Hepatocyte lineage cells derived from pluripotent stem cells

• US 6,458,589 B1
• Filed: 11/20/2000
• Issued: 10/01/2002
• 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): Crouch, Deborah
• Assistant Examiner(s): Ton, Thaian N.
• Application Number:
  US09/718,308
• Time in Patent Office: 680 Days
• Field of Search: 435/325
• US Class Current: 435/370
• 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
• 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 2533/90 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 set of isolated cell populations, consisting of:

a first cell population that comprises undifferentiated cells from a line of human embryonic stem (hES) cells; and

a second cell population, wherein at least ˜

60% of the cells in the second population are progeny of said hES cell line, and 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;

or

the morphological features of hepatocytes.

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

3. The set of claim 1, wherein at least about 80% of the calls in the second population have at least seven of said characteristics.

4. The set of claim 1, wherein cells in the second population have been genetically altered to express telomerase at an elevated level.

5. The set of claim 1, wherein the first and second cell populations are in separate containers.