METHODS AND COMPOSITIONS FOR THE DIFFERENTIATION OF STEM CELLS

• US 20100216181A1
• Filed: 02/22/2010
• Published: 08/26/2010
• Est. Priority Date: 02/20/2009
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Salvagiotto, Giorgia, Fuhrken, Peter, Daigh, Christine
• Granted Patent: US 8,557,580 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells

Abstract

The present invention provides methods and compositions for the production of hematopoietic progenitor cells or endothelial progenitor cells from human pluripotent stem cells using a defined cell culture medium without the need to utilize feeder cells or serum. In some embodiments, differentiation is accomplished using hypoxic atmospheric conditions. The defined medium of the present invention may contain growth factors and a matrix component. The hematopoietic progenitor cells may be further differentiated into cell lineages including red blood cells, macrophages, granulocytes, and megakaryocytes. The endothelial progenitor cells may be further differentiated into endothelial cells. Also disclosed are screening assays for identification of candidate substances that affect differentiation of pluripotent stem cells into progenitor cells.

Claims

1. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising:

a) culturing a pluripotent stem cell in a culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and

b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD34+ progenitor cells.

2. The method of claim 1, wherein the pluripotent stem cell is an embryonic stem cell or an induced embryonic stem cell.

3. The method of claim 1, wherein the hypoxic atmosphere comprises from about 0.5% oxygen gas to about 5.5% oxygen gas.

4. The method of claim 1, wherein the hypoxic atmosphere comprises from about 1.5% oxygen gas to about 5.3% oxygen gas.

5. The method of claim 1, wherein the matrix component comprises fibronectin, collagen, or an RGD peptide.

6. The method of claim 1, wherein the method further comprises harvesting the CD34+ progenitor cell or its progeny at 8 days to 12 days of culturing.

7. The method of claim 1, wherein the method further comprises harvesting the CD34+ progenitor cell or its progeny at 6 days to 9 days of culturing.

8. The method of claim 1, wherein the culture medium comprises BMP-4, VEGF, and bFGF.

9. The method of claim 1, wherein the culture medium comprises BMP-4 in an amount of from about 5 ng/mL to about 200 ng/mL.

10. The method of claim 1, wherein the culture medium comprises VEGF in an amount of from about 5 ng/mL to about 200 ng/mL.

11. The method of claim 1, wherein the culture medium comprises bFGF in an amount of from about 5 ng/mL to about 200 ng/mL.

12. The method of claim 1, wherein the culture medium further comprises a survival factor.

13. The method of claim 12, wherein the survival factor is an inhibitor of a Rho-associated kinase (ROCK) or an inhibitor of myosin II.

14. The method of claim 1, wherein the culture medium is free or essentially free of serum or feeder cells.

15. The method of claim 1, wherein the method further comprises culturing or maintaining the pluripotent stem cells in a first medium prior to differentiation, wherein the first medium comprises TeSR.

16. The method of claim 15, wherein the first medium comprises a TeSR medium and a survival factor.

17. The method of claim 16, wherein the survival factor is an inhibitor of a Rho-associated kinase (ROCK) or an inhibitor of myosin II.

18. The method of claim 1, wherein the method further comprises differentiating the CD34+ progenitor cells into one or more of the group consisting of erythrocytes, macrophages, granulocytes, megakaryocytes, dendritic cells, mast cells, or endothelial cells.

19. The method of claim 18, wherein the further differentiation occurs in culture medium comprising one or more of the group consisting of FMS-like tyrosine kinase 3 (FLT-3), stem cell factor (SCF), thrombopoietic (TPO), interleukin 3 (IL-3), interleukin 6 (IL-6), and heparin.

20. The method of claim 18, wherein the further differentiation occurs in culture medium comprising FMS-like tyrosine kinase 3 (FLT-3), stem cell factor (SCF), thrombopoietic (TPO), interleukin 3 (IL-3), interleukin 6 (IL-6), and heparin.

