positive control wells contained the same base medium, substituting 10-20 ng/ml Wnt3a (R&D Biosystems) for the test compound. Negative control wells contained base medium with 0.5% FCS and activin A alone (AA only) or alternatively, 0.5% FCS without activin A or Wnt3a (no treatment). Wells were aspirated and fed again with identical solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and converted to DMEM:F12 supplemented with 2% FCS and 100 ng/ml activin A (without test compound or Wnt3a); parallel negative control wells were maintained in DMEM:F12 base medium with 2% FCS and activin A (AA only) or alternatively, 2% FCS without activin A (no treatment).
At the end of culture, cells in 96-well plates were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. Alternatively, cells were fixed with ice cold 70% ethanol overnight at −20° C., washed three times with PBS, and then permeabilized with Triton X-100 for 5 minutes at 4° C. After fixing and permeabilizing, cells were washed again three times with PBS and then blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibodies (goat anti-human Sox17 and goat anti-human HNF-3beta; R&D Systems) were diluted 1:100 in 4% chicken serum and added to cells for one hour at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat IgG; Molecular Probes) was diluted 1:200 in PBS and added after washing the cells three times with PBS. To counterstain nuclei, 5 mM Draq5 (Alexis Biochemicals) was added for five minutes at room temperature. Cells were washed once with PBS and left in 100 ml/well PBS for imaging.
Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Draq5 and Alexa Fluor 488. Exposure times were optimized using a positive control wells and wells with secondary only for untreated negative controls. Twelve fields per well were obtained to compensate for any cell loss during the treatment and staining procedures. Total cell numbers and total cell intensity for Sox-17 and HNF-3beta were measured using the IN Cell Developer Toolbox 1.6 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for replicates. Total protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000 and form factors greater than or equal to 0.4. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data was calculated for averages and standard deviation for each replicate set.
Table IV is a representative summary of all screening results. Table V is a list of hits from this screening. Strong hits are defined as greater than or equal to 120% of control values; moderate hits are defined as falling within the interval of 60-120% of control values. A significant number of compounds induce both a proliferative response in this assay. In parallel, a significant number of compounds induce differentiation in this assay, as measured by the protein expression of Sox17 and Hnf-3b transcription factors.
Example 6
Effects of Inhibitors of GSK-3β Enzyme Activity on the Proliferation of Human Embryonic Stem Cells Determined Using a Plate Reader Assay
Maintenance of human embryonic stem cells (H9 or H1 lines) was conducted as described in Example 1. Colonies of cells were maintained in an undifferentiated, pluripotent state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37° C. followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment and washed to remove residual collagenase. Cell clusters were split at a ratio of 1:3 monolayer area for routine culture or a 1:1 ratio for immediate assay. The human embryonis stem cell lines used for these examples were maintained at passage numbers less than 50 and routinely evaluated for normal karyotypic phenotype as well as absence of mycoplasm contamination.
Cell clusters used in assay were evenly resuspended in normal culture medium and plated into MATRIGEL-coated 96-well Packard VIEWPLATES (PerkinElmer) in volumes of 100 μl/well. MEF conditioned medium supplemented with 8 ng/ml bFGF) was used for initial plating and recovery. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Plates were maintained at 37° C. in a humidified box, 5% CO2 throughout the duration of assay.
Primary screening assays were initiated by aspirating culture medium from each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 80-100 μl per well were added back containing DMEM:F12 base medium (Invitrogen) supplemented with 0.5% FCS (HyClone) and 100 ng/ml activin A (R&D Biosystems) and 10 μM test compound. Positive control wells contained the same medium substituting 10-20 ng/ml Wnt3a (R&D Biosystems). Negative control wells contained base medium with 0.5% FCS without activin A or Wnt3a. Screening compounds were tested in triplicate. Wells were aspirated and fed again with identical solutions on day 2 of the assay. On days 3 and 4, all assay wells were aspirated and converted to DMEM:F12 supplemented with 2% FCS and 100 ng/ml activin A with the exception of negative control wells which were maintained in DMEM:F12 base medium with 2% FCS.
On day 4 of assay, 15-20111 of MTS (Promega) was added to each well and plates were incubated at 37° C. for 1.5 to 4 hours. Densitometric readings at OD490 were determined using a Molecular Devices spectrophotometer plate reader. Average readings for replicate sets were calculated along with standard deviation and coefficient of variation. Experimental wells were compared to the Activin A/Wnt3a positive control to calculate a percent control value as a measure of proliferation.
Table VI is a representative summary of all screening results. Table VII is a list of hits from this screening. Strong hits are defined as greater than or equal to 120% of control values; moderate hits are defined as falling within the interval of 60-120% of control values. A significant number of compounds induce a proliferative response in this assay.
Example 7
Effects of GSK-3β Enzyme Inhibitors on the Differentiation and Proliferation of Human Embryonic Stem Cells: Dose Titration of Lead Compounds
It was important to confirm the activity of hits identified from primary screening and further analyze the range of activity by dose titration. New samples of a selective subset of primary screening hits were obtained as dry powders, solubilized to make fresh stock reagents, and diluted into secondary confirmation assays to evaluate effects on human embryonic stem cells.
Culture of two human embryonic stem cells (H1 and H9) was conducted as described in Example 1. Colonies of cells were maintained in an undifferentiated, pluripotent state on MatrigelTM (Invitrogen) coated polystyrene plastic, using a 1:30 dilution of MatrigelTM in DMEM:F12 to coat the surface. Cells were split by enzymatic passage every four days on average. Passage was performed by exposing cell monolayers to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 60 minutes at 37° C. followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, then washed to remove residual collagenase. Cell clusters were split at a 1:3 ratio for maintenance culture or a 1:1 ratio for subsequent assay. The human embryonic stem cell lines were maintained at less than passage 50 and routinely evaluated for normal karyotypic phenotype and absence of mycoplasma contamination.
Preparation of Cells for Assay:
Cell clusters of the H1 or H9 human embryonic stem cell lines used in the assay were evenly resuspended in culture medium and plated onto MatrigelTM-coated 96-well Packard VIEWPLATES (PerkinElmer) in volumes of 100 μl/well. MEF conditioned medium supplemented with 8 ng/ml bFGF was used for initial plating and expansion. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37° C., 5% CO2 in a humidified box for the duration of assay.
Preparation of Compounds and Assay Medium:
A subset of hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Twenty compounds available as dry powders were solubilized as 10 mM stocks in DMSO and stored dessicated at −20° C. until use Immediately prior to assay, compound stocks were diluted 1:1000 to make 10 μM test compound in DMEM:F12 base medium (Invitrogen) supplemented with 0.5% FCS (HyClone) and 100 ng/ml Activin A (R&D Biosystems). This was further diluted two-fold in series to make a seven point dilution curve for each compound, also in DMEM:F12 base medium with 0.5% FCS and 100 ng/ml Activin A.
Secondary Screening Assay:
Assay was initiated by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 100 μl per well were added back containing medium with 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium with 0.5% FCS and with 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS.
Assay Evaluation:
At the end of culture, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and then blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibodies (goat anti-human Sox17; R&D Systems) were diluted 1:100 in 4% chicken serum and added to the cells for one hour at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat IgG; Molecular Probes) was diluted 1:200 in PBS and added to each well after washing the cells three times with PBS. To counterstain nuclei, 2 μg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μl/well PBS for imaging.
Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone as an untreated negative control. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total Sox-17 intensity were obtained for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for each replicate data set. Total Sox17 protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000 and form factors greater than or equal to 0.4. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each replicate set.
Results
Results are shown for eight GSK-3B enzyme inhibitors where activity was confirmed and potency was determined by titration in this secondary assay. Data presented show compound effects on cell number and Sox17 intensity where respective data points were averaged from a duplicate set and mined for each parameter from identical fields and wells. In this example, Sox17 expression is indicative of definitive endoderm differentiation. Results for cell number and Sox17 intensity, respectively, using the H1 human embryonic stem cell line are shown in Tables VIII and IX. Results for the H9 human embryonic stem cell line are shown in Tables X and XI. Positive control values were normalized to 1.000 for cell number and Sox17 intensity. Negative control values were less-than 0.388 for cell number and less-than 0.065 for Sox17 intensity with both cell lines. A graphic portrayal of these data, comparing both human embryonic stem cell lines and including a dose titration of each compound, is provided in FIGS. 1 to 8. Cell number is presented in panel A; Sox 17 intensity is shown in panel B. These data confirm that each compound can promote hES cell proliferation and definitive endoderm differentiation and identify an optimal range of activity.
Example 8
Effects of GSK-3β Enzyme Inhibitors on the Expression of Additional Markers Associated with Definitive Endoderm
It was important to demonstrate that lead compounds could also induce other markers indicative of definitive endoderm differentiation, in addition to the transcription factor Sox17. A select subset of hits was tested for their ability to promote expression of CXCR4, a surface receptor protein, and HNF-3 beta, a transcription factor also associated with definitive endoderm differentiation.
Preparation of Cells for Assay:
Cell clusters from the H1 human embryonis stem cell line used in the assay were evenly resuspended in culture medium and plated onto MATRIGELTM-coated (1:30 dilution) 6-well plates (Corning) in volumes of 2 ml/well. MEF conditioned medium supplemented with 8 ng/ml bFGF was used for initial plating and expansion. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37° C., 5% CO2 for the duration of assay.
Preparation of Compounds and Assay Medium:
A subset of seven hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Neat compounds were solubilized as 10 mM stocks in DMSO and stored dessicated at −20° C. until use. Immediately prior to assay, compound stocks were diluted to a final concentration ranging between 1 μM and 5 μM in DMEM:F12 base medium (Invitrogen) supplemented with 0.5% FCS (HyClone) and 100 ng/ml Activin A (R&D Biosystems).
Assay:
The assay was initiated by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 2 ml per well were added back containing medium with 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium and 0.5% FCS with 100 ng/ml Activin A and 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS.
Assay Evaluation:
At the end of culture, cell monolayers were washed with PBS and harvested from culture plates by incubating 5 minutes with TrypLETM Express solution (Invitrogen, CA). Cells were resuspended in MEF conditioned medium and split into two equal samples. One set of samples was further stained with various fluorescent labeled antibodies and subjected to flow cytometric (FACS) analysis. A second parallel set of samples was subjected to quantitative PCR.
Cells for FACS analysis were washed into PBS and blocked for 15 minutes at 4° C. in 0. 125% human gamma-globulin (Sigma cat# G-4386) diluted in PBS and BD FACS staining buffer. Aliquots of cells (approximately 105 cells each) were stained for 30 minutes at 4° C. with antibodies directly conjugated to a fluorescent tag and having specificity for CD9 PE (BD#555372), CD99 PE (Caltag#MHCD9904), or CXCR-4 APC (R&D Systems cat# FAB173A). After a series of washes in BD FACS staining buffer, cells were stained with 7-AAD (BD#559925) to assess viability and analyzed on a BD FACS Array instrument (BD Biosciences), collecting at least 10,000 events. Mouse IgG1k isotype control antibodies for both PE and APC were used to gate percent positive cells.
Cells for quantitative PCR were processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, Inc.), and high-quality RNA was eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc. (ABI, CA) high capacity cDNA archive kit.
Unless otherwise stated, all reagents for real-time PCR amplification and quantitation were purchased from ABI. Real-time PCR reactions were performed using the ABI PRISM 7900 Sequence Detection System. TAQMAN UNIVERSAL PCR MASTER MIX (ABI, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μl. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by ABI. Primer and probe sets are listed as follows: CXCR4 (Hs00237052), GAPDH (4310884E), HNF3b (Hs00232764), SOX17 (probe part #450025, forward and reverse part #4304971).
After an initial incubation at 50° C. for 2 min followed by 95° C. for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95° C. for 15 sec followed by an annealing/extension step at 60° C. for 1 min. Data analysis was carried out using GENEAMP 7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ΔCt). The normalized amount of target was calculated as 2-ΔCt, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.
Results
FIG. 9 displays the FACS analysis of percent positive cells expressing CXCR4 surface receptor after treatment with various GSK3 inhibitors. Two concentrations of each compound, ranging between 1 μM and 5 μM, are shown relative to an untreated population of cells (negative control) or cells treated with Activin A and Wnt3 (positive control). FIG. 10 panels a, b, and c show real-time PCR data for CXCR4, Sox17, and HNF3beta, which are also considered to be markers of definitive endoderm. Both FACS and real-time PCR analysis demonstrate a significant increase in each of these markers observed in differentiated cells relative to untreated control cells. Expression levels of these definitive endoderm markers were equivalent in some cases to the positive control, demonstrating that a GSK3 inhibitor can substitute for Wnt3a at this stage of differentiation.
Example 9
Effects of GSK-3β Enzyme Inhibitors on the Formation of Pancreatic Endoderm
It was important to demonstrate that treatment with GSK3β inhibitors during induction of definitive endoderm did not prevent the subsequent differentiation of other cell types, such as pancreatic endoderm, for example. A select subset of hits was tested for their ability to promote expression of PDX1 and HNF6, key transcription factors associated with pancreatic endoderm.
Maintenance of human embryonic stem cells (H1 and H9 lines) was conducted as described in Example 1. Colonies of cells were maintained in an undifferentiated, pluripotent state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37° C., followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual collagenase. Cell clusters were split at a 1:3 ratio for routine maintenance culture or a 1:1 ratio for subsequent assay. The human embryonic stem cell lines used were maintained at less than passage 50 and routinely evaluated for normal karyotypic phenotype and absence of mycoplasma contamination.
Cell Preparation of Assay:
Cell clusters of the H1 human embryonis stem cell line used in the assay were evenly resuspended in culture medium and plated onto MATRIGELTM-coated (1:30 dilution) 24-well plates (black well; Arctic White) in volumes of 1 ml/well. MEF conditioned medium supplemented with 8 ng/ml bFGF was used for initial plating and expansion. In a second experiment, clusters of hES cells from the H9 line were plated in 96-well plates on mouse embryonic feeder (MEF) layers, previously inactivated by treating with mitomycin C (Sigma Chemical Co). Culture medium for hES cells on MEF monolayers consisted of DMEM:F12 with 20% Knockout Serum Replacer (Invitrogen) supplemented with minimal essential amino acids (Invitrogen), L-glutamine, and 2-mercaptoethanol. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37° C., 5% CO2 for the duration of assay.
Preparation of Compounds and Assay Medium:
A subset of eight hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Neat compounds were solubilized as 10 mM stocks in DMSO and stored dessicated at −20° C. until use. Immediately prior to assay, compound stocks were diluted to a final concentration ranging between 1 μM and 5 μM in base medium with additives.
Assay:
In this assay, GSK3 inhibitors were included only on days 1 and 2 of the definitive endoderm differentiation step, substituting for Wnt3a. Embryonic stem cell cultures on MATRIGELTM were initiated as described in Examples 7 and 8 above by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes (0.5 ml per well for 24-well plates, 100 ill per well for 96-well plates) were added containing DMEM:F12 medium with) 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium with 0.5% FCS and with 100 ng/ml Activin A and 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.2511M KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were then treated for an additional four days, feeding daily with DMEM:F12 containing 1% B27 (Invitrogen), 0.2511M KAAD cyclopamine, 2 μM Retinoic Acid (RA; Sigma-Aldrich) and 20 ng/ml FGF7. Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 2% FCS (stage 2) or 1% B27 (stage 3) and without any other additives.
Parallel cultures of H9 human embryonic cells were grown on MEF feeder layers, and differentiated to pancreatic endoderm. Definitive endoderm differentiation was achieved by culturing the cells in medium consisting of RPMI-1640 (Invitrogen) containing no serum on day 1 and 0.2% FCS on days 2 and 3 along with different concentrations of inhibitor compounds and 100 ng/ml Activin A. Positive control wells contained the same base medium (with or without serum) with 100 ng/ml Activin A and 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with or without serum, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On day 3, all assay wells were aspirated and fed with RPMI-1640 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound and Wnt3a. Parallel negative control wells were maintained on day 3 in RPMI-1640 base medium with 2% FCS. Cells were differentiated into pancreatic endoderm by treating the cells for four days, feeding daily with RPMI-1640 base medium containing 2% FCS with 0.25 mM KAAD cyclopamine (EMD Biosciences) and 50 ng/ml FGF10 (R&D Biosystems). Subsequently, cells were treated for three days duration, feeding daily with RPMI-1640 containing 1% B27 (Invitrogen), 0.25 mM KAAD cyclopamine, 2 mM Retinoic Acid (RA; Sigma-Aldrich) and 50 ng/ml FGF10. Parallel negative control wells were maintained throughout in RPMI-1640 base medium with 2% FCS (stage 2) or 1% B27 (stage 3) and without any other additives.
Assay Evaluation:
At the end the differentiation, cells were examined as described in Example 8 for gene expression by real-time PCR. For high content fluorescence staining, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (goat anti-human Pdx1; Santa Cruz) was diluted 1:100 in 4% chicken serum and added to cells for two hours at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat IgG; Molecular Probes) was diluted 1:200 in PBS and added to each well after washing the cells three times with PBS. To counterstain nuclei, 2 μg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μl/well PBS for imaging.
Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total Pdx1 intensity were obtained for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for each replicate data set. Total Pdx1 protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each replicate set.
Cells for quantitative PCR were lysed in RLT buffer (Qiagen) and then processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, Inc.), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc. (ABI, CA) high capacity cDNA archive kit.
Unless otherwise stated, all reagents for real-time PCR amplification and quantitation were purchased from ABI. Real-time PCR reactions were performed using the ABI PRISM 7900 Sequence Detection System. TAQMAN UNIVERSAL PCR MASTER MIX was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μl. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by ABI. Primer and probe sets are listed as follows: PDX1 (Hs00236830_ml), GAPDH (4310884E), and HNF6 (Hs00413554_ml).
After an initial incubation at 50° C. for 2 min followed by 95° C. for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95° C. for 15 sec followed by an annealing/extension step at 60° C. for 1 min. Data analysis was carried out using GENEAMPÒ7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ΔCt). The normalized amount of target was calculated as 2-ΔCt, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.
Results
Results are shown for eight GSK-3β enzyme inhibitors. Data presented in FIG. 11 from high content analysis show effects on cell number (panel A) and Pdx1 intensity (panel B) for the H1 hES cell line, where respective data points were averaged from a duplicate sample set and mined for each parameter from identical fields and wells. Data presented in FIG. 12 from real-time PCR show effects of these small molecule inhibitors on induced expression of two transcription factors, Pdx1 and HNF6. In these examples, Pdx1 and HNF6 expression are indicative of pancreatic endoderm differentiation. GSK3β inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment; resulting cells sustain a capacity to form pancreatic endoderm during later sequential stages of differentiation.
Example 10
Effects of GSK-3β Enzyme Inhibitors on the Formation of Pancreatic Endocrine Cells
It was important to demonstrate that treatment with GSK3 inhibitors during induction of definitive endoderm did not prevent the subsequent differentiation of other cell types, such as pancreatic endocrine cells, or insulin producing cells, for example. A select subset of hits was tested for their ability to promote expression of pancreatic hormones.
Cell Preparation for Assay:
Pancreatic endoderm cells obtained according to the methods described in Example 9 (cultured on 96-wellplates and 24-well plates) were subsequently subjected to agents that cause the cells to differentiate into pancreatic hormone expressing cells.
Assay for cultures of the H1 human embryonic stem cell line on MATRIGELTM was initiated as described in Examples 7-9 above by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes (0.5 ml per well for 24-well plates, 100 μl per well for 96-well plates) were added containing medium with 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium and 0.5% FCS with 100 ng/ml Activin A and 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3, 4, and 5, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3, 4, and 5 in DMEM:F12 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.25 μM KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were subsequently treated for four days, feeding daily with DMEM:F12 containing 1% B27 (Invitrogen), 0.25 μM KAAD cyclopamine, 2 μM Retinoic Acid (RA; Sigma-Aldrich) and 20 ng/ml FGF7. Parallel negative control wells during stages 2 and 3 were maintained throughout in DMEM:F12 base medium with 2% FCS or 1% B27 and without any other additives. After formation of pancreatic endoderm, cells were treated further for six days duration, feeding daily with DMEM:F12 base medium containing 1% B27 with 1 μM DAPT (gamma secretase inhibitor: EMD Biosciences) and 50 ng/ml Exendin 4 (Sigma-Aldrich). Cells were then treated for another three days duration, feeding daily with DMEM:F12 base medium containing 1% B27, 50 ng/ml Exendin 4, 50 ng/ml IGF (R&D Biosystems) and 50 ng/ml HGF (R&D Biosystems). Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 1% B27 and without any other additives.
Assay Evaluation:
At the end of culture, cells were treated as in Examples 7 and 8 above for evaluation by high content analysis or real-time PCR.
For high content fluorescence staining, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (guinea pig anti-swine insulin, cross-reactive with human insulin; DakoCytomation) was diluted 1:500 in 4% goat serum and added to cells for one hour at room temperature. Cells were washed three times with PBS and then stained with Alexa Fluor 488 conjugated secondary antibody (goat anti-guinea pig IgG; Molecular Probes) diluted 1:100 in 4% goat serum. To counterstain nuclei, 2 μg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μl/well PBS for imaging.
Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total insulin intensity were obtained for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for each replicate data set. Total insulin protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each triplicate set.
Cells for quantitative PCR were lysed in RLT buffer (Qiagen) and then processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc. (ABI, CA) high capacity cDNA archive kit.
Unless otherwise stated, all reagents for real-time PCR amplification and quantitation were purchased from ABI. Real-time PCR reactions were performed using the ABI PRISM® 7900 Sequence Detection System. TAQMAN® UNIVERSAL PCR MASTER MIX® (ABI, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μl. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN®probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by ABI. Primer and probe sets are listed as follows: PDX1 (Hs00236830_ml), Insulin (Hs00355773), and GAPDH (4310884E).
After an initial incubation at 50° C. for 2 min followed by 95° C. for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95° C. for 15 sec followed by an annealing/extension step at 60° C. for 1 min. Data analysis was carried out using GENEAMP®7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ΔCt). The normalized amount of target was calculated as 2−Δct, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.
Results
Results are shown for eight GSK-3B enzyme inhibitors. Data presented in FIG. 13 from high content analysis show compound effects on cell number (panel A) and insulin intensity (panel B) for the H1 hES cell line where respective data points were averaged from a triplicate set and mined for each parameter from identical fields and wells. Data presented in FIG. 14 from real-time PCR show compound effects for Pdx1 and insulin. In these examples, Pdx1 and insulin expression are indicative of pancreatic endoderm differentiation and generation of hormonal positive cells. Selective GSK3β inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment and can induce and sustain pancreatic beta cell formation during later sequential stages of differentiation, as evident from both insulin immunostaining and real-time PCR.
Example 11
Additive Effects of GSK-3β Enzyme Inhibitors on the Formation of Pancreatic Endocrine Cells
It was important to demonstrate that treatment with GSK3β inhibitors could improve pancreatic beta cell differentiation if added during multiple phases of cell fate commitment. A select subset of hits was tested by sequential timed addition to enhance insulin expression associated with pancreatic hormonal positive cells.
Preparation of Cells for Assay: Cell Preparation for Assay:
Pancreatic endoderm cells obtained according to the methods described in Example 9 and 10 (cultured on 96-wellplates) were subsequently subjected to agents that cause the cells to differentiate into pancreatic hormone expressing cells.
Assay for cultures of the H1 human embryonic stem cell line on MATRIGELTM was initiated as described in Examples 7-9 above by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes (100 μl per well for 96-well plates) were added containing medium with 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium and 0.5% FCS with 100 ng/ml Activin A and 20 ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3, 4, and 5, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and 100 ng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3, 4, and 5 in DMEM:F12 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.25 μM KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were subsequently treated for four days, feeding daily with DMEM:F12 containing 1% B27 (Invitrogen), 0.25 μM KAAD cyclopamine, 2 μM Retinoic Acid (RA; Sigma-Aldrich) and 20 ng/ml FGF7. Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 2% FCS or 1% B27 and without any other additives. After formation of pancreatic endoderm, cells were treated further for six days duration, feeding alternating days with DMEM:F12 base medium containing 1% B27 with 1 μM DAPT (gamma secretase inhibitor: EMD Biosciences) and 50 ng/ml Exendin 4 (Sigma-Aldrich) and 1 μM TGFbeta R1 inhibitor II (ALK5 inhibitor; EMD Biosciences). During this six day period, GSK3β inhibitors were added back to respective wells, using the same concentration as previous treatment at the initiation of differentiation. Cells were then treated for another three days duration, feeding alternating days with DMEM:F12 base medium containing 1% B27, 50 ng/ml Exendin 4, 50 ng/ml IGF (R&D Biosystems) and 50 ng/ml HGF (R&D Biosystems), and 1 μM TGFbeta R1 inhibitor II (ALK5 inhibitor; EMD Biosciences). During this three day period, GSK3β inhibitors were added back to respective wells, using the same concentration as previous treatment at the initiation of differentiation. Parallel sets of positive control wells were treated in the presence or absence of 20 ng/ml Wnt3a. Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 1% B27 and without any other additives.
Assay Evaluation:
At the end of culture, cells were treated as in Examples 10 above for evaluation by high content analysis.
For high content fluorescence staining, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (guinea pig anti-swine insulin, cross-reactive with human insulin; DakoCytomation) was diluted 1:500 in 4% goat serum and added to cells for one hour at room temperature. Cells were washed three times with PBS and then stained with Alexa Fluor 488 conjugated secondary antibody (goat anti-guinea pig IgG; Molecular Probes) diluted 1:100 in 4% goat serum. To counterstain nuclei, 2 μg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μl/well PBS for imaging.
Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total insulin intensity were obtained for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for each replicate data set. Total insulin protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each triplicate set.
Results
Results are shown for eight GSK-3B enzyme inhibitors. Data presented in FIG. 15 from high content analysis show compound effects on cell number (panel A) and insulin intensity (panel B) for the H1 hES cell line, where respective data points were averaged from a triplicate set and mined for each parameter from identical fields and wells. In this example, insulin expression is indicative of differentiation to hormonal positive pancreatic cells. Selective GSK3β inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment and, when added at later stages of differentiation, appear to promote enhanced insulin expression relative to a positive control sample.
Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law.







