Protocols for making hepatocytes from embryonic stem cells

• US 20050037493A1
• Filed: 03/26/2004
• Published: 02/17/2005
• Est. Priority Date: 04/27/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Mandalam, Ramkumar, Carpenter, Melissa K., Rambhatla, Lakshmi, Nadeau, Isabelle, Chiu, Choy-Pik, Pfendler-Bonham, Kristina, Faouzi, Saadia, Rao, Namitha
• Granted Patent: US 7,473,555 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 2500/62 DMSO
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• 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 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• 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

This disclosure provides a newly developed strategy and particular options for differentiating pluripotent stem cells into cells of the hepatocyte lineage. Many of the protocols are based on a strategy in which the cells are first differentiated into early germ layer cells, then into hepatocyte precursors, and then into mature cells. The cells obtained have morphological features and phenotypic markers characteristic of human adult hepatocytes. They also show evidence of cytochrome p450 enzyme activity, validating their utility for commercial applications such as drug screening, or use in the manufacture of medicaments and medical devices for clinical therapy.

Claims

1. A process for differentiating primate pluripotent stem (pPS) cells into hepatocyte lineage cells in at least three discrete steps, comprising:

a) culturing undifferentiated pPS cells with a means for causing differentiation of the cells into cells having characteristics of fetal endoderm;

b) culturing the cells from a) with a means for causing differentiation of fetal endoderm cells into cells having characteristics of hepatocyte progenitor cells;

c) culturing the cells from b) with a means for causing differentiation of hepatocyte progenitor cells into cells having characteristics of mature hepatocytes;

thereby producing a cell population that has at least five 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 enzyme activity;

evidence of glucose-6-phosphatase enzyme activity; and

the morphological features of hepatocytes.

2. The process of claim 1, wherein the culturing in a) produces a cell population in which most of the cells express at least three markers selected from Hex, Soxl7, HNF-3a, HNF-3b, and α

-fetoprotein (AFP).

3. The process of claim 1, wherein the culturing in b) produces a cell population in which most of the cells express at least three markers selected from y-glutamyl tranpeptidase, HNF-4a isomer a7/a8, albumin, α

₁-antitrypsin (AAT), and Matripase 2.

4. The process of claim 1, wherein the culturing in c) produces a cell population in which most of the cells express at least three markers selected from ApoCII, tyrosine oxygenase (TO), CYP3A4, CYP3A7, HNF-4a isomer a1/a2, and LST-1.

5. The process of claim 1, wherein a) comprises culturing the cells with DMSO, fibroblast growth factor 8 (FGF-8), or a bone morphogenic protein (BMP).

6. The process of claim 1, wherein b) comprises culturing the cells with a histone deacetylase inhibitor, a BMP, epidermal growth factor (EGF), a corticosteroid, or Oncostatin M.

7. The process of claim 1, wherein c) comprises culturing the cells with hepatocyte growth factor (HGF), or with one or more growth factors in combination with a histone deacetylase inhibitor.

8. The process of claim 1, comprising culturing the cells with DMSO, then with butyrate in the absence of growth factors, then with butyrate in combination with one or more growth factors.

9. The process of claim 1, comprising culturing the cells with DMSO, then with one or more growth factors in combination with Oncostatin M, optionally in the presence of a corticosteroid, then with HGF.

10. The process of claim 1, comprising culturing the cells with a BMP, then with Oncostatin M, optionally in the presence of a BMP or a corticosteroid, then with HGF.

19. A system for generating hepatocyte lineage cells, comprising a cell population produced by the process of claim 1, wherein the system further comprises the pPS cell line used to produce said cell population according to the process.

21. The process of claim 1, wherein the pPS cells are obtained from a human blastocyst, or are the progeny of such cells.

22. The process of claim 1, wherein the pPS cells are human embryonic stem cells.

11. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:

a) culturing the undifferentiated pluripotent cells with DMSO,

b) culturing the cells from a) with a histone deacetylase inhibitor in the absence of growth factors; and

then

c) culturing the cells from b) with a histone deacetylase inhibitor in combination with one or more growth factors.

12. The process of claim 11, wherein the histone deacetylase inhibitor is n-butyrate.

13. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:

a) culturing undifferentiated hES cells with DMSO;

b) culturing the cells from a) with one or more growth factors in combination with Oncostatin M; and

then

c) culturing the cells from b) with hepatocyte growth factor (HGF).

14. The process of claim 13, wherein a) comprises culturing the cells with FGF-1, FGF-2, FGF-4 or FGF-8.

15. The process of claim 13, wherein b) comprises culturing the cells with EGF or FGF in combination with Oncostatin M and dexamethasone.

16. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:

a) culturing undifferentiated hES cells with FGF-8 or a bone morphogenic protein (BMP);

b) culturing the cells from a) with Oncostatin M, and then

c) culturing the cells from b) with HGF.

17. The process of claim 16, wherein a) comprises culturing the cells with at least two bone morphogenic proteins selected from BMP-2, BMP-4, and BMP-7, optionally in the presence of dexamethasone.

18. The process of claim 16, wherein b) comprises culturing the cells with a BMP, dexamethasone, or nerve growth factor (NGF).

20. A system for generating hepatocyte lineage cells, comprising a cell population produced by the process of claim 16, wherein the system further comprises the pPS cell line used to produce said cell population according to the process.