Methods for producing hepatocytes

• US 10,683,486 B2
• Filed: 10/29/2016
• Issued: 06/16/2020
• Est. Priority Date: 10/30/2015
• Status: Active Grant

• Current Assignee: BioLamina AB, University of Edinburgh
• Original Assignee: BioLamina AB
• Inventors: Hagbard, Louise Kristina, Ericsson, Carl Gunnar Jesper, Cameron, Katherine Rachel, Hay, David Colin, Forbes, Stuart John, Rashidi, Hassan
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US15/338,352
• Publication Number:
  US 20170121686A1
• Time in Patent Office: 1,326 Days
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/62 DMSO
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/76 Agarose, agar-agar
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0695 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods for producing hepatocytes from pluripotent human stem cells are disclosed herein. The stem cells are plated on a cell culture substrate comprising two laminins. The stem cells are then exposed to different cell culture mediums to induce differentiation. The resulting hepatocytes have higher metabolic capacity compared to hepatocytes cultured on different substrates.

Claims

1. A method for producing hepatocytes, comprising:

plating pluripotent human stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin selected from the group consisting of laminin-111 and laminin-221, wherein the laminin-521 and the second laminin are each an intact protein; and

culturing the pluripotent human stem cells to obtain the hepatocytes;

wherein the culturing of the pluripotent human stem cells is performed by;

culturing the cells on the cell culture substrate in an endoderm differentiation medium containing activin A and Wnt3a;

culturing the cells on the cell culture substrate in a hepatoblast differentiation medium;

culturing the cells on the cell culture substrate in a hepatocyte maturation medium containing hydrocortisone (HC), hepatocyte growth factor (HGF), and endostatin m (OSM); and

wherein the resulting hepatocytes exhibit CYP1A2 activity of at least 600,000 RLU/mg/mL or CYP3A activity of at least 800,000 RLU/mg/mL.

2. The method of claim 1, wherein the first laminin and the second laminin are present in the cell culture substrate in a weight ratio from about 1:

4 to about 1;

1.

3. The method of claim 1, wherein the cells are cultured in the endoderm differentiation medium for a period of about 60 hours to about 84 hours.

4. The method of claim 1, wherein the cells are cultured in the hepatoblast differentiation medium for a period of about 73 hours to about 180 hours.

5. The method of claim 1, wherein the cells are cultured in the hepatocyte maturation medium for at least 216 hours.

6. The method of claim 1, wherein the activin A is present in the endoderm differentiation medium in an amount of about 50 ng/mL to about 150 ng/mL.

7. The method of claim 1, wherein the Wnt3a is present in the endoderm differentiation medium in an amount of about 20 ng/mL to about 100 ng/mL.

8. The method of claim 1, wherein the endoderm differentiation medium includes RPMI 1640 and B27.

9. The method of claim 1, wherein the hepatoblast differentiation medium comprises about 20% Serum Replacement, 1% non-essential amino acids, 0.1 mM beta-mercaptoethanol, and 1% dimethyl sulfoxide.

10. The method of claim 1, wherein the hepatocyte growth factor is present in the hepatocyte maturation medium in an amount of about 5 ng/mL to about 20 ng/mL.

11. The method of claim 1, wherein the oncostatin m is present in the hepatocyte maturation medium in an amount of about 10 ng/mL to about 30 ng/mL.

12. The method of claim 1, wherein the cell culture substrate further comprises a cadherin.

13. The method of claim 12, wherein a weight ratio of a sum of the first laminin and the second laminin to the cadherin is from about 5:

1 to about 15;

1.

14. The method of claim 1, wherein the cell culture substrate does not contain any differentiation inhibitors, feeder cells, differentiation inductors, or apoptosis inhibitors besides the laminin-521 and the second laminin.

15. The method of claim 1, wherein the pluripotent human stem cells have a three-dimensional structure prior to culturing.

16. A method for producing hepatocytes, comprising:

plating pluripotent human stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin which is laminin-221, wherein the laminin-521 and the second laminin are each an intact protein; and

culturing the pluripotent human stem cells on the cell culture substrate to obtain the hepatocytes;

wherein the culturing of the pluripotent human stem cells is performed by;

culturing the cells in an endoderm differentiation medium containing activin A and Wnt3a;

culturing the cells in a hepatoblast differentiation medium; and

culturing the cells in a hepatocyte maturation medium containing hydrocortisone (HC), hepatocyte growth factor (HGF), and oncostatin m (OSM).

17. The method of claim 16, wherein the first laminin and the second laminin are present in the cell culture substrate in a weight ratio from about 1:

4 to about 1.1.

18. The method of claim 16, wherein the cells are cultured in the endoderm differentiation medium for a period of about 60 hours to about 84 hours.

19. The method of claim 16, wherein the cells are cultured in the hepatoblast differentiation medium for a period of about 73 hours to about 180 hours.

20. The method of claim 16, wherein the cells are cultured in the hepatocyte maturation medium for at least 216 hours.

21. The method of claim 16, wherein the activin A is present in the endoderm differentiation medium in an amount of about 50 ng/mL to about 150 ng/mL.

22. The method of claim 16, wherein the Wnt3a is present in the endoderm differentiation medium in an amount of about 20 ng/mL to about 100 ng/mL.

23. The method of claim 16, wherein the endoderm differentiation medium includes RPMI 1640 and B27.

24. The method of claim 16, wherein the hepatoblast differentiation medium comprises about 20% Serum Replacement, 1% non-essential amino acids, 0.1 mM beta-mercaptoethanol, and 1% dimethyl sulfoxide.

25. The method of claim 16, wherein the hepatocyte growth factor is present in the hepatocyte maturation medium in an amount of about 5 ng/mL to about 20 ng/mL.

26. The method of claim 16, wherein the oncostatin m is present in the hepatocyte maturation medium in an amount of about 10 ng/mL to about 30 ng/mL.

27. The method of claim 16, wherein the cell culture substrate further comprises a cadherin.

28. The method of claim 27, wherein a weight ratio of a sum of the first laminin and the second laminin to the cadherin is from about 5:

1 to about 15;

1.

29. The method of claim 16, wherein the cell culture substrate does not contain any differentiation inhibitors, feeder cells, differentiation inductors, or apoptosis inhibitors besides the laminin-521 and the second laminin.

30. The method of claim 16, wherein the pluripotent human stem cells have a three-dimensional structure prior to culturing.