TABLE IA







LIST OF PRIMARY ANTIBODIES USED FOR


FACS AND IMMUNOSTAINININGANALYSIS.












Antibody
Supplier
Isotype
Clone







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



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



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



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



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



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



AP
R&D Systems
Mouse IgG1
B4-78



HNF3β
R&D Systems
Goat IgG



PDX1
Santa Cruz
Goat IgG
A-17




Biotechnology,




INC



GATA4
R&D Systems
Goat IgG



Sox 17
R&D Systems
Goat IgG



CD 9
BD
Mouse IgG1
M-L13

















TABLE IB







LIST OF SECONDARY CONJUGATED ANTIBODIES USED


FOR FACS AND IMMUNOSTAINININGANALYSIS.









Secondary Conjugated




Antibody
Supplier
Dilution





Goat Anti-Mouse IgG APC
Jackson ImmunoResearch
1:200


conjugated
(PA)


Goat Anti-Mouse IgG PE
Jackson ImmunoResearch
1:200


conjugated
(PA)


Donkey anti-rabbit PE or -
Jackson ImmunoResearch
1:200


APC conjugated
(PA)


Donkey anti-goat PE or -
Jackson ImmunoResearch
1:200


APC conjugated
(PA)


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


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


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
















TABLE II







EFFECTS OF INHIBITORS OF GSK-3B ENZYME ACTIVITY ON


THE VIABILITY OF CELLS EXPRESSING PLURIPOTENCY


MARKERS.













