Claims
- 1. A cell population comprising at least ˜60% differentiated cells that are progeny of primate pluripotent stem (pPS) cells and have at least three of the following characteristics:
antibody-detectable expression of α1-antitrypsin (AAT); antibody-detectable expression of albumin; absence of antibody-detectable expression of α-fetoprotein; RT-PCR detectable expression of asialoglycoprotein receptor (ASGR); evidence of glycogen storage; evidence of cytochrome p450 activity; evidence of glucose-6-phosphatase activity; and the morphological features of hepatocytes; wherein the cell population is cultured or maintained in a medium containing a histone deacetylase inhibitor so as to maintain said characteristics.
- 2. A cell population comprising at least ˜60% differentiated cells that are progeny of primate pluripotent stem (pPS) cells and have at least three of the following characteristics:
antibody-detectable expression of α1-antitrypsin (AAT); antibody-detectable expression of albumin; absence of antibody-detectable expression of α-fetoprotein; RT-PCR detectable expression of asialoglycoprotein receptor (ASGR); evidence of glycogen storage; evidence of cytochrome p450 activity; evidence of glucose-6-phosphatase activity; and the morphological features of hepatocytes; wherein the differentiated cells are karyotypically normal and non-malignant, and wherein the cell population comprises less than 0.1% endothelial cells or Kupffer cells.
- 3. The cell population of claim 1, wherein at least about 60% of the cells have at least five of said characteristics.
- 4. The cell population of claim 1, wherein at least about 80% of the cells have at least seven of said characteristics.
- 5. The cell population of claim 1, wherein the level of cytochrome p450 enzyme 1A1/1A2 activity is at least as high as in primary human adult hepatocytes.
- 6. The cell population of claim 1, wherein the pPS cells are embryonic stem cells.
- 7. The cell population of claim 6, wherein the embryonic stem cells are human.
- 8. The cell population of claim 7, which is essentially free of undifferentiated hES cells.
- 9. A method for obtaining the cell population of claim 1, comprising culturing cells from the stem cell line in a growth environment that comprises a hepatocyte differentiation agent which is a histone deacetylase inhibitor.
- 10. The method of claim 9, wherein the hepatocyte differentiation agent is n-butyrate.
- 11. The method of claim 9, comprising pre-differentiating the cells by forming embryoid bodies, or by pre-differentiating the cells by culturing in a medium containing dimethyl sulfoxide (DMSO), dimethylacetamide (DMA); hexmethylene bisacetamide, or another polymethylene bisacetamide.
- 12. The method of claim 1, comprising further culturing the cells in a medium containing a cytokine or hormone selected from glucocorticoids, epidermal growth factor (EGF), insulin, TGF-α, TGF-β, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), IL-1, IL-6, IGF-I, IGF-II, and HBGF-1.
- 13. A method for obtaining the cell population of claim 1, comprising culturing cells from the stem cell line in a growth environment that comprises one or more hepatocyte maturation factors that are either:
a) an organic solvent selected from dimethyl sulfoxide (DMSO), dimethylacetamide (DMA); hexmethylene bisacetamide, and other polymethylene bisacetamides; or b) a cytokine or hormone selected from glucocorticoids, epidermal growth factor (EGF), insulin, TGF-α, TGF-β, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), IL-1, IL-6, IGF-I, IGF-II, and HBGF-1.
- 14. A method of screening a compound for hepatocellular toxicity, comprising combining a cell according to claim 1 with the compound, and determining whether the compound is toxic to the cell.
- 15. A method of screening a compound for its ability to modulate hepatocellular function, comprising combining a cell according to claim 1 with the compound, determining any phenotypic or metabolic changes in the cell that result from contact with the compound, and correlating the change with an ability to modulate hepatocellular function.
- 16. The method of claim 15, comprising determining whether the compound changes enzyme activity or enzyme secretion by the cell.
Priority Claims (2)
Number |
Date |
Country |
Kind |
PCT/US01/13471 |
Apr 2001 |
US |
|
01/81549 |
Nov 2000 |
WO |
|
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of pending U.S. utility application 10/001,267, filed Oct. 31, 2001, which is a continuation-in-part of U.S. Ser. No. 09/872,182, filed May 31, 2001; and International Patent Application PCT/US01/13471, filed Apr. 26, 2001 (designating the U.S. and published as WO 01/81549 on Nov. 1, 2001); which in turn claims priority to U.S. provisional patent application No. 60/200,095, filed Apr. 27, 2000.
[0002] This application claims the priority benefit of all the aforelisted applications. The four priority documents are hereby incorporated herein by reference in their entirety, along with U.S. utility application 09/718,308, and International Patent Publication WO 01/51616.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60200095 |
Apr 2000 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
10001267 |
Oct 2001 |
US |
Child |
10087142 |
Mar 2002 |
US |
Parent |
09872182 |
May 2001 |
US |
Child |
10001267 |
Oct 2001 |
US |