21. The method of claim 1, wherein the culture medium comprises one or more amino acids, antibiotics, vitamins, salts, minerals, or lipids.

22. The method of claim 1, wherein the culture medium comprises BIT 9500, BMP4, VEGF, bFGF, L-glutamine, non-essential amino acids, monothioglycerol, penicillin, and streptomycin.

23. The method of claim 1, wherein the culture medium comprises two or more recombinant growth factors selected from the group consisting of BMP-4, VEGF, and bFGF.

24. The method of claim 1, wherein the method comprises using a robot to automate at least a portion of the method.

25. The method of claim 1, wherein a plurality of the pluripotent stem cells are cultured using a bioreactor.

26. The method of claim 1, further comprising sorting the CD34+ progenitor cells using magnetic-activated cell sorting (MACS), flow cytometry, or fluorescence-activated cell sorting (FACS).

27. The method of claim 1, further comprising sorting the CD34+ progenitor cells based on the expression of one or more of the group consisting of CD34, CD43, and CD31.

28. The method of claim 1, further comprising:

a) dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells, and

b) seeding the dispersed cells into a culture medium at a density of from about 10,000 stem cells per square centimeter of culturing surface to about 70,000 stem cells per square centimeter of culturing surface.

29. The method of claim 28, wherein the cells are dispersed into a culture medium comprising a survival factor.

30. The method of claim 28, wherein the dispersed cells are seeded at a density of from about 10,000 stem cells per square centimeter of culturing surface to about 50,000 stem cells per square centimeter of culturing surface.

31. The method of claim 28, wherein the pluripotent stem cells are dispersed by treatment with an effective amount of one or more enzymes.

32. The method of claim 31, wherein at least one of the enzymes is trypsin or trypLE.

33. The method of claim 1, wherein the differentiation medium is a defined differentiation medium.

63. The method of claim 1, wherein the differentiation medium further comprises one or more of the group consisting of SCF, TPO, FLT-3, IL-3, IL-6, and heparin.

34. A method for differentiating a human pluripotent stem cell into a CD43+ progenitor cell comprising:

a) culturing a pluripotent stem cell in a culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and

b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD43+ progenitor cells.

35. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising:

a) culturing a pluripotent stem cell in a culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and

b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD31+ progenitor cells.

36. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising growing a pluripotent stem cell in a culture comprising:

a) a matrix component; and

b) a differentiation medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, wherein the culture is free or essentially free of non-human animal serum, feeder cells, and Matrigel™

.

37. (canceled)

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39. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising growing a pluripotent stem cell in a culture comprising:

a) a matrix component; and

b) a differentiation medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, wherein the culture is free or essentially free of non-human animal serum, feeder cells, and Matrigel™

.

40. (canceled)

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42. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.

43. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.

44. A method for differentiating a human pluripotent stem cell into a CD43+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.

45. A differentiation culture medium comprising BMP-4, VEGF, bFGF, and a matrix component, wherein the medium is free or essentially free of feeder cells, serum, and Matrigel™

.

57. A kit comprising the differentiation culture medium of claim 45 in one or more sealed vials.

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60. A method of screening a candidate substance for ability to promote differentiation of a pluripotent cell into a CD34+ progenitor cell, comprising:

a) culturing a pluripotent stem cell in a culture medium that is free or essentially free of feeder cells, wherein the medium comprises a matrix component, at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, and a candidate substance;

b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD34+ progenitor cells; and

c) assessing the pluripotent cell for differentiation into a CD34+ progenitor cell.

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64. A clonal cell population comprising CD34+ progenitor cells, wherein at least 5% of the cells are CD34+ or CD43+ progenitor cells and the cell population is in a defined medium that is free or essentially free of serum and feeder cells.

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66. A clonal cell population comprising CD31+ progenitor cells, wherein at least 20% of the cells are CD31+ or CD34+ progenitor cells and the cell population is in a defined medium that is free or essentially free of serum and feeder cells.

67. (canceled)