Raw data







(duplicate)
Average
S.D.
% CV
% Control
















JNJ5226780
0.785
0.790
0.788
0.00382
0.48
94.0


JNJ10179026
0.148
0.152
0.150
0.00247
1.65
4.8


JNJ17154215
0.427
0.462
0.444
0.02496
5.62
46.0


JNJ17205955
0.643
0.638
0.641
0.00368
0.57
73.5


JNJ180125
0.762
0.762
0.762
0.00007
0.01
90.4


JNJ18157646
0.850
0.824
0.837
0.01824
2.18
101.0


JNJ19370026
0.911
0.884
0.898
0.01881
2.10
109.5


JNJ19567340
0.723
0.743
0.733
0.01421
1.94
86.4


JNJ7830433
0.161
0.169
0.165
0.00559
3.39
6.9


JNJ10179130
0.767
0.789
0.778
0.01556
2.00
92.6


JNJ17154215
0.512
0.555
0.533
0.03048
5.72
58.4


JNJ17205955
0.282
0.293
0.288
0.00792
2.75
24.1


JNJ18014061
0.764
0.723
0.743
0.02892
3.89
87.9


JNJ18157698
0.853
0.858
0.855
0.00382
0.45
103.5


JNJ19376240
0.832
0.837
0.834
0.00361
0.43
100.6


JNJ19567405
0.726
0.725
0.725
0.00042
0.06
85.3


JNJ8706646
0.132
0.137
0.134
0.00368
2.74
2.6


JNJ10182562
0.797
0.793
0.795
0.00346
0.44
95.1


JNJ17157659
0.776
0.787
0.782
0.00792
1.01
93.2


JNJ17205994
0.164
0.148
0.156
0.01131
7.24
5.6


JNJ18014074
0.475
0.383
0.429
0.06548
15.26
43.8


JNJ18157698
0.823
0.774
0.798
0.03444
4.31
95.6


JNJ19386042
0.781
0.729
0.755
0.03649
4.83
89.5


JNJ19573541
0.143
0.149
0.146
0.00396
2.72
4.2


JNJ8710481
0.716
0.716
0.716
0.00014
0.02
84.1


JNJ10182562
0.804
0.802
0.803
0.00148
0.18
96.2


JNJ17163042
0.900
0.877
0.888
0.01626
1.83
108.2


JNJ17226703
0.824
0.799
0.812
0.01725
2.13
97.4


JNJ18018338
0.744
0.819
0.781
0.05261
6.73
93.2


JNJ18157711
0.952
0.966
0.959
0.00933
0.97
118.1


JNJ19410833
0.952
0.919
0.935
0.02277
2.43
114.8


JNJ19574867
0.776
0.777
0.777
0.00042
0.05
92.5


JNJ8710481
0.691
0.617
0.654
0.05254
8.03
75.4


JNJ10184655
0.162
0.134
0.148
0.02022
13.66
4.5


JNJ10166565
0.791
0.608
0.700
0.12947
18.50
81.8


JNJ17982133
0.153
0.129
0.141
0.01676
11.87
3.5


JNJ18018351
0.731
0.585
0.658
0.10317
15.68
75.9


DMSO
0.789
0.700
0.744
0.06279
8.44
88.0


JNJ19410859
0.909
0.675
0.792
0.16546
20.88
94.7


JNJ19574880
0.164
0.151
0.157
0.00926
5.89
5.8


JNJ10148307
0.706
0.672
0.689
0.02404
3.49
83.9


JNJ10222784
0.641
0.601
0.621
0.02878
4.63
73.7


JNJ17174664
0.882
0.748
0.815
0.09504
11.66
102.5


JNJ17989049
0.822
0.802
0.812
0.01400
1.72
102.1


JNJ18047991
0.777
0.764
0.771
0.00919
1.19
95.9


DMSO
0.798
0.771
0.785
0.01916
2.44
98.0


JNJ19410872
0.791
0.789
0.790
0.00134
0.17
98.7


JNJ20948798
0.628
0.640
0.634
0.00806
1.27
75.6


JNJ10164830
0.149
0.135
0.142
0.00969
6.81
2.7


JNJ10222927
0.803
0.782
0.792
0.01492
1.88
99.1


JNJ17187027
0.125
0.129
0.127
0.00318
2.51
0.4


JNJ17994873
0.315
0.542
0.428
0.15995
37.34
45.2


JNJ18055726
0.820
0.748
0.784
0.05091
6.49
97.9


JNJ18157711
0.154
0.165
0.160
0.00806
5.05
5.3


JNJ19558929
0.737
0.730
0.734
0.00481
0.66
90.4


JNJ21192730
0.659
0.647
0.653
0.00813
1.25
78.5


JNJ10164895
0.165
0.154
0.159
0.00785
4.93
5.2


JNJ10231273
0.637
0.554
0.595
0.05876
9.87
69.9


JNJ17187053
0.684
0.588
0.636
0.06809
10.71
76.0


JNJ17994899
0.750
0.624
0.687
0.08945
13.02
83.5


JNJ18077800
0.678
0.618
0.648
0.04285
6.61
77.8


JNJ19363357
0.777
0.667
0.722
0.07757
10.74
88.7


DMSO
0.799
0.649
0.724
0.10564
14.59
89.0


JNJ21194667
0.648
0.625
0.636
0.01662
2.61
76.0


JNJ10172058
0.601
0.620
0.611
0.01308
2.14
72.2


JNJ10259847
0.695
0.702
0.698
0.00552
0.79
85.2


JNJ17193774
0.568
0.709
0.639
0.09956
15.59
76.4


JNJ17994912
0.623
0.765
0.694
0.10041
14.46
84.6


JNJ18157074
0.758
0.762
0.760
0.00297
0.39
94.3


JNJ19369233
0.487
0.434
0.461
0.03769
8.18
49.9


JNJ19567314
0.690
0.686
0.688
0.00262
0.38
83.7


JNJ21196227
0.535
0.550
0.543
0.01089
2.01
62.1


JNJ10178727
0.743
0.638
0.691
0.07446
10.78
84.1


JNJ10259847
0.694
0.603
0.649
0.06449
9.94
77.8


JNJ17200976
0.160
0.186
0.173
0.01824
10.56
7.2


JNJ17994925
0.662
0.566
0.614
0.06788
11.05
72.7


JNJ18157087
0.600
0.514
0.557
0.06102
10.96
64.2


JNJ19369246
0.685
0.524
0.605
0.11427
18.90
71.3


JNJ19567327
0.731
0.525
0.628
0.14552
23.18
74.7


JNJ24843611
0.715
0.596
0.655
0.08436
12.87
78.8


JNJ24843611
0.592
0.572
0.582
0.01393
2.39
70.0


JNJ25758785
0.614
0.611
0.613
0.00177
0.29
74.6


JNJ26064571
0.766
0.849
0.807
0.05869
7.27
104.3


JNJ26130403
0.830
0.813
0.822
0.01195
1.45
106.5


JNJ26170794
0.727
0.730
0.728
0.00198
0.27
92.2


JNJ26241774
0.713
0.836
0.774
0.08733
11.28
99.3


JNJ26367991
0.726
0.719
0.722
0.00523
0.72
91.3


JNJ26483310
0.646
0.681
0.663
0.02510
3.78
82.4


JNJ24326185
0.651
0.649
0.650
0.00120
0.19
80.3


JNJ25758850
0.642
0.622
0.632
0.01407
2.23
77.5


JNJ26067626
0.843
0.672
0.758
0.12099
15.97
96.7


JNJ26134771
0.734
0.815
0.774
0.05728
7.40
99.3


JNJ26170820
0.823
0.743
0.783
0.05699
7.28
100.6


JNJ26241917
0.871
0.874
0.872
0.00219
0.25
114.2


JNJ26714220
0.652
0.642
0.647
0.00721
1.12
79.8


JNJ26483223
0.617
0.633
0.625
0.01174
1.88
76.5


JNJ24843572
0.657
0.655
0.656
0.00134
0.20
81.2


JNJ25758863
0.684
0.809
0.746
0.08803
11.80
95.0


JNJ26067652
0.901
0.735
0.818
0.11731
14.34
106.0


JNJ26150202
0.791
0.768
0.779
0.01591
2.04
100.1


JNJ26170833
0.948
0.764
0.856
0.12982
15.17
111.7


JNJ26243204
0.821
0.608
0.714
0.15033
21.05
90.1


JNJ26399906
0.745
0.685
0.715
0.04243
5.94
90.2


JNJ26483236
0.624
0.618
0.621
0.00417
0.67
76.0


JNJ24843585
0.652
0.624
0.638
0.01916
3.00
78.5


JNJ25873419
0.773
0.662
0.718
0.07792
10.86
90.6


JNJ26069901
0.856
0.834
0.845
0.01570
1.86
110.1


JNJ26153647
0.828
0.800
0.814
0.02008
2.47
105.4


JNJ26177086
0.821
0.841
0.831
0.01421
1.71
108.0


JNJ26247143
0.816
0.787
0.802
0.02072
2.58
103.5


JNJ26399906
0.744
0.737
0.741
0.00453
0.61
94.1


JNJ26483249
0.699
0.679
0.689
0.01464
2.12
86.3


JNJ25753520
0.186
0.208
0.197
0.01541
7.83
11.3


JNJ25887537
0.665
0.699
0.682
0.02432
3.57
85.2


JNJ26077883
0.810
0.683
0.746
0.09030
12.10
95.0


JNJ26158015
0.141
0.162
0.151
0.01506
9.95
4.3


DMSO
0.784
0.605
0.695
0.12671
18.25
87.1


JNJ26248729
0.726
0.590
0.658
0.09624
14.63
81.5


JNJ26399945
0.635
0.620
0.628
0.01068
1.70
76.9


JNJ26483249
0.697
0.695
0.696
0.00113
0.16
87.3


JNJ25753403
0.154
0.153
0.154
0.00042
0.28
4.5


JNJ25900641
0.616
0.645
0.630
0.02072
3.29
82.1


JNJ22791671
0.909
0.830
0.869
0.05614
6.46
121.0


JNJ26158054
0.150
0.150
0.150
0.00028
0.19
3.9


JNJ26177762
0.981
1.056
1.018
0.05303
5.21
145.3


JNJ26261105
0.166
0.189
0.177
0.01626
9.19
8.3


JNJ26399971
0.718
0.451
0.584
0.18887
32.34
74.6


JNJ26483262
0.652
0.647
0.649
0.00389
0.60
85.2


JNJ25757173
0.503
0.529
0.516
0.01860
3.61
63.5


JNJ25900654
0.603
0.609
0.606
0.00424
0.70
78.1


JNJ26116922
0.856
0.793
0.824
0.04419
5.36
113.7


JNJ26893438
0.883
0.848
0.866
0.02503
2.89
120.5


JNJ26184457
0.779
0.784
0.781
0.00368
0.47
106.7


JNJ26361712
0.892
0.914
0.903
0.01591
1.76
126.6


JNJ26399984
0.544
0.537
0.540
0.00460
0.85
67.5


JNJ26511901
0.532
0.682
0.607
0.10543
17.37
78.3


JNJ25757173
0.665
0.645
0.655
0.01400
2.14
86.1


JNJ25900706
0.676
0.677
0.677
0.00035
0.05
89.7


JNJ26120601
0.935
0.807
0.871
0.09115
10.47
121.3


JNJ26158093
0.916
0.859
0.887
0.03981
4.49
124.0


JNJ26219050
0.907
0.891
0.899
0.01124
1.25
125.9


JNJ26361725
0.909
0.896
0.902
0.00919
1.02
126.4


JNJ26399997
0.682
0.797
0.740
0.08118
10.98
99.9


JNJ26511927
0.679
0.644
0.661
0.02510
3.80
87.2


JNJ25757238
0.300
0.223
0.261
0.05452
20.88
22.0


JNJ26047723
0.183
0.175
0.179
0.00573
3.20
8.6


JNJ26120614
0.741
0.728
0.734
0.00884
1.20
99.1


JNJ26158106
0.935
0.906
0.921
0.02051
2.23
129.4


JNJ26219063
0.131
0.128
0.129
0.00212
1.64
0.5


JNJ26366730
0.138
0.137
0.138
0.00092
0.67
1.9


JNJ26400049
0.241
0.227
0.234
0.01032
4.41
17.6


JNJ26941226
0.604
0.639
0.622
0.02475
3.98
80.7


JNJ25758707
0.247
0.182
0.215
0.04617
21.52
14.4


JNJ26054912
0.659
0.634
0.647
0.01718
2.66
84.8


JNJ26128726
0.758
0.575
0.667
0.12961
19.44
88.1


JNJ26161343
0.166
0.170
0.168
0.00276
1.64
6.9


JNJ26220454
0.651
0.559
0.605
0.06541
10.81
78.0


JNJ26367991
0.803
0.694
0.748
0.07693
10.28
101.3


JNJ26483197
0.823
0.634
0.728
0.13378
18.37
98.1


JNJ26511953
0.624
0.618
0.621
0.00431
0.69
80.6


RWJ351001
0.639
0.603
0.621
0.02553
4.11
73.6


RWJ382867
0.143
0.149
0.146
0.00403
2.76
2.9


RWJ682205
0.817
0.818
0.818
0.00071
0.09
102.8


RWJ665862
0.742
0.752
0.747
0.00679
0.91
92.2


RWJ670804
0.856
0.905
0.881
0.03479
3.95
112.1


RWJ673829
0.650
0.576
0.613
0.05268
8.59
72.4


RWJ675260
0.768
0.724
0.746
0.03097
4.15
92.2


RWJ675946
0.556
0.549
0.553
0.00537
0.97
63.4


RWJ351958
0.227
0.242
0.235
0.01103
4.70
16.1


RWJ395477
0.634
0.663
0.649
0.02044
3.15
77.7


RWJ447228
0.141
0.128
0.135
0.00919
6.83
1.3


RWJ666167
0.847
0.832
0.840
0.01110
1.32
106.0


RWJ670908
0.803
0.845
0.824
0.02998
3.64
103.7


RWJ673830
0.860
0.860
0.860
0.00035
0.04
109.1


RWJ675261
0.528
0.497
0.513
0.02227
4.34
57.5


RWJ675948
0.683
0.688
0.686
0.00332
0.48
83.1


RWJ447228
0.611
0.628
0.620
0.01202
1.94
73.3


RWJ414342
0.719
0.749
0.734
0.02143
2.92
90.3


RWJ553709
0.916
0.838
0.877
0.05487
6.26
111.6


RWJ666168
0.771
0.740
0.755
