Process for making hepatocytes from pluripotent stem cells

• US 20020160511A1
• Filed: 10/31/2001
• Published: 10/31/2002
• Est. Priority Date: 04/27/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Carpenter, Melissa K., Rambhatla, Lakshmi
• Granted Patent: US 7,256,042 B2
• Time in Patent Office: Days
• US Class Current: 435/370
• 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 2533/92 Amnion; Decellularised dermis or mucosa
• 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 cell population cultured in vitro, in which at least ˜

60% cells have the same genome as an established line of primate embryonic stem cells, and that 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);

evidence of glycogen storage;

evidence of cytochrome p450 activity;

evidence of glucose-6-phosphatase activity; and

the morphological features of hepatocytes.

2. The cell population of claim 1, wherein at least about 60% of the cells have at least five of said characteristics.

3. The cell population of claim 1, wherein at least about 80% of the cells have at least seven of said characteristics.

4. 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.

5. The cell population of claim 1, which has been genetically altered to express telomerase at an elevated level.

6. 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.

7. The method of claim 6, wherein the hepatocyte differentiation agent is n-butyrate.

8. 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.

9. An isolated cell having at least three of the characteristics listed in claim 1, which is either harvested from the cell population of claim 1, or is the progeny of such a cell.

10. 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.

11. 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.

12. The method of claim 11, comprising determining whether the compound changes enzyme activity or secretion by the cell.