Differentiation of Pluripotent Cells into Primary Germ Layer Progenitors

• US 20100034785A1
• Filed: 11/09/2007
• Published: 02/11/2010
• Est. Priority Date: 11/09/2006
• Status: Active Grant

• Current Assignee: Cambridge Enterprises Limited
• Original Assignee: Cambridge Enterprises Limited
• Inventors: Vallier, Ludovic, Pedersen, Roger
• Granted Patent: US 8,323,971 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/35 Polyols, e.g. glycerin, inositol
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

This invention relates to the culture of pluripotent cells in a fully humanised chemically defined medium. Cells may be cultured over a prolonged period of time without losing their pluripotent status or may be controllably induced to differentiate into progenitor cells of the three primary germ layers by the addition of differentiation factors, for example differentiation factors which modulate one or more of the Activin/Nodal, FGF, Wnt or BMP signalling pathways.

Claims

1. A method of producing a population of partially differentiated progenitor cells comprising;

culturing human pluripotent cells in a humanised chemically defined medium (CDM) supplemented with one or more differentiation factors, and,

allowing said human pluripotent cells to differentiate into said progenitor cells.

2. A method according to claim 1 wherein said one or more differentiation factors modulate one or more of the Activin/Nodal, FGF, Wnt or BMP signalling pathways in the human pluripotent cells.

3. A method according to claim 1 or claim 2 wherein the progenitor cells are ectoderm progenitor cells and one or more differentiation factors are FGF2 and an activin antagonist.

4. A method according to claim 3 wherein the progenitor cells are neuroectoderm progenitor cells.

6. A method according to any one of claims 3 to 5 wherein the activin antagonist is SB431542.

7. A method according to any one of claims 3 to 6 wherein the humanised CDM is supplemented with 1 to 100 μ

M of the activin antagonist.

8. A method according to any one of claims 1 to 7 wherein the humanised CDM is supplemented with 1 to 100 ng/ml of FGF2.

9. A method according to any one of claims 3 to 8 wherein the ectoderm progenitor cells express Sox2 in absence of Oct4.

10. A method according to any one of claims 3 to 9 further comprising allowing the cells to differentiate into neurons, astrocytes, or oligodendrocytes.

11. A method according to claim 1 or claim 2 wherein the partially differentiated progenitor cells are mesoderm/endoderm progenitor cells and the one or more differentiation factors are activin, FGF2 and BMP4.

12. A method according to claim 11 wherein the humanised CDM-PVA is further supplemented with a phosphatidyl-3-inositol kinase inhibitor.

13. A method according to claim 11 wherein the human pluripotent cells are cultured by a method comprising;

(i) culturing said human pluripotent cells in humanised CDM comprising polyvinyl alcohol (CDM-PVA) and supplemented with activin and FGF2,

(ii) further culturing said cells in humanised CDM-PVA supplemented with an FGF2 antagonist and activin, and

(iii) further culturing said cells in humanised CDM-PVA supplemented with FGF2, BMP4 and activin.

14. A method according to claim 13 wherein the concentration of activin in step (ii) is less than the concentration of activin in step (i).

15. A method according to claim 13 or claim 14 wherein the humanised CDM-PVA in step (i) is supplemented with 5 to 20 ng/ml of activin A and 1 to 50 ng/ml of FGF2.

16. A method according to any one of claims 13 to 15 wherein the humanised CDM in step (ii) is supplemented with 1 to 50 M of the FGF2 antagonist and 1 to 10 ng/ml activin.

17. A method according to any one of claims 13 to 16 wherein the humanised CDM in step (iii) is supplemented with 1 to 100 ng/ml of FGF2, 1 to 100 ng/ml of BMP4 and 1 to 200 ng/ml of activin.

18. A method according to claim 17 wherein the humanised CDM in step (iii) is supplemented with more than 50 ng/ml activin and the mesoderm/endoderm progenitor cells are endoderm progenitors.

19. A method according to claim 17 wherein the humanised CDM is supplemented with less than 50 ng/ml activin and the mesoderm/endoderm progenitor cells are mesoderm progenitors.

20. A method according to any one of claims 11 to 19 further comprising allowing the cells to differentiate into liver cells or liver cell progenitors.

21. A method according to any one of the preceding claims wherein the human pluripotent cells are selected from the group consisting of embryonic stem cells, fetal and adult somatic stem cells or neural crest cells.

22. A method according to any one of the preceding claims wherein the humanised CDM comprises a basal defined culture medium, insulin, transferrin, 1-thiolglycerol and one or more of human serum albumin, polyvinyl alcohol, Plasmanate and Buminate.

23. A method according to claim 22 wherein the basal defined culture medium is 50% IMDM, 50% F12 NUT-MIX.

24. A method according to claim 22 or 23 wherein the humanised chemically defined medium comprises 50% IMDM, 50% F12 NUT-MIX, 7 μ

g/ml insulin, 15 μ

g/ml transferrin, 1% chemically defined lipid concentrate, 5 mg/ml human serum albumin or Polyvinyl Alcohol (PVA) and 450 μ

M 1-thiolglycerol.

25. A method according to any one of the preceding claims comprising isolating and/or purifying said progenitor cells.

26. A method according to claim 25 comprising formulating said cells with a pharmaceutically acceptable carrier.

27. A population of progenitor cells produced by a method according to any one of claims 1 to 26.

28. A population of progenitor cells produced by a method according to any one of claims 1 to 26 for use in a method of treatment of the human or animal body.

29. A population of progenitor cells produced by a method according to any one of claims 1 to 26 for use in a method of treatment of damaged or dysfunctional tissue.

30. Use of a population of progenitor cells produced by a method according to any one of claims 1 to 26 in the manufacture of a medicament for the treatment of damaged or dysfunctional tissue.

31. A method of treatment of damaged or dysfunctional tissue comprising administering a population of cells according to claim 27 to an individual in need thereof.

32. A method of screening for a compound useful in the treatment of damaged or dysfunctional tissue may comprise;

contacting a population of progenitor cells produced by a method according to any one of claims 1 to 26 with a test compound and

determining the growth or differentiation of the progenitor cells.

5. A method of producing a population of neuroectoderm progenitor cells comprising;

culturing human pluripotent cells in a humanised chemically defined medium (CDM) supplemented with FGF2 and an activin antagonist, and,

allowing said human pluripotent cells to differentiate into said neuroectoderm progenitor cells.

33. A method of expanding a population of human pluripotent cells comprising;

culturing one or more human pluripotent cells in a humanised CDM supplemented with FGF2 and Activin, and,

thereby expanding said population of human pluripotent cells.

34. A method according to claim 33 wherein the humanised chemically defined medium comprises a basal defined culture medium supplemented with insulin, transferrin, 1-thiolglycerol and one or more of human serum albumin, polyvinyl alcohol, Plasmanate and Buminate.

35. A method according to claim 34 wherein the basal defined culture medium is 50% IMDM, 50% F12 NUT-MIX.

36. A method according to claim 35 wherein the humanised chemically defined medium comprises 50% IMDM, 50% F12 NUT-MIX, 7 μ

g/ml insulin, 15 μ

g/ml transferrin, 5 mg/ml human serum albumin or polyvinyl alcohol (PVA), 1% chemically defined lipid concentrate and 450 μ

M 1-thiolglycerol.

37. A method according to any one of claims 33 to 36 wherein the humanised CDM is supplemented with 1 to 100 ng/ml of FGF2 and 1 to 200 ng/ml of activin.