0.02178
2.88
93.5


RWJ670984
0.820
0.852
0.836
0.02305
2.76
105.5


RWJ674239
0.971
0.913
0.942
0.04137
4.39
121.2


RWJ675430
0.839
0.743
0.791
0.06746
8.53
98.8


RWJ676061
0.562
0.527
0.544
0.02440
4.48
62.2


RWJ352190
0.678
0.661
0.670
0.01195
1.78
80.8


RWJ414984
0.722
0.713
0.717
0.00658
0.92
87.9


RWJ659780
0.802
0.801
0.802
0.00106
0.13
100.4


RWJ666205
0.854
0.857
0.855
0.00205
0.24
108.4


RWJ671232
0.767
0.798
0.782
0.02157
2.76
97.5


RWJ674240
0.789
0.776
0.782
0.00870
1.11
97.5


RWJ675266
0.720
0.709
0.714
0.00764
1.07
87.4


RWJ676085
0.641
0.618
0.630
0.01619
2.57
74.9


RWJ352244
0.603
0.584
0.593
0.01372
2.31
69.4


RWJ425264
0.135
0.158
0.146
0.01633
11.18
3.0


RWJ662440
0.792
0.572
0.682
0.15563
22.83
82.6


RWJ666213
0.752
0.593
0.673
0.11292
16.79
81.2


RWJ672667
0.805
0.598
0.702
0.14644
20.87
85.5


RWJ674241
0.599
0.504
0.552
0.06682
12.11
63.2


RWJ675366
0.714
0.593
0.654
0.08549
13.08
78.4


RWJ676137
0.699
0.698
0.698
0.00099
0.14
85.0


RWJ352628
0.690
0.674
0.682
0.01131
1.66
83.3


RWJ425268
0.616
0.634
0.625
0.01301
2.08
74.8


RWJ663860
0.809
0.817
0.813
0.00552
0.68
103.0


RWJ667045
0.128
0.133
0.131
0.00361
2.76
0.7


RWJ672932
0.821
0.811
0.816
0.00721
0.88
103.4


RWJ674320
0.456
0.474
0.465
0.01223
2.63
50.8


RWJ675369
0.762
0.766
0.764
0.00304
0.40
95.7


RWJ676139
0.680
0.663
0.671
0.01195
1.78
81.8


RWJ353258
0.615
0.635
0.625
0.01400
2.24
74.8


RWJ355923
0.681
0.698
0.689
0.01266
1.84
84.5


RWJ664545
0.830
0.807
0.818
0.01584
1.94
103.8


RWJ667046
0.869
0.849
0.859
0.01442
1.68
109.9


RWJ672934
0.821
0.841
0.831
0.01428
1.72
105.7


RWJ674817
0.819
0.840
0.830
0.01485
1.79
105.5


RWJ675430
0.795
0.793
0.794
0.00078
0.10
100.1


RWJ676431
0.640
0.636
0.638
0.00283
0.44
76.7


RWJ355131
0.610
0.628
0.619
0.01266
2.05
73.9


RWJ425271
0.143
0.144
0.144
0.00035
0.25
2.6


RWJ353709
0.804
0.903
0.853
0.07000
8.20
109.0


RWJ667069
0.918
0.854
0.886
0.04483
5.06
113.9


RWJ673313
0.105
1.080
0.593
0.68971
116.37
70.0


RWJ674855
0.877
0.860
0.868
0.01209
1.39
111.3


RWJ675578
0.808
0.695
0.751
0.07941
10.57
93.8


RWJ676432
0.720
0.697
0.709
0.01648
2.33
87.3


RWJ355923
0.636
0.621
0.629
0.01054
1.68
75.4


RWJ425348
0.640
0.634
0.637
0.00474
0.74
76.6


RWJ665436
0.833
0.833
0.833
0.00000
0.00
106.0


RWJ669182
0.887
0.846
0.866
0.02934
3.39
111.0


RWJ673515
0.845
0.877
0.861
0.02326
2.70
110.2


RWJ674855
0.794
0.784
0.789
0.00686
0.87
99.4


RWJ675605
0.770
0.786
0.778
0.01138
1.46
97.8


RWJ67657
0.629
0.659
0.644
0.02128
3.30
77.7


RWJ356205
0.584
0.558
0.571
0.01817
3.18
66.8


RWJ445224
0.707
0.679
0.693
0.01987
2.87
85.0


RWJ665588
0.727
0.578
0.652
0.10536
16.15
78.9


RWJ669327
0.742
0.629
0.685
0.07969
11.63
83.8


DMSO
0.653
0.507
0.580
0.10310
17.78
68.0


RWJ675104
0.722
0.568
0.645
0.10904
16.90
77.9


RWJ675881
0.643
0.581
0.612
0.04384
7.16
72.9


RWJ676639
0.608
0.590
0.599
0.01245
2.08
70.9


JNJ26511966
0.597
0.610
0.603
0.00926
1.54
71.2


JNJ26511979
0.687
0.668
0.677
0.01336
1.97
82.4


JNJ26512005
0.840
0.832
0.836
0.00594
0.71
106.1


JNJ26533065
0.831
0.822
0.826
0.00587
0.71
104.7


JNJ26533091
0.863
0.856
0.860
0.00509
0.59
109.7


JNJ26533104
0.886
0.802
0.844
0.05954
7.05
107.3


JNJ26533156
0.753
0.687
0.720
0.04660
6.47
88.8


JNJ26714181
0.455
0.463
0.459
0.00587
1.28
49.6


JNJ26714194
0.668
0.678
0.673
0.00764
1.13
81.7


JNJ26714207
0.181
0.171
0.176
0.00658
3.74
7.2


JNJ26714220
0.832
0.842
0.837
0.00658
0.79
106.3


JNJ26875563
0.795
0.802
0.798
0.00445
0.56
100.5


JNJ22791671
0.157
0.140
0.148
0.01202
8.11
3.0


JNJ26893438
0.153
0.153
0.153
0.00035
0.23
3.7


JNJ26941226
0.168
0.154
0.161
0.00969
6.02
4.9


JNJ28572128
0.670
0.641
0.655
0.02079
3.17
79.1


RWJ67694
0.706
0.679
0.693
0.01888
2.73
84.7


RWJ676940
0.788
0.666
0.727
0.08627
11.86
89.8


RWJ677545
0.879
0.785
0.832
0.06640
7.98
105.6


RWJ678986
0.168
0.176
0.172
0.00537
3.13
6.6


RWJ680665
0.946
0.848
0.897
0.06972
7.77
115.3


RWJ680667
0.187
0.202
0.194
0.01089
5.61
9.9


RWJ680668
0.906
0.688
0.797
0.15394
19.31
100.3


RWJ680669
0.715
0.674
0.694
0.02850
4.10
84.9


RWJ680858
0.695
0.700
0.697
0.00339
0.49
85.3


RWJ680858
0.665
0.631
0.648
0.02369
3.66
78.0


RWJ680879
0.590
0.613
0.601
0.01655
2.75
71.0


RWJ680885
0.681
0.687
0.684
0.00382
0.56
83.3


RWJ680991
0.829
0.821
0.825
0.00530
0.64
104.5


RWJ680992
0.822
0.790
0.806
0.02270
2.82
101.6


RWJ680993
0.671
0.684
0.677
0.00912
1.35
82.3


RWJ681140
0.686
0.668
0.677
0.01266
1.87
82.3


RWJ681142
0.212
0.197
0.204
0.01047
5.12
11.5


RWJ681146
0.666
0.666
0.666
0.00007
0.01
80.7


RWJ681945
0.736
0.656
0.696
0.05643
8.11
85.1


RWJ68198
0.726
0.610
0.668
0.08217
12.30
81.0


JNJ28850601
0.303
0.310
0.306
0.00488
1.59
26.7


DMSO
0.786
0.659
0.722
0.09001
12.46
89.1


DMSO
0.673
0.649
0.661
0.01676
2.53
79.9


DMSO
0.701
0.686
0.693
0.01011
1.46
84.8
















TABLE III







EFFECTS OF INHIBITORS OF GSK-3B ENZYME ACTIVITY ON


THE VIABILITY OF CELLS EXPRESSING PLURIPOTENCY MARKERS.














cmpd conc
Raw data







(uM)
(duplicate)
Average
S.D.
% CV
% Control

















EXPRES 01 medium

0.6379
0.6180
0.6280
0.0141
2.2
74.6


no treatment

0.7412
0.7038
0.7225
0.0264
3.7
88.7


AA only

0.7674
0.8047
0.7861
0.0264
3.4
98.3


AA + Wnt3a

0.7754
0.8200
0.7977
0.0315
4.0
100.0


JNJ26512005
10
0.1412
0.1515
0.1464
0.0073
5.0
2.4


JNJ26512005
5
0.1501
0.1444
0.1473
0.0040
2.7
2.5


JNJ26512005
2.5
0.1541
0.4254
0.2898
0.1918
66.2
23.9


JNJ26533065
10
0.1272
0.1282
0.1277
0.0007
0.6
−0.4


JNJ26533065
5
0.5862
0.5880
0.5871
0.0013
0.2
68.4


JNJ26533065
2.5
0.7613
0.7603
0.7608
0.0007
0.1
94.5


JNJ26533156
10
0.1481
0.1592
0.1537
0.0078
5.1
3.5


JNJ26533156
5
0.1479
0.1639
0.1559
0.0113
7.3
3.8


JNJ26533156
2.5
0.2861
0.2477
0.2669
0.0272
10.2
20.4


JNJ26714194
10
0.2092
0.2426
0.2259
0.0236
10.5
14.3


JNJ26714194
5
0.6815
0.7128
0.6972
0.0221
3.2
84.9


JNJ26714194
2.5
0.7389
0.7870
0.7630
0.0340
4.5
94.8


JNJ26150202
10
0.1381
0.1398
0.1390
0.0012
0.9
1.3


JNJ26150202
5
0.7826
0.7578
0.7702
0.0175
2.3
95.9


JNJ26150202
2.5
0.8231
0.7742
0.7987
0.0346
4.3
100.1


JNJ26158015
10
0.1352
0.1326
0.1339
0.0018
1.4
0.5


JNJ26158015
5
0.2632
0.2604
0.2618
0.0020
0.8
19.7


JNJ26158015
2.5
0.4160
0.5314
0.4737
0.0816
17.2
51.4


RWJ670804
10
0.4447
0.4791
0.4619
0.0243
5.3
49.7


RWJ670804
5
0.6902
0.6884
0.6893
0.0013
0.2
83.8


RWJ670804
2.5
0.7476
0.7483
0.7480
0.0005
0.1
92.5


JNJ26170833
10
0.6790
0.6704
0.6747
0.0061
0.9
81.6


JNJ26170833
5
0.7833
0.7924
0.7879
0.0064
0.8
98.5


JNJ26170833
2.5
0.8155
0.8389
0.8272
0.0165
2.0
104.4


JNJ26177086
10
0.6533
0.6884
0.6709
0.0248
3.7
81.0


JNJ26177086
5
0.7697
0.7738
0.7718
0.0029
0.4
96.1


JNJ26177086
2.5
0.8119
0.8219
0.8169
0.0071
0.9
102.9


JNJ26177762
10
0.1242
0.1323
0.1283
0.0057
4.5
−0.4


JNJ26177762
5
0.1263
0.1303
0.1283
0.0028
2.2
−0.3


JNJ26177762
2.5
0.8480
0.7725
0.8103
0.0534
6.6
101.9


RWJ673515
10
0.1695
0.1890
0.1793
0.0138
7.7
7.3


RWJ673515
5
0.7217
0.7435
0.7326
0.0154
2.1
90.2


RWJ673515
2.5
0.7812
0.7221
0.7517
0.0418
5.6
93.1


EXPRES 01 medium

0.6294
0.6363
0.6329
0.0049
0.8
70.3


no treatment

0.7156
0.7356
0.7256
0.0141
1.9
83.3


AA only

0.8732
0.9046
0.8889
0.0222
2.5
106.0


AA + Wnt3a

0.8415
0.8500
0.8458
0.0060
0.7
100.0


JNJ19370026
10
0.1403
0.1493
0.1448
0.0064
4.4
2.3


JNJ19370026
5
0.4434
0.3878
0.4156
0.0393
9.5
40.1


JNJ19370026
2.5
0.7734
0.8038
0.7886
0.0215
2.7
92.0


JNJ26483197
10
0.2993
0.3026
0.3010
0.0023
0.8
24.1


JNJ26483197
5
0.7023
0.6299
0.6661
0.0512
7.7
75.0


JNJ26483197
2.5
0.7835
0.8043
0.7939
0.0147
1.9
92.8


RWJ675605
10
0.7205
0.7369
0.7287
0.0116
1.6
83.7


RWJ675605
5
0.7769
0.8272
0.8021
0.0356
4.4
93.9


RWJ675605
2.5
0.8214
0.8640
0.8427
0.0301
3.6
99.6


RWJ675430
10
0.6275
0.5980
0.6128
0.0209
3.4
67.5


RWJ675430
5
0.7159
0.7222
0.7191
0.0045
0.6
82.3


RWJ675430
2.5
0.9245
0.9403
0.9324
0.0112
1.2
112.1


RWJ675948
10
0.7220
0.6670
0.6945
0.0389
5.6
78.9


RWJ675948
5
0.7526
0.7486
0.7506
0.0028
0.4
86.7


RWJ675948
2.5
0.7557
0.7390
0.7474
0.0118
1.6
86.3


JNJ26483249
10
0.8214
0.8636
0.8425
0.0298
3.5
99.5


JNJ26483249
5
0.7996
0.7873
0.7935
0.0087
1.1
92.7


JNJ26483249
2.5
0.8669
0.8195
0.8432
0.0335
4.0
99.6


RWJ67657
10
0.6195
0.5908
0.6052
0.0203
3.4
66.5


RWJ67657
5
0.8047
0.8319
0.8183
0.0192
2.4
96.2


RWJ67657
2.5
0.8041
0.7900
0.7971
0.0100
1.3
93.2


RWJ676639
10
0.1261
0.1520
0.1391
0.0183
13.2
1.5


RWJ676639
5
0.1303
0.1263
0.1283
0.0028
2.2
0.0


RWJ676639
2.5
0.4482
0.4051
0.4267
0.0305
7.1
41.6
















TABLE IV







EFFECTS OF INHIBITORS OF GSK-3B ENZYME ACTIVITY ON THE DIFFERENTIATION AND


PROLIFERATION OF HUMAN EMBRYONIC STEM CELLS.












Proliferative Response
SOX-17 Expression
Proliferative Response
HNF-3b Expression















Compound

Fold over Wnt

Fold over Wnt

Fold over Wnt
Total
Fold over Wnt


name
Total cells
3a/AA control
Total Intensity
3a/AA control
Total cells
3a/AA control
Intensity
3a/AA control


















