Protocols for making hepatocytes from embryonic stem cells

• US 9,587,223 B2
• Filed: 08/27/2013
• Issued: 03/07/2017
• Est. Priority Date: 04/27/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Mandalam, Ramkumar, Faouzi, Saadia, Nadeau, Isabelle, Pfendler-Bonham, Kristina, Rao, Namitha, Carpenter, Melissa K., Rambhatla, Lakshmi, Chiu, Choy-Pik
• Primary Examiner(s): Gamett, Daniel C
• Application Number:
  US14/010,578
• Publication Number:
  US 20140106343A1
• Time in Patent Office: 1,288 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/30 Organic components
• 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
• 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 method for screening for a response of cultured hepatocyte lineage cells to an administered chemical compound, the method comprising:

a) obtaining a population of cultured cells that are in vitro differentiated progeny of cells expressing SSEA3, SSEA4, TRA-1-60 and TRA-1-81 and have morphological features of hepatocytes;

b) administering the chemical compound to the population of cultured cells; and

c) measuring a response of the population of cultured cells to the administered compound;

wherein the response is selected from a change in the viability, survival, morphology, marker phenotype, metabolic activity, and/or leakage of enzymes into the culture medium of the cultured cells.

2. The method of claim 1, further comprising:

d) obtaining a second population of cultured cells that are in vitro differentiated progeny of cells expressing SSEA3, SSEA4, TRA-1-60 and TRA-1-81 and have morphological features of hepatocytes;

wherein the second population of cultured cells are genetically identical to the population of cultured cells of claim 1, except for a difference at a single polymorphic locus that encodes a drug metabolizing enzyme;

e) administering the chemical compound to the second population of cultured cells; and

f) measuring a response of the second population of cultured cells to the administered compound;

wherein the response is the same response measured in c).

3. The method of claim 2, wherein the single polymorphic locus is associated with altered toxicity or metabolism.

4. The method of claim 3, wherein the single polymorphic locus encodes a cytochrome P450 enzyme.

5. The method of claim 1, wherein the response is a change in the viability of the cultured cells.

6. The method of claim 1, wherein the response is a change in the survival of the cultured cells.

7. The method of claim 1, wherein the response is a change in the morphology of the cultured cells.

8. The method of claim 1, wherein the response is a change in the marker phenotype of the cultured cells.

9. The method of claim 1, wherein the response is a change in the metabolic activity of the cultured cells.

10. The method of claim 1, wherein the response is a change in the leakage of enzymes into the culture medium of the cultured cells.