Process for making hepatocytes from pluripotent stem cells

• US 7,256,042 B2
• Filed: 10/31/2001
• Issued: 08/14/2007
• 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: Carpenter, Melissa K., Rambhatla, Lakshmi
• Primary Examiner(s): Ton; Thaian N.
• Application Number:
  US10/001,267
• Publication Number:
  US 20020160511A1
• Time in Patent Office: 2,113 Days
• Field of Search: 435/370, 435/366, 435/377, 435/405, 435/325, 435/363, 435/374, 435/404, 435/395
• US Class Current: 435/377
• 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 method for producing hepatocyte lineage cells from primate pluripotent stem (pPS) cells, comprising culturing the pPS cells in a medium comprising a hepatocyte lineage differentiation agent selected from sodium butyrate, n-butyric acid, trichosratin A, propionic acid, isobutyric acid, and isovaleric acid, wherein the method produces hepatocyte lineage cells;

wherein the hepatocyte lineage cells have at least three of the following characteristics;

antibody-detectable expression of α

₁-antitrypsin;

antibody-detectable expression of albumin;

absence of antibody-detectable expression of α

-fetoprotein;

RT-PCR detectable expression of asialoglycoprotein receptor;

evidence of glycogen storage;

evidence of cytochrome p450 activity;

evidence of glucose-6-phosphatase activity;

or the morphological features of hepatocytes.

2. The method of claim 1, wherein the hepatocyte lineage differentiation agent is sodium butyrate.

3. The method of claim 1, wherein differentiation is initiated in the pPS cells before the cells are cultured with the hepatocyte lineage differentiation agent.

4. The method of claim 3, wherein differentiation of the pPS cells is initiated by forming embryoid bodies.

5. The method of claim 3, wherein differentiation of the pPS cells is initiated by culturing in a medium containing dimethyl sulfoxide (DMSO), dimethylacetamide (DMA);

hexamethylene bisacetamide, or another polymethylene bisacetamide.

6. The method of claim 1, comprising further culturing the hepatocyte lineage cells in a medium containing a cytokine or hormone selected from glucocorticoids, epidermal growth factor (EGF), insulin, TGF-α

, TGF-β

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

7. The method of claim 6, wherein the hepatocyte lineage cells are cultured in a medium containing at least three of said cytokines or hormones.

8. The method of claim 7, wherein the hepatocyte lineage cells are cultured in a medium containing EGF, TGF-α

, and HGF.

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

10. The method of claim 1, further comprising maintaining the hepatocyte lineage cells by culturing them in a medium containing sodium butyrate.