JNJ26511966
1723
0.11244207
68870409
0.0708
1645
0.10460717
50143628
0.0453


JNJ26511979
1110
0.07245904
42978557
0.0442
94
0.00597755
0
0.0000


JNJ26512005
7990
0.52154188
339840000
0.3494
6833
0.43448539
231745000
0.2092


JNJ26533065
4914
0.32074548
238555000
0.2453
2907
0.18485899
82808745
0.0747


JNJ26533091
3056
0.19945819
153145000
0.1575
2643
0.16807097
122246784
0.1103


JNJ26533104
3960
0.25850251
47669463
0.0490
4641
0.29512575
210730000
0.1902


JNJ26533156
12243
0.79917096
699160000
0.7189
6536
0.41559887
248855000
0.2246


JNJ26714181
401
0.02614400
25580022
0.0263
27
0.00168516
0
0.0000


JNJ26714194
7958
0.51948561
351070000
0.3610
6992
0.44459636
288075000
0.2600


JNJ26714207
277
0.01808212
6558563
0.0067
12
0.00073130
535481
0.0005


JNJ26714220
1327
0.08662445
69037756
0.0710
1194
0.07589584
40478497
0.0365


JNJ26875563
791
0.05160259
24732475
0.0254
64
0.00406982
1092011
0.0010


JNJ22791671
0
0.00000000
0
0.0000
3
0.00019077
95784
0.0001


JNJ26893438
2
0.00013056
0
0.0000
0
0.00000000
0
0.0000


JNJ26941226
6
0.00035903
1092432
0.0011
2
0.00009539
150222
0.0001


JNJ28572128
2742
0.17899341
122926199
0.1264
3166
0.20132905
120729987
0.1090


JNJ28850601
33
0.00212155
3855900
0.0040
8
0.00050873
208129
0.0002


RWJ674817
2000
0.13055682
110080123
0.1132
116
0.00737655
4290889
0.0039


RWJ674855
3495
0.22814805
110559816
0.1137
438
0.02782105
24450647
0.0221


RWJ674855
3107
0.20278739
120998421
0.1244
6177
0.39276971
273965000
0.2473


RWJ675104
658
0.04295320
37841044
0.0389
646
0.04107977
31352380
0.0283


RWJ675260
5991
0.39108297
252690000
0.2598
8479
0.53915615
306520000
0.2767


RWJ675261
1953
0.12745610
88653625
0.0912
641
0.04076182
18162585
0.0164


RWJ675266
2024
0.13209087
128395000
0.1320
4923
0.31302661
232020000
0.2094


RWJ675366
2979
0.19446439
93454696
0.0961
3582
0.22775110
137054653
0.1237


RWJ675369
3703
0.24169332
138180000
0.1421
3980
0.25306032
139550000
0.1260


RWJ675430
21070
1.37538351
1089750000
1.1205
21203
1.34831961
1281000000
1.1562


RWJ675578
1297
0.08466610
47445962
0.0488
30
0.00190773
0
0.0000


RWJ675605
14529
0.94839741
1013360000
1.0419
9871
0.62767480
540725000
0.4881


RWJ675881
4063
0.26522619
207891758
0.2137
3973
0.25264697
177190000
0.1599


RWJ675946
1
0.00006528
0
0.0000
7
0.00041334
0
0.0000


RWJ675948
9716
0.63421242
572520000
0.5887
7650
0.48643922
329425000
0.2973


RWJ676061
916
0.05979503
0
0.0000
1076
0.06839210
40211776
0.0363


RWJ676085
738
0.04817547
30943000
0.0318
503
0.03198626
0
0.0000


RWJ676137
8367
0.54618448
373185000
0.3837
7976
0.50720168
260000000
0.2347


RWJ676139
20079
1.31069260
1104750000
1.1359
16884
1.07363836
1052345000
0.9499


RWJ676431
13789
0.90012403
789085000
0.8113
11369
0.72296588
547055000
0.4938


RWJ676432
16652
1.08698348
1045395000
1.0749
14950
0.95065340
854325000
0.7711


RWJ676657
6376
0.41618252
324450000
0.3336
6058
0.38523417
269025000
0.2428


RWJ676639
6470
0.42231869
327055000
0.3363
4357
0.27706591
109160000
0.0985


RWJ674817
2000
0.13055682
110080123
0.1132
116
0.00737655
4290889
0.0039


RWJ674855
3495
0.22814805
110559816
0.1137
438
0.02782105
24450647
0.0221


RWJ674855
3107
0.20278739
120998421
0.1244
6177
0.39276971
273965000
0.2473


RWJ675104
658
0.04295320
37841044
0.0389
646
0.04107977
31352380
0.0283


RWJ675260
5991
0.39108297
252690000
0.2598
8479
0.53915615
306520000
0.2767


RWJ675261
1953
0.12745610
88653625
0.0912
641
0.04076182
18162585
0.0164


RWJ675266
2024
0.13209087
128395000
0.1320
4923
0.31302661
232020000
0.2094


RWJ675366
2979
0.19446439
93454696
0.0961
3582
0.22775110
137054653
0.1237


RWJ675369
3703
0.24169332
138180000
0.1421
3980
0.25306032
139550000
0.1260


RWJ675430
21070
1.37538351
1089750000
1.1205
21203
1.34831961
1281000000
1.1562


RWJ675578
1297
0.08466610
47445962
0.0488
30
0.00190773
0
0.0000


RWJ675605
14529
0.94839741
1013360000
1.0419
9871
0.62767480
540725000
0.4881


RWJ675881
4063
0.26522619
207891758
0.2137
3973
0.25264697
177190000
0.1599


RWJ675946
1
0.00006528
0
0.0000
7
0.00041334
0
0.0000


RWJ675948
9716
0.63421242
572520000
0.5887
7650
0.48643922
329425000
0.2973


RWJ676061
916
0.05979503
0
0.0000
1076
0.06839210
40211776
0.0363


RWJ676085
738
0.04817547
30943000
0.0318
503
0.03198626
0
0.0000


RWJ676137
8367
0.54618448
373185000
0.3837
7976
0.50720168
260000000
0.2347


RWJ676139
20079
1.31069260
1104750000
1.1359
16884
1.07363836
1052345000
0.9499


RWJ676431
13789
0.90012403
789085000
0.8113
11369
0.72296588
547055000
0.4938


RWJ676432
16652
1.08698348
1045395000
1.0749
14950
0.95065340
854325000
0.7711


RWJ67657
6376
0.41618252
324450000
0.3336
6058
0.38523417
269025000
0.2428


RWJ676639
6470
0.42231869
327055000
0.3363
4357
0.27706591
109160000
0.0985


No treatment
3891
0.25396566
97657703
0.1004
6091
0.38733268
109336609
0.0987


AA
4348
0.28379790
104735084
0.1077
122
0.00775810
5341271
0.0048


AA/3a
15319
1.00000000
972595000
1.0000
15726
1.00000000
1107900000
1.0000


RWJ351001
738
0.44211577
0
0.0000
0
0.00000000
0
0.0000


RWJ351958
0
0.00000000
0
0.0000
0
0.00000000
0
0.0000


DMSO
56
0.03353293
454796
0.0148
211
0.16644754
4455058
0.1626


RWJ352190
1313
0.78642715
28506437
0.9266
5485
4.32684722
85245671
3.1115


RWJ352244
12
0.00738523
85949
0.0028
67
0.05259006
1300640
0.0475


RWJ352628
2899
1.73612774
32703235
1.0630
7460
5.88456482
149772525
5.4668


RWJ353258
562
0.33632735
11388240
0.3702
787
0.62108861
10743082
0.3921


RWJ355131
118
0.07045908
2574279
0.0837
57
0.04522745
2584708
0.0943


RWJ355923
136
0.08163673
410648
0.0133
0
0.00000000
0
0.0000


RWJ356205
19
0.01137725
0
0.0000
0
0.00000000
0
0.0000


RWJ382867
3
0.00159681
431883
0.0140
31
0.02419143
847186
0.0309


RWJ395477
33
0.01976048
0
0.0000
225
0.17749145
5223879
0.1907


RWJ414342
16
0.00978044
0
0.0000
496
0.39127005
8966327
0.3273


RWJ414984
26
0.01556886
459801
0.0149
189
0.14935577
1819533
0.0664


RWJ425264
1
0.00039920
0
0.0000
42
0.03339469
1605538
0.0586


RWJ425268
22
0.01297405
82062
0.0027
311
0.24506968
5749996
0.2099


RWJ425271
0
0.00000000
0
0.0000
0
0.00000000
0
0.0000


RWJ425348
26
0.01556886
0
0.0000
0
0.00000000
0
0.0000


RWJ445224
202
0.12095808
627280
0.0204
1079
0.85143308
14326715
0.5229


RWJ447228
3
0.00179641
0
0.0000
4
0.00315540
101114
0.0037


RWJ553709
1310
0.78423154
24382455
0.7926
3249
2.56323955
75834631
2.7680


RWJ659780
20
0.01177645
0
0.0000
425
0.33526164
8880858
0.3242


RWJ663860
9
0.00538922
37140
0.0012
134
0.10570602
2144545
0.0783


RWJ662440
7
0.00419162
48154
0.0016
5
0.00420720
170177
0.0062


RWJ664545
70
0.04191617
589594
0.0192
0
0.00000000
0
0.0000


RWJ665436
1215
0.72774451
7568849
0.2460
0
0.00000000
0
0.0000


no Treatment
1145
0.68542914
6979814
0.2269
not done





AA
100
0.05988024
1264807
0.0411
51
0.04049435
923625
0.0337


AA/3a
1670
1.00000000
30764293
1.0000
1268
1.00000000
27396787
1.0000


RWJ665588
43
0.00510815
706614
0.0055
0
0.00000000
0
0.0000


RWJ665862
7
0.00079815
102445
0.0008
0
0.00000000
0
0.0000


RWJ666167
46
0.00546732
0
0.0000
46
0.00548446
818478
0.0044


RWJ666168
5
0.00059861
284777
0.0022
32
0.00385502
2309043
0.0124


RWJ666205
258
0.03092825
4009395
0.0312
391
0.04665766
14340307
0.0769


RWJ666213
62
0.00742278
782261
0.0061
112
0.01335347
2792473
0.0150


RWJ667045
36
0.00431000
312039
0.0024
2
0.00027820
1731575
0.0093


RWJ667046
59
0.00702371
397711
0.0031
103
0.01232017
3561761
0.0191


RWJ667069
22
0.00267380
770128
0.0060
0
0.00000000
0
0.0000


RWJ669182
77
0.00925852
1631067
0.0127
0
0.00000000
0
0.0000


RWJ669327
129
0.01540426
997629
0.0078
98
0.01164454
4138261
0.0222


RWJ670804
2386
0.28565728
20866647
0.1625
2594
0.30931563
61161468
0.3280


RWJ670908
172
0.02063213
625299
0.0049
133
0.01589699
3578458
0.0192


RWJ670984
8
0.00099769
394948
0.0031
530
0.06319053
16678849
0.0894


RWJ671232
17
0.00207519
0
0.0000
53
0.00627931
2270954
0.0122


RWJ672667
11
0.00127704
0
0.0000
36
0.00433193
2287281
0.0123


RWJ672932
2
0.00023944
0
0.0000
0
0.00000000
0
0.0000


RWJ672934
174
0.02087158
1451727
0.0113
0
0.00000000
0
0.0000


RWJ673313
80
0.00961769
940367
0.0073
333
0.03970273
5586343
0.0300


RWJ673515
11886
1.42305850
223646667
1.7415
10331
1.23173834
309900000
1.6618


RWJ673829
545
0.06524862
5849381
0.0455
404
0.04820761
6738305
0.0361


RWJ673830
10
0.00115732
315367
0.0025
35
0.00421270
3072013
0.0165


RWJ674239
2473
0.29603320
80676667
0.6282
4209
0.50182815
143916667
0.7718


RWJ674240
8
0.00091787
233687
0.0018
6
0.00071536
0
0.0000


RWJ674241
1
0.00007981
1309298
0.0102
0
0.00000000
0
0.0000


RWJ674320
0
0.00003991
0
0.0000
0
0.00000000
0
0.0000


No treatment
7653
0.91619443
26272707
0.2046
12050
1.43665050
74453588
0.3993


AA
15
0.00175593
0
0.0000
210
0.02503776
3777945
0.0203


AA/3a
8353
1.00000000
128424304
1.0000
8387
1.00000000
186480000
1.0000


RWJ355923
7319
0.91843393
387695000
1.0342
5436
1.07644321
437495000
0.9520


RWJ664545
6620
0.83065629
333205000
0.8889
4767
0.94395485
397435000
0.8649


RWJ353709
6217
0.78014807
337920000
0.9014
5013
0.99277156
437235000
0.9515


reference cmpd
5934
0.74463546
363935000
0.9708
4122
0.81621943
348135000
0.7576


JNJ18157698
10447
1.31089221
382680000
1.0208
6908
1.36805624
560475000
1.2196


JNJ5226780
10963
1.37570586
296920000
0.7921
5679
1.12456679
463525000
1.0087


JNJ7830433
1766
0.22160873
162790000
0.4343
2184
0.43241905
189875000
0.4132


JNJ8706646
2914
0.36566696
230965000
0.6161
2776
0.54975740
125125000
0.2723


JNJ8710481
3600
0.45175053
276080000
0.7365
4121
0.81612041
294665000
0.6412


JNJ8710481
1977
0.24808633
164760000
0.4395
2266
0.44865828
152060000
0.3309


JNJ10148307
9964.5
1.25040783
363855000
0.9706
9728
1.92642836
635655000
1.3832


JNJ10164830
2536.5
0.31829590
179185000
0.4780
2397
0.47460145
150600000
0.3277


JNJ10164895
5706.5
0.71608734
319930000
0.8534
5096
1.00920883
341360000
0.7428


JNJ10172058
4645.5
0.58294642
257295000
0.6864
4507
0.89256362
312605000
0.6803


JNJ10178727
2892.5
0.36296900
213165000
0.5686
3043
0.60253490
269570000
0.5866


JNJ10179026
2460.5
0.30875894
203350000
0.5425
2410
0.47727498
209795000
0.4565


JNJ10179130
4783
0.60020078
306085000
0.8165
4556
0.90226755
326475000
0.7104


JNJ10182562
6916.5
0.86792571
377885000
1.0080
4504
0.89196950
365090000
0.7945


JNJ10182562
7370.5
0.92489647
365075000
0.9739
5300
1.04950985
399265000
0.8688


JNJ10184655
10533
1.32174677
475250000
1.2678
5186
1.02693336
404710000
0.8807


JNJ10222784
3513
0.44083323
242750000
0.6476
2522
0.49945539
214575000
0.4669


No Treatment
not done



not done





AA
not done



not done





AA/3a
7969
1.00000000
374870000
1.0000
5050
1.00000000
459540000
1.0000


JNJ10222784
563
0.31250000
57351132
0.3295
1744
0.03386884
165365000
1.1010


JNJ10222927
158
0.08777778
14786632
0.0850
83
0.00161234
14201404
0.0946


JNJ10231273
3
0.00166667
0
0.0000
4
0.00007770
28439
0.0002


JNJ10259847
5
0.00277778
0
0.0000
10
0.00019426
0
0.0000


JNJ10259847
15
0.00805556
548982
0.0032
0
0.00000000
0
0.0000


JNJ17154215
24
0.01305556
689535
0.0040
11
0.00021368
0
0.0000


JNJ17154215
94
0.05194444
11142426
0.0640
12
0.00022340
1767033
0.0118


JNJ17157659
15
0.00805556
0
0.0000
21
0.00039823
4567590
0.0304


JNJ17163042
33
0.01805556
2188847
0.0126
69
0.00134038
13689421
0.0911


JNJ10166565
4
0.00194444
0
0.0000
3
0.00005828
291660
0.0019


JNJ17174664
88
0.04888889
7121122
0.0409
399
0.00774117
65100086
0.4335


JNJ17187027
11
0.00583333
1073763
0.0062
5
0.00008742
0
0.0000


JNJ17187053
8
0.00444444
0
0.0000
9
0.00016512
0
0.0000


JNJ17193774
109
0.06027778
15714170
0.0903
136
0.00263219
15725984
0.1047


JNJ17200976
5
0.00250000
125443
0.0007
5
0.00009713
0
0.0000


JNJ17205955
20
0.01083333
3135653
0.0180
8
0.00015541
0
0.0000


JNJ17205955
9
0.00472222
72387
0.0004
17
0.00033024
736311
0.0049


JNJ17205994
6
0.00305556
644015
0.0037
4
0.00007770
0
0.0000


JNJ17226703
77
0.04277778
12632849
0.0726
28
0.00054392
9312311
0.0620


JNJ17982133
14
0.00750000
887585
0.0051
1
0.00001943
52047
0.0003


JNJ17989049
23
0.01277778
2117429
0.0122
13
0.00024282
0
0.0000


No Treatment
not done



432
0.00838222
42987388
0.2862


AA
147
0.08138889
20330009
0.1168
8
0.00014569
87206
0.0006


AA/3a
1800
1.00000000
174052346
1.0000
1478
0.02870158
150190000
1.0000
















TABLE V







EFFECTS OF INHIBITORS OF GSK-3B ENZYME ACTIVITY


ON THE DIFFERENTIATION AND PROLIFERATION


OF HUMAN EMBRYONIC STEM CELLS.










