Hepatocyte lineage cells derived from pluripotent stem cells

• US 6,506,574 B1
• Filed: 05/31/2001
• Issued: 01/14/2003
• Est. Priority Date: 04/27/2000
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Rambhatla, Lakshmi, Carpenter, Melissa K.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/872,182
• Time in Patent Office: 593 Days
• Field of Search: 435/21, 435/15, 435/23, 435/24, 435/29, 435/363, 435/366
• US Class Current: 435/21
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

It has been discovered that when pluripotent stem cells are cultured in the presence of a hepatocyte differentiation agent, a population of cells is derived that has a remarkably high proportion of cells with phenotypic characteristics of liver cells. In one example, human embryonic stem cells are allowed to form embryoid bodies, and then combined with the differentiation agent n-butyrate, optionally supplemented with maturation factors. In another example, n-butyrate is added to human embryonic stem cells in feeder-free culture. Either way, a remarkably uniform cell population is obtained, which is predominated by cells with morphological features of hepatocytes, expressing surface markers characteristic of hepatocytes, and having enzymatic and biosynthetic activity important for liver function. Since stem cells readily proliferate in culture, this system provides an abundant source of cells of the hepatocyte lineage for a variety of applications, such as drug screening, and replenishing liver function in the context of clinical treatment.

Claims

1. A method of screening a compound for its effect on hepatocytes or a hepatocyte activity, comprising:

a) combining the compound with a cell population obtained by differentiating primate pluripotent stem (pPS) cells, wherein at least ˜

60% of cells in the population have at least three of the following characteristics;

antibody-detectable expression of α

₁-antitrypsin (AAT);

antibody-detectable expression of albumin;

absence of antibody-detectable expression of α

-fetoprotein;

RT-PCR detectable expression of asialoglycoprotein receptor (ASGR);

ability to store glycogen;

cytochrome p450 activity;

glucose-6-phosphatase activity;

or

the morphological features of hepatocytes;

b) determining any change to cells in the population or their activity that results from being combined with the compound; and

c) correlating the change with the effect of the compound on hepatocytes or a hepatocyte activity.

4. The method of claim 1, comprising determining whether the compound is toxic to cells in the population.

5. The method of claim 1, comprising determining whether the compound affects ability of cells in the population to proliferate or be maintained in culture.

6. The method of claim 1, comprising determining whether the compound changes enzyme activity or secretion.

7. The method of claim 6, comprising determining whether the compound changes activity of a hepatocyte Phase I metabolizing enzyme.

8. The method of claim 6, comprising determining whether the compound changes activity of a hepatocyte Phase II metabolizing enzyme.

9. The method of claim 6, comprising determining whether the compound changes cytochrome p450 expression or activity.

10. The method of claim 9, comprising determining whether the compound changes CYP3A3-5 activity, CYP2D activity, or CYP2C9 activity.

11. The method of claim 9, comprising determining whether the compound affects CYP1A1 or CYP1A2 activity.

12. The method of claim 6, comprising determining whether the compound affects the activity of 7-ethoxycoumarin O-de-ethylase, aloxyresorufin O-de-alkylase, coumarin 7-hydroxylase, p-nitrophenol hydroxylase, testosterone hydroxylation, UDP-glucuronyltransferase, glutathione S-transferase, γ

-glutamyl tranpeptidase, or glucose-6-phosphatase.

13. The method of claim 1, comprising determining whether the compound affects the synthesis of a plasma protein.

14. The method of claim 13, wherein the plasma protein is albumin, transferrin, α

₁-antitrypsin (AAT), or α

-fetoprotein.

15. The method of claim 1, comprising determining whether the compound affects gluconeogenesis, ureagenesis, bilirubin conjugation, or bile acid conjugation.

16. The method of claim 1, comprising determining whether the compound affects synthesis or secretion of cholesterol or lipoprotein, the level of glutathione, nucleoside phosphate metabolism, intracellular K²⁺ or Ca⁺ concentration, the release of nuclear matrix proteins or oligonucleosomes, induction of apoptosis, or glycogen storage.

17. The method of claim 1, wherein the pPS cells are human embryonic stem cells.

18. The method of claim 1, wherein cells in the population have been genetically altered.

19. The method of claim 18, wherein cells in the population have been genetically altered to express telomerase reverse transcriptase.

20. The method of claim 1, wherein at least ˜

60% of the cells in the population have at least five of said characteristics.

21. The method of claim 1, wherein at least ˜

80% of the cells in the population have at least seven of said characteristics.

22. The method of claim 1, wherein the level of cytochrome p450 enzyme 1A1/1A2 activity in the cell population is at least as high as in primary human adult hepatocytes.

23. The method of claim 1, wherein the cell population has been obtained by culturing the pPS cells in a growth environment that comprises a hepatocyte differentiating agent.

24. The method of claim 23, wherein the hepatocyte differentiating agent is n-butyrate or an inhibitor of histone deacetylase.

25. The method of claim 1, wherein the cell population has been obtained by culturing progeny of the pPS cells in a medium containing one or more hepatocyte maturation factors.

26. The method of claim 25, wherein at least one hepatocyte maturation factor is either:

an organic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylacetamide (DMA);

hexmethylene bisacetamide, and other polymethylene bisacetamides;

or

a cytokine or hormone selected from the group consisting of glucocorticoids, epidermal growth factor (EGF), insulin, TGF-α

, TGF-β

, fibroblast growth factor (FGF), heparin, and hepatocyte growth factor (HGF), IL-1, IL-6, IGF-I, IGF-II, and HBGF-1.

27. The method of claim 1, comprising providing the cell population with which the compound is subsequently combined.

28. The method of claim 1, comprising differentiating an hES cell line to obtain the cell population with which the compound is subsequently combined.

2. A method of screening a compound for its effect on hepatocytes or a hepatocyte activity, comprising:

a) combining the compound with a cell population obtained by differentiating primate pluripotent stem (pPS) cells in a medium containing butyrate or an inhibitor of histone deacetylase;

b) determining any change to cells in the population or their activity that results from being combined with the compound; and

c) correlating the change with the effect of the compound on hepatocytes or a hepatocyte activity.

3. A method of screening a compound for its effect on hepatocytes or a hepatocyte activity, comprising:

a) combining the compound with a cell population containing cells that have the same genome as an established human embryonic stem (hES) cell line, wherein at least 60% of cells in the population have at least three of the following characteristics;

antibody-detectable expression of α

₁-antitrypsin (AAT);

antibody-detectable expression of albumin;

absence of antibody-detectable expression of α

-fetoprotein;

RT-PCR detectable expression of asialoglycoprotein receptor (ASGR);

ability to store glycogen;

cytochrome p450 activity;

glucose-6-phosphatase activity;

or

the morphological features of hepatocytes;

b) determining any change to cells in the population or their activity that results from being combined with the compound; and

c) correlating the change with the effect of the compound on hepatocytes or a hepatocyte activity.