Hepatocytes for therapy and drug screening made from embryonic stem cells

• US 7,282,366 B2
• Filed: 03/01/2002
• Issued: 10/16/2007
• Est. Priority Date: 04/27/2000
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Carpenter, Melissa K., Rambhatla, Lakshmi
• Primary Examiner(s): Falk; Anne-Marie
• Assistant Examiner(s): Ton; Thaian N.
• Application Number:
  US10/087,142
• Publication Number:
  US 20030003573A1
• Time in Patent Office: 2,055 Days
• Field of Search: 435/325, 435/363, 435/366, 435/374, 435/377, 435/404, 435/405, 435/395, 435/401, 435/370
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/30 Organic components
• 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
• 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 2533/92 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 obtaining hepatocyte lineage cells, comprising culturing primate pluripotent stem;

pPS;

cells in a growth environment that comprises butyrate or an analog of butyrate and also 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.

2. The method of claim 1, wherein the growth environment comprises DMSO.

3. The method of claim 1, wherein the growth environment comprises a cytokine.

4. The method of claim 1, wherein the growth environment comprises HGF.

5. The method of claim 1, wherein the growth environment comprises insulin.

6. The method of claim 1, wherein the growth environment comprises FGF.

7. The method of claim 1, wherein the growth environment comprises dimethylacetamide.

8. The method of claim 1, wherein the growth environment comprises hexmethylene bisacetamide.

9. The method of claim 1, wherein the growth environment comprises EGF.