Compound name
Fold over Wnt 3a/AA control











Proliferative Response - Strong Hits










RWJ352628
5.8846



RWJ352190
4.3268



RWJ553709
2.5632



JNJ10148307
1.9264



RWJ673515
1.4231



JNJ5226780
1.3757



RWJ675430
1.3754



JNJ18157698
1.3681



JNJ10184655
1.3217



RWJ676139
1.3107







Proliferative Response - Moderate Hits










JNJ5226780
1.1246



RWJ676432
1.0870



RWJ355923
1.0764



RWJ676139
1.0736



JNJ10182562
1.0495



JNJ10184655
1.0269



JNJ10164895
1.0092



RWJ353709
0.9928



RWJ675605
0.9484



RWJ664545
0.9440



JNJ10182562
0.9249



JNJ10179130
0.9023



RWJ676431
0.9001



JNJ10172058
0.8926



RWJ445224
0.8514



reference cmpd
0.8162



JNJ8710481
0.8161



JNJ26533156
0.7992



RWJ352190
0.7864



RWJ553709
0.7842



RWJ665436
0.7277



RWJ675948
0.6342



RWJ353258
0.6211



JNJ10173727
0.6025







SOX17 Expression - Strong Hits










RWJ673515
1.7415



JNJ10184655
1.2678







SOX17 Expression - Moderate Hits










RWJ676139
1.1359



RWJ675430
1.1205



RWJ676432
1.0749



RWJ352628
1.0630



RWJ675605
1.0419



RWJ355923
1.0342



JNJ18157698
1.0208



JNJ10182562
1.0030



reference cmpd
0.9708



JNJ10148307
0.9706



RWJ352190
0.9266



RWJ353709
0.9014



RWJ664545
0.8889



JNJ10164895
0.8534



JNJ10179130
0.8165



RWJ676431
0.8113



RWJ553709
0.7926



JNJ5226780
0.7921



JNJ8710481
0.7365



JNJ26533156
0.7189



JNJ10172058
0.6864



JNJ10222784
0.6476



RWJ674239
0.6282



JNJ8706646
0.6161



RWJ675948
0.5887



JNJ10178727
0.5686







HNF3β Expression - Strong Hits










RWJ352628
5.4668



RWJ352190
3.1115



RWJ553709
2.7680



RWJ673515
1.6618



JNJ10148307
1.3832



JNJ18157698
1.2196







HNF3b Expression - Moderate Hits










RWJ675430
1.1562



JNJ10222784
1.1010



JNJ5226780
1.0087



RWJ355923
0.9520



RWJ353709
0.9515



RWJ676139
0.9499



JNJ10184655
0.8807



JNJ10182562
0.8683



RWJ664545
0.8649



RWJ674239
0.7718



RWJ676432
0.7711



reference cmpd
0.7576



JNJ10164895
0.7428



JNJ10179130
0.7104



JNJ10172058
0.6803



JNJ8710481
0.6412



JNJ10178727
0.5866

















TABLE VI







EFFECTS OF INHIBITORS OF GSK-3B ENZYME ACTIVITY ON


THE PROLIFERATION OF HUMAN EMBRYONIC STEM CELLS.












JNJ number
Raw Data
Average
S.D.
% CV
% Control

















conditioned medium
1.1348
1.0099
1.1092
1.0846
0.0660
6.1
116.5


no treatment
0.9344
0.5977
0.8454
0.7925
0.1745
22.0
85.2


AA/DMSO
0.3878
0.2434
0.2252
0.2855
0.0891
31.2
30.7


AA/Wnt3a/DMSO
0.6098
1.0804
0.7635
0.8179
0.2403
25.8
100.0


RWJ351001
0.3418
0.4276
0.5751
0.4482
0.1180
26.3
48.2


RWJ351958
0.1362
0.1531
0.1532
0.1475
0.0098
6.6
15.8


RWJ352190
1.3764
1.2753
1.3208
1.3242
0.0506
3.8
142.3


RWJ352244
0.6923
0.5994
0.6134
0.6350
0.0501
7.9
68.2


RWJ352628
1.7896
1.4721
2.1908
1.8175
0.3602
19.8
195.3


RWJ353258
1.7591
1.6274
1.6518
1.6794
0.0701
4.2
180.4


RWJ355131
0.3702
0.3193
0.3368
0.3421
0.0259
7.6
36.8


RWJ355923
0.5876
0.6384
0.9154
0.7138
0.1764
24.7
76.7


RWJ356205
0.3074
0.2328
0.2920
0.2774
0.0394
14.2
29.8


RWJ382867
0.1311
0.1245
0.1288
0.1281
0.0034
2.6
13.8


RWJ395477
0.1270
0.2778
0.1916
0.1988
0.0757
38.1
21.4


RWJ414342
0.2166
0.3062
0.2915
0.2714
0.0481
17.7
29.2


RWJ414984
0.4362
0.3728
0.2481
0.3524
0.0957
27.2
37.9


RWJ425264
0.1560
0.1481
0.1359
0.1467
0.0101
6.9
15.8


RWJ425268
0.2932
0.3883
0.6258
0.4358
0.1713
39.3
46.8


RWJ425271
0.1362
0.1479
0.1298
0.1380
0.0092
6.7
14.8


RWJ425348
0.2198
0.2159
0.2300
0.2219
0.0073
3.3
23.8


RWJ445224
0.7624
0.2705
0.2478
0.4269
0.2908
68.1
45.9


RWJ447228
0.1239
0.1233
0.1269
0.1247
0.0019
1.5
13.4


RWJ553709
0.1277
0.1254
0.6980
0.3170
0.3299
104.1
34.1


RWJ659780
0.2665
0.3215
0.2605
0.2828
0.0336
11.9
30.4


RWJ662440
0.2395
0.3235
0.1333
0.2321
0.0953
41.1
24.9


RWJ663860
0.2646
0.1873
0.1293
0.1937
0.0679
35.0
20.8


RWJ664545
0.3590
0.2790
0.1515
0.2632
0.1047
39.8
28.3


RWJ665436
0.4690
0.5805
0.3349
0.4615
0.1230
26.6
49.6


conditioned medium
1.1525
1.1269
1.1140
1.1311
0.0196
1.7
71.0


no treatment
1.2057
1.2358
1.3132
1.2516
0.0555
4.4
78.6


AA/DMSO
0.2622
0.2073
0.2830
0.2508
0.0391
15.6
15.8


AA/Wnt3a/DMSO
1.3943
1.7976
1.8000
1.5922
0.2136
13.4
100.0


RWJ665588
0.1930
0.2223
0.2167
0.2107
0.0156
7.4
13.2


RWJ665862
0.1757
0.1813
0.1835
0.1802
0.0040
2.2
11.3


RWJ666167
0.1473
0.1880
0.1732
0.1695
0.0206
12.2
10.6


RWJ666168
0.1330
0.1362
0.1867
0.1520
0.0301
19.8
9.5


RWJ666205
0.8191
0.5493
0.6526
0.6737
0.1361
20.2
42.3


RWJ666213
0.4008
0.2779
0.3869
0.3552
0.0673
18.9
22.3


RWJ667045
0.1220
0.1248
0.1251
0.1240
0.0017
1.4
7.8


RWJ667046
0.2883
0.3308
0.5503
0.3898
0.1406
36.1
24.5


RWJ667069
0.2835
0.4024
0.5698
0.4186
0.1438
34.4
26.3


RWJ669182
0.3704
0.6073
0.5280
0.5019
0.1206
24.0
31.5


RWJ669327
0.2266
0.1815
0.2289
0.2123
0.0267
12.6
13.3


RWJ670804
1.0820
1.1862
1.1076
1.1253
0.0543
4.8
70.7


RWJ670908
0.3590
0.5457
0.6123
0.5057
0.1313
26.0
31.8


RWJ670984
0.2198
0.3564
0.3202
0.2988
0.0708
23.7
18.8


RWJ671232
0.2928
0.2920
0.3659
0.3169
0.0424
13.4
19.9


RWJ672667
0.3349
0.3013
0.3507
0.3290
0.0252
7.7
20.7


RWJ672932
0.1852
0.1924
0.2349
0.2042
0.0269
13.2
12.8


RWJ672934
0.2170
0.3003
0.1877
0.2350
0.0584
24.9
14.8


RWJ673313
0.3094
0.2515
0.1881
0.2497
0.0607
24.3
15.7


RWJ673515
1.8452
1.7710
1.5591
1.7251
0.1485
8.6
108.3


RWJ673829
0.7305
0.7067
0.6250
0.6874
0.0553
8.0
43.2


RWJ673830
0.2113
0.1800
0.1547
0.1820
0.0284
15.6
11.4


RWJ674239
1.5225
1.5912
0.1081
1.0739
0.8371
78.0
67.4


RWJ674240
0.4006
1.2807
0.1162
0.5992
0.6071
101.3
37.6


RWJ674241
0.1972
0.1839
0.1162
0.1658
0.0434
26.2
10.4


RWJ674320
0.1351
0.1318
0.1169
0.1279
0.0097
7.6
8.0


conditioned medium
1.0568
1.0604

1.0586
0.0025
0.2
71.9


no treatment
1.1544
0.9576

1.0560
0.1392
13.2
71.7


AA only+ DMSO
0.6329
0.8434

0.7382
0.1488
20.2
47.1


AA + Wnt3a + DMSO
1.2704
1.8669

1.4229
0.2960
20.8
100.0


RWJ674817
0.5617
0.2098

0.3858
0.2488
64.5
19.9


RWJ674855
0.6850
0.5853

0.6352
0.0705
11.1
39.2


RWJ674855
0.7496
0.9187

0.8342
0.1196
14.3
54.5


RWJ675104
0.2320
0.2124

0.2222
0.0139
6.2
7.3


RWJ675260
0.8079
1.4391

1.1235
0.4463
39.7
76.9


RWJ675261
0.8310
0.7318

0.7814
0.0701
9.0
50.5


RWJ675266
1.0646
1.1384

1.1015
0.0522
4.7
75.2


RWJ675366
0.6344
1.0400

0.8372
0.2868
34.3
54.8


no cells
0.1335
0.2070

0.1703
0.0520
30.5
3.3


RWJ675369
0.8643
0.4060

0.6352
0.3241
51.0
39.2


RWJ675430
1.7922
1.8533

1.8228
0.0432
2.4
130.9


RWJ675578
0.1914
0.2371

0.2143
0.0323
15.1
6.7


RWJ675605
1.8401
1.7563

1.7982
0.0593
3.3
129.0


RWJ675881
1.0301
1.0356

1.0329
0.0039
0.4
69.9


RWJ675946
0.1306
0.1338

0.1322
0.0023
1.7
0.3


RWJ675948
1.7143
1.6506

1.6825
0.0450
2.7
120.0


RWJ676061
0.4170
0.4956

0.4563
0.0556
12.2
25.4


RWJ676085
0.1772
0.2348

0.2060
0.0407
19.8
6.0


RWJ676137
1.0231
1.2392

1.1312
0.1528
13.5
77.5


RWJ676139
1.9718
2.0997

2.0358
0.0904
4.4
147.3


RWJ676431
1.5168
1.6872

1.6020
0.1205
7.5
113.8


RWJ676432
1.6935
1.9710

1.8323
0.1962
10.7
131.6


RWJ67657
1.2655
1.1829

1.2242
0.0584
4.8
84.7


RWJ676639
1.3481
1.3168

1.3325
0.0221
1.7
93.0


JNJ26511966
0.6444
0.7239

0.6842
0.0562
8.2
43.0


JNJ26511979
0.2046
0.3076

0.2561
0.0728
28.4
9.9


JNJ26512005
1.3627
1.0693

1.2160
0.2075
17.1
84.0


JNJ26533065
0.8722
0.9660

0.9191
0.0663
7.2
61.1


JNJ26533091
1.0332
0.4554

0.7443
0.4086
54.9
47.6


JNJ26533104
0.8775
0.7347

0.8061
0.1010
12.5
52.4


JNJ26533156
1.7865
1.2008

1.4937
0.4142
27.7
105.5


JNJ26714181
0.2396
0.1584

0.1990
0.0574
28.9
5.5


JNJ26714194
0.8122
1.0827

0.9475
0.1913
20.2
63.3


JNJ26714207
0.1342
0.1363

0.1353
0.0015
1.1
0.6


JNJ26714220
1.0462
0.5838

0.8150
0.3270
40.1
53.1


JNJ26875563
0.4586
0.2903

0.3745
0.1190
31.8
19.0


JNJ22791671
0.1277
0.1402

0.1340
0.0088
6.6
0.5


JNJ26893438
0.1258
0.1324

0.1291
0.0047
3.6
0.1


JNJ26941226
0.1219
0.1216

0.1218
0.0002
0.2
−0.5


JNJ28572128
0.4223
0.4721

0.4472
0.0352
7.9
24.7


JNJ28850601
0.1514
0.1396

0.1455
0.0083
5.7
1.4


conditioned medium
0.7423
0.7081

0.7252
0.0242
3.3
87.7


no treatment
0.4936
0.5689

0.5313
0.0532
10.0
59.8


AA only + DMSO
0.1433
0.1939

0.1686
0.0358
21.2
7.6


AA + Wnt3a + DMSO
0.6808
0.9406

0.8107
0.1837
22.7
100.0


JNJ17994873
0.2447
0.1331

0.1889
0.0789
41.8
10.6


JNJ17994899
0.1537
0.1302

0.1420
0.0166
11.7
3.8


no cells
0.1163
0.1147

0.1155
0.0011
1.0
0.0


JNJ17994912
0.2994
0.2592

0.2793
0.0284
10.2
23.6


JNJ17994925
0.1353
0.2121

0.1737
0.0543
31.3
8.4


JNJ180125
0.1267
0.1419

0.1343
0.0107
8.0
2.7


JNJ18014061
0.1376
0.1676

0.1526
0.0212
13.9
5.3


JNJ18014074
0.1134
0.1103

0.1119
0.0022
2.0
−0.5


JNJ18018338
0.1318
0.1478

0.1398
0.0113
8.1
3.5


JNJ18018351
0.2569
0.2124

0.2347
0.0315
13.4
17.1


JNJ18047991
0.2674
0.2636

0.2655
0.0027
1.0
21.6


JNJ18055726
0.4357
0.3467

0.3912
0.0629
16.1
39.7


JNJ18077800
0.1265
0.1588

0.1427
0.0228
16.0
3.9


JNJ18157074
0.1662
0.2521

0.2092
0.0607
29.0
13.5


JNJ18157087
0.1596
0.1566

0.1581
0.0021
1.3
6.1


JNJ18157646
0.2725
0.1636

0.2181
0.0770
35.3
14.8


JNJ18157711
1.2256
1.0636

1.1446
0.1146
10.0
148.0


JNJ18157711
0.1134
0.1070

0.1102
0.0045
4.1
−0.8


JNJ19363357
0.1469
0.1495

0.1482
0.0018
1.2
4.7


JNJ19369233
0.1169
0.1122

0.1146
0.0033
2.9
−0.1


JNJ19369246
0.1595
0.1422

0.1509
0.0122
8.1
5.1


JNJ19370026
1.0484
1.0749

1.0617
0.0187
1.8
136.1


JNJ19376240
0.3012
0.2347

0.2680
0.0470
17.5
21.9


JNJ19386042
0.1267
0.1510

0.1389
0.0172
12.4
3.4


JNJ19410833
1.1902
1.1487

1.1695
0.0293
2.5
151.6


JNJ19410859
0.6400
0.7076

0.6738
0.0478
7.1
80.3


JNJ19410872
0.1701
0.1752

0.1727
0.0036
2.1
8.2


JNJ19558929
0.3435
0.3488

0.3462
0.0037
1.1
33.2


JNJ19567314
0.4032
0.3548

0.3790
0.0342
9.0
37.9


JNJ19567327
0.1602
0.1502

0.1552
0.0071
4.6
5.7


JNJ19567340
0.1604
0.2079

0.1842
0.0336
18.2
9.9


JNJ19567405
0.1646
0.1592

0.1619
0.0038
2.4
6.7


JNJ19573541
0.1779
0.2273

0.2026
0.0349
17.2
12.5


JNJ19574867
0.1225
0.1443

0.1334
0.0154
11.6
2.6


JNJ19574880
0.1300
0.1291

0.1296
0.0006
0.5
2.0


JNJ20948798
0.1263
0.1336

0.1300
0.0052
4.0
2.1


JNJ21192730
0.2778
0.1326

0.2052
0.1027
50.0
12.9


JNJ21194667
0.2569
0.1219

0.1894
0.0955
50.4
10.6


JNJ21196227
0.1640
0.1158

0.1399
0.0341
24.4
3.5


JNJ24843611
1.1486
0.8970

1.0228
0.1779
17.4
130.5


JNJ24843611
0.1358
0.1201

0.1280
0.0111
8.7
1.8


JNJ24326185
0.1257
0.1257

0.1257
0.0000
0.0
1.5


JNJ24843572
0.4676
0.4803

0.4740
0.0090
1.9
51.6


conditioned medium
0.6935
0.7803

0.7369
0.0614
8.3
104.8


no treatment
0.4735
0.6069

0.5402
0.0943
17.5
71.5


AA only+ DMSO
0.1428
0.1656

0.1542
0.0161
10.5
6.3


AA + Wnt3a + DMSO
0.5702
0.8468

0.7085
0.1956
27.6
100.0


JNJ24843585
0.1599
0.2380

0.1990
0.0552
27.8
13.8


JNJ25753520
0.1287
0.1244

0.1266
0.0030
2.4
1.6


no cells
0.1241
0.1100

0.1171
0.0100
8.5
0.0


JNJ25753403
0.1235
0.1152

0.1194
0.0059
4.9
0.4


JNJ25757173
0.1199
0.1278

0.1239
0.0056
4.5
1.1


JNJ25757173
0.1174
0.1162

0.1168
0.0008
0.7
−0.1


JNJ25757238
1.1100
0.9464

1.0282
0.1157
11.3
154.1


JNJ25758707
0.1247
0.1115

0.1181
0.0093
7.9
0.2


JNJ25758785
0.2640
0.1688

0.2164
0.0673
31.1
16.8


JNJ25758850
0.2313
0.1307

0.1810
0.0711
39.3
10.8


JNJ25758863
0.8639
0.9218

0.8929
0.0409
4.6
131.2


JNJ25873419
0.2540
0.2320

0.2430
0.0156
6.4
21.3


JNJ25887537
0.1809
0.3077

0.2443
0.0897
36.7
21.5


JNJ25900641
0.1892
0.1872

0.1882
0.0014
0.8
12.0


JNJ25900654
0.1967
0.2492

0.2230
0.0371
16.7
17.9


JNJ25900706
0.3346
0.1619

0.2483
0.1221
49.2
22.2


JNJ26047723
0.1106
0.1138

0.1122
0.0023
2.0
−0.8


JNJ26054912
0.1224
0.1445

0.1335
0.0156
11.7
2.8


JNJ26064571
0.1312
0.1270

0.1291
0.0030
2.3
2.0


JNJ26067626
0.1653
0.2114

0.1884
0.0326
17.3
12.0


JNJ26067652
0.1732
0.1467

0.1600
0.0187
11.7
7.2


JNJ26069901
0.1618
0.2754

0.2186
0.0803
36.7
17.2


JNJ26077883
1.0006
0.9631

0.9819
0.0265
2.7
146.2


JNJ26116922
0.6472
0.4319

0.5396
0.1522
28.2
71.4


JNJ26120601
0.1539
0.1469

0.1504
0.0049
3.3
5.6


JNJ26120614
0.1127
0.1309

0.1218
0.0129
10.6
0.8


JNJ26128726
0.6887
0.5860

0.6374
0.0726
11.4
88.0


JNJ26130403
0.1141
0.1094

0.1118
0.0033
3.0
−0.9


JNJ26134771
0.2774
0.1690

0.2232
0.0767
34.3
17.9


JNJ26150202
0.9482
1.1150

1.0316
0.1179
11.4
154.6


JNJ26153647
0.7687
0.6804

0.7246
0.0624
8.6
102.7


JNJ26158015
0.7125
0.3347

0.5236
0.2671
51.0
68.7


JNJ26158054
0.1446
0.1221

0.1334
0.0159
11.9
2.7


JNJ26158093
1.0968
1.3108

1.2038
0.1513
12.6
183.8


JNJ26158106
0.3167
0.3415

0.3291
0.0175
5.3
35.8


JNJ26161343
0.1261
0.1144

0.1203
0.0083
6.9
0.5


JNJ26170794
0.2223
0.2930

0.2577
0.0500
19.4
23.8


JNJ26170820
0.1265
0.1236

0.1251
0.0021
1.6
1.3


JNJ26170833
1.1940
0.9431

1.0686
0.1774
16.6
160.9


JNJ26177086
1.0689
0.6879

0.8784
0.2694
30.7
128.7


JNJ26177762
1.0444
0.7603

0.9024
0.2009
22.3
132.8


JNJ26184457
0.1443
0.1209

0.1326
0.0165
12.5
2.6


JNJ26219050
0.1152
0.1309

0.1231
0.0111
9.0
1.0


conditioned medium
0.7590
0.7451

0.7521
0.0098
1.3
98.0


no treatment
0.5687
0.4490

0.5089
0.0846
16.6
60.4


AA only + DMSO
0.1988
0.1522

0.1755
0.0330
18.8
8.9


AA + Wnt3a + DMSO
0.6837
0.8460

0.7649
0.1148
15.0
100.0


JNJ26219063
0.1911
0.1101

0.1506
0.0573
38.0
5.0


JNJ26220454
0.2772
0.1151

0.1962
0.1146
58.4
12.1


no cells
0.1278
0.1084

0.1181
0.0137
11.6
0.0


JNJ26241774
0.1443
0.2120

0.1782
0.0479
26.9
9.3


JNJ26241917
0.4413
0.2238

0.3326
0.1538
46.2
33.2


JNJ26243204
0.1098
0.1085

0.1092
0.0009
0.8
−1.4


JNJ26247143
0.1389
0.2147

0.1768
0.0536
30.3
9.1


JNJ26248729
0.1852
0.1342

0.1597
0.0361
22.6
6.4


JNJ26261105
0.1114
0.1295

0.1205
0.0128
10.6
0.4


JNJ26361712
0.5375
0.6158

0.5767
0.0554
9.6
70.9


JNJ26361725
0.1259
0.1441

0.1350
0.0129
9.5
2.6


JNJ26366730
0.1206
0.1312

0.1259
0.0075
6.0
1.2


JNJ26367991
0.2269
0.2857

0.2563
0.0416
16.2
21.4


JNJ26367991
0.1140
0.1079

0.1110
0.0043
3.9
−1.1


JNJ26399906
0.9589
0.8868

0.9229
0.0510
5.5
124.4


JNJ26399906
1.0442
0.9622

1.0032
0.0580
5.8
136.8


JNJ26399945
0.1961
0.1735

0.1848
0.0160
8.6
10.3


JNJ26399971
0.5732
0.5216

0.5474
0.0365
6.7
66.4


JNJ26399984
0.1273
0.1217

0.1245
0.0040
3.2
1.0


JNJ26399997
0.5932
0.6671

0.6302
0.0523
8.3
79.2


JNJ26400049
0.1444
0.1368

0.1406
0.0054
3.8
3.5


JNJ26483197
1.0786
1.0891

1.0839
0.0074
0.7
149.3


JNJ26483310
0.5418
0.2338

0.3878
0.2178
56.2
41.7


JNJ26483223
0.1268
0.2052

0.1660
0.0554
33.4
7.4


JNJ26483236
0.1169
0.1184

0.1177
0.0011
0.9
−0.1


JNJ26483249
0.8618
1.0400

0.9509
0.1260
13.3
128.8


JNJ26483249
0.8430
1.0187

0.9309
0.1242
13.3
125.7


JNJ26483262
0.3659
0.3168

0.3414
0.0347
10.2
34.5


JNJ26511901
0.9184
0.8116

0.8650
0.0755
8.7
115.5


JNJ26511927
0.2384
0.3156

0.2770
0.0546
19.7
24.6


JNJ26511953
0.2297
0.1469

0.1883
0.0585
31.1
10.9


RWJ67694
0.1955
0.1256

0.1606
0.0494
30.8
6.6


RWJ676940
0.1658
0.1704

0.1681
0.0033
1.9
7.7


RWJ677545
0.1399
0.1303

0.1351
0.0068
5.0
2.6


RWJ678986
0.1234
0.1236

0.1235
0.0001
0.1
0.8


RWJ680665
0.1397
0.2147

0.1772
0.0530
29.9
9.1


RWJ680667
0.1218
0.1310

0.1264
0.0065
5.1
1.3


RWJ680668
0.1456
0.1981

0.1719
0.0371
21.6
8.3


RWJ680669
0.5412
0.1898

0.3655
0.2485
68.0
38.2


RWJ680858
0.1996
0.1245

0.1621
0.0531
32.8
6.8


RWJ680858
0.1418
0.2014

0.1716
0.0421
24.6
8.3


RWJ680879
0.1106
0.1197

0.1152
0.0064
5.6
−0.5


RWJ680885
0.1159
0.1272

0.1216
0.0080
6.6
0.5


conditioned medium
0.8077
0.7210

0.7644
0.0613
8.0
74.7


no treatment + DMSO
0.4638
0.4073

0.4356
0.0400
9.2
36.7


AA/Wnt3a
0.8466
0.9935

0.9830
0.2592
26.4
100.0


JNJ10222784
0.8095
0.9055

0.8575
0.0679
7.9
85.5


JNJ10222927
0.3519
0.4708

0.4114
0.0841
20.4
33.9


JNJ10231273
0.1609
0.1275

0.1442
0.0236
16.4
3.1


JNJ10259847
0.5020
0.2733

0.3877
0.1617
41.7
31.2


JNJ10259847
0.3413
0.4146

0.3780
0.0518
13.7
30.1


JNJ17154215
0.1176
0.1174

0.1175
0.0001
0.1
0.0


JNJ17154215
0.1148
0.1410

0.1279
0.0185
14.5
1.2


JNJ17157659
0.2394
0.2450

0.2422
0.0040
1.6
14.4


JNJ17163042
0.3672
0.3098

0.3385
0.0406
12.0
25.5


JNJ10166565
0.2722
0.1593

0.2158
0.0798
37.0
11.3


JNJ17174664
0.5079
0.4349

0.4714
0.0516
11.0
40.9


JNJ17187027
0.1076
0.1168

0.1122
0.0065
5.8
−0.6


JNJ17187053
0.2569
0.2151

0.2360
0.0296
12.5
13.7


JNJ17193774
0.2846
0.4376

0.3611
0.1082
30.0
28.1


JNJ17200976
0.1168
0.1136

0.1152
0.0023
2.0
−0.3


JNJ17205955
0.1168
0.1152

0.1160
0.0011
1.0
−0.2


JNJ17205955
0.1137
0.1195

0.1166
0.0041
3.5
−0.1


JNJ17205994
0.1154
0.1152

0.1153
0.0001
0.1
−0.3


JNJ17226703
0.2188
0.2353

0.2271
0.0117
5.1
12.6


JNJ17982133
0.4588
0.2521

0.3555
0.1462
41.1
27.5


JNJ17989049
0.3081
0.1961

0.2521
0.0792
31.4
15.5


conditioned medium
0.7914
1.1189

0.9552
0.2316
24.2
93.3


no treatment
0.4215
0.5259

0.4737
0.0738
15.6
39.8


no cells
0.1152
0.1160

0.1156
0.0006
0.5
0.0


AA/Wnt3a
0.7168
0.8836

1.0151
0.2016
19.9
100.0


RWJ680991
0.2882
0.2308

0.2844
0.0499
17.6
18.8


RWJ680992
0.3049
0.2845

0.3127
0.0282
9.0
21.9


RWJ680993
0.5403
0.2570

0.3855
0.1332
34.6
30.0


RWJ681140
0.7323
0.3034

0.4388
0.2041
46.5
35.9


RWJ681142
0.1185
0.1216

0.1199
0.0018
1.5
0.5


RWJ681146
0.2496
0.2683

0.2302
0.0376
16.3
12.7


RWJ681945
0.1548
0.1356

0.1513
0.0134
8.8
4.0


RWJ68198
0.1555
0.1450

0.1581
0.0161
10.2
4.7


RWJ682205
0.2347
0.1920

0.3785
0.2589
68.4
29.2


RWJ447228
0.1842
0.2093

0.3793
0.2585
68.2
29.3


RWJ675430
0.7223
0.8707

0.4291
0.2452
57.2
34.8


RWJ355923
0.6268
0.3192

0.3354
0.1667
49.7
24.4
















TABLE VII







EFFECTS OF INHIBITORS OF GSK-3B ENZYME


ACTIVITY ON THE PROLIFERATION OF


HUMAN EMBRYONIC STEM CELLS.










JNJ Number
% Control Value











List Strong Hits


>= 120% control










RWJ352628
195.3



JNJ26158093
183.8



RWJ353258
180.4



JNJ26170833
160.9



JNJ26150202
154.6



JNJ25757238
154.1



JNJ19410833
151.6



JNJ26483197
149.3



JNJ18157711
148.0



RWJ676139
147.3



JNJ26077883
146.2



RWJ352190
142.3



JNJ26399906
136.8



JNJ19370026
136.1



JNJ26177762
132.8



RWJ676432
131.6



JNJ25758863
131.2



RWJ675430
130.9



JNJ24843611
130.5



RWJ675605
129.0



JNJ26483249
128.8



JNJ26177086
128.7



JNJ26483249
125.7



JNJ26399906
124.4



RWJ675948
120.0







List Moderate Hits


60-120% control










JNJ26511901
115.5



RWJ676431
113.8



RWJ673515
108.3



JNJ26533156
105.5



JNJ26153647
102.7



RWJ676639
93.0



JNJ26128726
88.0



JNJ10222784
85.5



RWJ67657
84.7



JNJ26512005
84.0



JNJ19410859
80.3



JNJ26399997
79.2



RWJ676137
77.5



RWJ675260
76.9



RWJ355923
76.7



RWJ675266
75.2



JNJ26116922
71.4



JNJ26361712
70.9



RWJ670804
70.7



RWJ675881
69.9



JNJ26158015
68.7



RWJ352244
68.2



RWJ674239
67.4



JNJ26399971
66.4



JNJ26714194
63.3



JNJ26533065
61.1

















TABLE VIII







DOSE-DEPENDANT EFFECTS OF INHIBITORS OF GSK-3B


ENZYME ACTIVITY ON THE PROLIFERATION OF CELLS OF THE HUMAN


EMBRYONIC STEM CELL LINE H1.

















Concentration [uM]
Cell number
SD
Cell number
SD
Cell number
SD
Cell number
SD
Cell number
SD
















JNJ10220067
JNJ17163796
JNJ17189731
JNJ17223375
JNJ18157698

















10
1.006
0.051
0.039
0.049
0.193
0.147
1.280
0.014
1.049
0.062


5
1.058
0.047
1.164
0.018
0.889
0.035
1.348
0.007
1.104
0.014


2.5
1.031
0.054
1.022
0.023
0.896
0.035
1.318
0.028
0.932
0.087


1.25
0.899
0.040
1.121
0.023
1.120
0.072
1.159
0.041
1.006
0.023


0.625
0.742
0.095
1.092
0.044
1.107
0.093
1.029
0.018
0.832
0.026


0.313
0.754
0.010
0.931
0.056
1.132
0.018
1.018
0.044
0.742
0.127


0.156
0.822
0.074
0.804
0.002
1.082
0.041
0.776
0.054
0.712
0.020













JNJ26158015
JNJ26483197
JNJ26483249
JNJ17225871
JNJ17228458

















10
0.001
0.001
0.096
0.103
0.058
0.074
0.290
0.307
0.000
0.000


5
0.034
0.035
0.262
0.268
0.173
0.207
0.458
0.263
0.089
0.067


2.5
0.566
0.461
0.592
0.019
0.428
0.326
0.640
0.104
0.438
0.050


1.25
0.897
0.103
1.124
0.101
0.850
0.238
0.739
0.129
0.636
0.016


0.625
0.921
0.122
1.106
0.056
0.910
0.061
0.805
0.036
0.736
0.025


0.313
1.028
0.069
0.888
0.213
0.868
0.131
0.785
0.094
0.791
0.038


0.156
1.027
0.067
0.890
0.079
0.742
0.051
0.774
0.027
0.832
0.005













JNJ19370026
JNJ26150202
JNJ26170833
JNJ26177086
JNJ26177762

















10
0.000
0.000
0.496
0.690
0.129
0.170
0.412
0.081
0.996
0.246


5
0.024
0.034
0.768
0.490
0.530
0.080
1.128
0.026
0.908
0.179


2.5
1.097
0.294
1.001
0.129
1.174
0.016
1.031
0.217
1.005
0.086


1.25
1.446
0.076
1.158
0.043
1.113
0.057
0.914
0.100
1.200
0.065


0.625
1.296
0.183
0.699
0.248
1.188
0.041
0.801
0.136
1.111
0.300


0.313
1.034
0.197
0.617
0.232
1.158
0.102
0.785
0.121
0.959
0.094


0.156
0.826
0.030
0.812
0.120
0.974
0.065
0.659
0.068
0.912
0.059













JNJ26512005
JNJ26533065
JNJ26533156
JNJ26714194
JNJ3026582

















10
0.000
0.000
0.021
0.027
0.002
0.002
0.052
0.067
0.053
0.024


5
0.000
0.000
0.339
0.254
1.011
0.499
1.161
0.134
0.905
0.036


2.5
0.192
0.233
1.350
0.170
1.724
0.042
1.293
0.020
1.019
0.015


1.25
0.552
0.458
1.277
0.101
1.652
0.032
1.213
0.087
1.163
0.062


0.625
0.895
0.054
0.713
0.151
1.357
0.023
1.025
0.045
1.231
0.152


0.313
0.734
0.075
0.665
0.207
1.213
0.177
1.241
0.031
1.216
0.007


0.156
0.594
0.078
0.469
0.465
1.206
0.142
1.041
0.007
1.103
0.065
















TABLE IX







DOSE-DEPENDANT EFFECTS OF INHIBITORS OF GSK-3B


ENZYME ACTIVITY ON THE DIFFERENTIATION OF CELLS OF THE


HUMAN EMBRYONIC STEM CELL LINE H1.

















Concentration












[uM]
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
















JNJ10220067
JNJ17163796
JNJ17189731
JNJ17223375
JNJ18157698

















10
0.889
0.144
0.029
0.034
0.140
0.095
1.183
0.044
0.969
0.040


5
1.004
0.021
0.824
0.035
0.785
0.077
1.171
0.010
1.013
0.002


2.5
1.023
0.092
0.849
0.003
0.842
0.032
1.169
0.031
0.838
0.068


1.25
0.954
0.100
0.985
0.082
1.028
0.043
1.106
0.006
0.940
0.071


0.625
0.793
0.135
0.986
0.059
1.016
0.000
0.931
0.033
0.767
0.014


0.313
0.803
0.048
0.916
0.028
1.058
0.017
0.943
0.056
0.692
0.167


0.156
0.941
0.106
0.822
0.036
1.039
0.015
0.789
0.074
0.651
0.032













JNJ26158015
JNJ26483197
JNJ26483249
JNJ17225871
JNJ17228458

















10
0.001
0.001
0.034
0.027
0.054
0.063
0.267
0.280
0.000
0.001


5
0.017
0.020
0.071
0.054
0.141
0.169
0.402
0.229
0.056
0.035


2.5
0.200
0.157
0.497
0.076
0.373
0.326
0.605
0.041
0.286
0.034


1.25
0.792
0.066
0.993
0.144
0.783
0.282
0.686
0.185
0.587
0.023


0.625
0.824
0.118
1.061
0.066
0.887
0.062
0.786
0.061
0.695
0.001


0.313
0.934
0.127
0.937
0.136
0.859
0.176
0.780
0.132
0.753
0.098


0.156
0.986
0.055
0.888
0.062
0.666
0.015
0.782
0.061
0.816
0.043













JNJ19370026
JNJ26150202
JNJ26170833
JNJ26177086
JNJ26177762

















10
0.000
0.000
0.491
0.681
0.281
0.358
0.330
0.059
0.701
0.307


5
0.035
0.049
0.158
0.224
0.460
0.189
0.846
0.036
0.728
0.146


2.5
1.336
0.192
0.800
0.201
1.018
0.139
0.887
0.191
0.928
0.019


1.25
1.238
0.030
0.910
0.045
0.960
0.106
0.819
0.179
1.159
0.093


0.625
0.997
0.095
0.567
0.190
1.050
0.038
0.755
0.126
1.136
0.186


0.313
0.791
0.172
0.515
0.276
1.032
0.063
0.667
0.125
1.006
0.009


0.156
0.669
0.037
0.708
0.148
0.950
0.087
0.628
0.053
0.922
0.096













JNJ26512005
JNJ26533065
JNJ26533156
JNJ26714194
JNJ3026582

















10
0.000
0.000
0.018
0.021
0.002
0.001
0.054
0.062
0.074
0.048


5
0.000
0.000
0.235
0.174
1.052
0.281
1.250
0.177
1.006
0.070


2.5
0.270
0.382
1.153
0.223
1.459
0.074
1.186
0.069
1.120
0.038


1.25
0.678
0.434
1.055
0.046
1.322
0.078
1.112
0.038
1.122
0.009


0.625
0.978
0.021
0.569
0.124
1.173
0.015
0.913
0.005
1.241
0.230


0.313
0.742
0.048
0.555
0.118
1.102
0.165
1.140
0.036
1.231
0.012


0.156
0.508
0.049
0.451
0.443
1.060
0.126
0.998
0.006
1.034
0.008
















TABLE X







DOSE-DEPENDANT EFFECTS OF INHIBITORS OF GSK-3B


ENZYME ACTIVITY ON THE PROLIFERATION OF CELLS OF THE HUMAN


EMBRYONIC STEM CELL LINE H9.

















Concentration [uM]
Cell number
SD
Cell number
SD
Cell number
SD
Cell number
SD
Cell number
SD
















JNJ10220067
JNJ17163796
JNJ17189731
JNJ17223375
JNJ18157698

















10
0.164
0.209
0.001
0.000
0.049
0.028
0.123
0.106
0.770
0.077


5
0.147
0.141
0.616
0.497
0.583
0.155
0.954
0.146
0.496
0.011


2.5
0.140
0.112
1.295
0.402
1.108
0.170
0.795
0.101
0.384
0.247


1.25
0.307
0.198
1.233
0.058
1.195
0.147
0.541
0.051
0.395
0.002


0.625
0.138
0.071
0.606
0.121
1.100
0.014
0.332
0.049
0.221
0.009


0.313
0.063
0.008
0.397
0.020
0.887
0.078
0.206
0.085
0.172
0.071


0.156
0.069
0.001
0.214
0.025
0.699
0.109
0.142
0.039
0.138
0.048













JNJ26158015
JNJ26483197
JNJ26483249
JNJ17225871
JNJ17228458

















10
0.001
0.000
0.785
0.192
0.208
0.134
0.377
0.040
0.000
0.000


5
0.023
0.024
1.067
0.236
0.320
0.087
0.336
0.081
0.052
0.009


2.5
0.681
0.223
1.368
0.025
0.388
0.019
0.296
0.016
0.089
0.003


1.25
1.011
0.461
1.477
0.147
0.334
0.113
0.222
0.035
0.106
0.003


0.625
0.927
0.108
0.899
0.108
0.267
0.148
0.282
0.096
0.169
0.041


0.313
0.686
0.022
0.540
0.094
0.192
0.056
0.208
0.003
0.119
0.026


0.156
0.458
0.001
0.206
0.089
0.147
0.067
0.174
0.051
0.067
0.015













JNJ19370026
JNJ26150202
JNJ26170833
JNJ26177086
JNJ26177762

















10
0.000
0.000
0.452
0.094
0.002
0.001
1.117
0.043
1.022
0.422


5
0.002
0.000
0.433
0.050
1.325
0.015
0.793
0.030
1.281
0.109


2.5
0.668
0.059
0.521
0.229
1.355
0.026
0.600
0.122
1.197
0.068


1.25
0.988
0.032
0.293
0.038
1.182
0.076
0.442
0.018
1.039
0.213


0.625
0.390
0.032
0.200
0.122
0.928
0.127
0.371
0.072
0.686
0.014


0.313
0.250
0.090
0.072
0.025
0.772
0.050
0.100
0.008
0.437
0.066


0.156
0.095
0.020
0.057
0.044
0.336
0.056
0.072
0.015
0.276
0.043













JNJ26512005
JNJ26533065
JNJ26533156
JNJ26714194
JNJ3026582

















10
0.007
0.002
0.000
0.000
0.000
0.000
0.044
0.038
0.004
0.001


5
0.002
0.001
0.127
0.069
0.415
0.023
0.382
0.110
0.017
0.003


2.5
0.001
0.001
0.151
0.059
0.425
0.082
0.345
0.001
0.033
0.037


1.25
0.090
0.097
0.108
0.051
0.325
0.042
0.284
0.076
0.044
0.028


0.625
0.248
0.058
0.230
0.168
0.314
0.062
0.266
0.021
0.100
0.099


0.313
0.264
0.048
0.086
0.033
0.267
0.098
0.347
0.084
0.057
0.032


0.156
0.133
0.069
0.063
0.004
0.218
0.012
0.192
0.014
0.070
0.048
















TABLE XI







DOSE-DEPENDANT EFFECTS OF INHIBITORS OF GSK-3B


ENZYME ACTIVITY ON THE DIFFERENTIATION OF CELLS OF THE


HUMAN EMBRYONIC STEM CELL LINE H9.

















Concentration












[μM]
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
Sox17 Intensity
SD
















JNJ10220067
JNJ17163796
JNJ17189731
JNJ17223375
JNJ18157698

















0.157
0.051
0.003
0.132
0.003
0.678
0.093
0.116
0.047
0.095
0.025


0.313
0.052
0.008
0.311
0.005
0.951
0.010
0.155
0.071
0.110
0.030


0.625
0.103
0.058
0.453
0.076
1.160
0.013
0.277
0.061
0.154
0.013


1.25
0.312
0.255
1.012
0.051
1.042
0.134
0.459
0.066
0.317
0.062


2.5
0.100
0.062
0.986
0.269
0.869
0.158
0.726
0.079
0.297
0.235


5
0.105
0.089
0.480
0.423
0.432
0.111
1.114
0.066
0.353
0.080


10
0.121
0.141
0.002
0.002
0.022
0.005
0.140
0.110
0.694
0.123













JNJ26158015
JNJ26483197
JNJ26483249
JNJ17225871
JNJ17228458

















0.157
0.364
0.044
0.149
0.058
0.125
0.051
0.132
0.063
0.039
0.010


0.313
0.577
0.062
0.398
0.166
0.129
0.018
0.146
0.005
0.070
0.027


0.625
0.985
0.072
0.678
0.197
0.212
0.134
0.196
0.084
0.137
0.049


1.25
0.943
0.419
1.110
0.042
0.202
0.103
0.129
0.029
0.075
0.017


2.5
0.559
0.238
0.857
0.012
0.209
0.045
0.177
0.030
0.053
0.005


5
0.019
0.019
0.194
0.007
0.154
0.023
0.174
0.070
0.038
0.001


10
0.001
0.001
0.129
0.037
0.129
0.067
0.200
0.022
0.000
0.000













JNJ19370026
JNJ26150202
JNJ26170833
JNJ26177086
JNJ26177762

















0.157
0.074
0.024
0.040
0.030
0.291
0.086
0.054
0.014
0.186
0.040


0.313
0.170
0.046
0.051
0.016
0.746
0.088
0.080
0.006
0.342
0.068


0.625
0.246
0.036
0.150
0.095
0.941
0.111
0.268
0.050
0.563
0.019


1.25
0.981
0.075
0.155
0.010
1.119
0.045
0.332
0.006
0.936
0.186


2.5
0.914
0.038
0.408
0.279
1.305
0.066
0.432
0.154
1.146
0.137


5
0.001
0.001
0.251
0.092
1.185
0.012
0.543
0.004
1.127
0.121


10
0.000
0.000
0.262
0.068
0.000
0.000
0.822
0.024
0.759
0.328













JNJ26512005
JNJ26533065
JNJ26533156
JNJ26714194
JNJ3026582

















0.157
0.085
0.041
0.049
0.011
0.173
0.009
0.146
0.041
0.059
0.051


0.313
0.240
0.030
0.068
0.010
0.203
0.061
0.282
0.135
0.054
0.040


0.625
0.165
0.043
0.222
0.201
0.220
0.070
0.202
0.013
0.073
0.066


1.25
0.114
0.134
0.076
0.034
0.202
0.002
0.165
0.030
0.053
0.035


2.5
0.001
0.001
0.120
0.066
0.299
0.019
0.205
0.002
0.042
0.049


5
0.001
0.001
0.087
0.036
0.300
0.095
0.234
0.078
0.016
0.001


10
0.009
0.003
0.000
0.000
0.000
0.000
0.042
0.028
0.004
0.003

## Claims

1. A method to expand and differentiate human embryonic stem cells, comprising the steps of:
a. Culturing human embryonic stem cells, and
b. Treating the human embryonic stem cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity, wherein the inhibitor is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.

2. The method of claim 1, wherein the human embryonic stem cells express at least one of the following pluripotency markers: ATP-binding cassette, subfamily G, member 2 (ABCG2), crypto, forkhead box D3 (FoxD3), Connexin43, Connexin45, POU domain transcription factor Oct4, sex-determining region Y-box2 (SOX-2), Nanog, human telomerase reverse transcriptase (hTERT), undifferentiated embryonic cell transcription factor 1 (UTF-1), zinc finger protein 42 (ZFP42), stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumor-related antigen 1-60 (Tra1-60), and tumor-related antigen 1-81 (Tra1-81).

3. The method of claim 1, wherein the human embryonic stem cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.

4. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours.

5. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours.

6. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.

7. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 10004.

8. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μM to about 10 μM.

9. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μM.