Methods and means for stem cell proliferation and subsequent generation and expansion of progenitor cells, as well as production of effector cells as clinical therapeutics

1. A medium for culturing, expanding and/or differentiating stem cells, said medium comprising a basic cell culture medium, 1-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin N-desulphated-N-acetylated heparin in relation to the volume of culture medium and a combination of cytokines.

2. A medium according to claim 1 , which medium comprises about 3-8 mmol/l of O-acetyl-L-carnitine in relation to the volume of culture medium.

3. A medium according to claim 1 , which comprises about 50-70 mg/l of N-desulphated-N-acetylated heparin in relation to the volume of culture medium.

4. A medium according to claim 1 for expanding stem cells, comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium and a combination of cytokines.

5. A medium for expansion according to claim 4 , comprising about 15% of human serum.

6. A medium for expansion according to claim 4 , comprising about 5 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium.

7. A medium for expansion according to claim 4 , comprising 60 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium.

8. A medium according to claim 4 wherein the basic medium is RPMI1640.

9. A medium for expansion according to claim 4 , which comprises thrombopoietin (TPO), flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7.

10. A medium according to claim 1 for differentiating stem cells into natural killer (NK) cell progenitors, comprising between 5-10% of human serum in relation to the volume of culture medium.

11. A medium according to claim 10 in which the amount of serum is around 8% in relation to the volume of culture medium.

12. A medium for differentiation according to claim 10 , which comprises about 5 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium.

13. A medium for differentiation according to claim 10 , which comprises 60 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium.

14. A medium according to claim 10 , wherein the basic medium is a mixture of 2:1 (v/v) DMEM and HAM-F12.

15. An initial differentiation medium according to claim 10 , wherein the combination of cytokines is TPO, FLT-3L, SCF, IL-7, VEGF, IL-2, GM-CSF, G-CSF, LIF MIP-Iα and IL-6.

16. A secondary differentiation medium according to claim 10 , wherein the combination of cytokines is TPO, FLT-3 ligand, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

17. A kit of parts for expanding and differentiating stem cells into NK progenitor cells, comprising an expansion medium comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines; and a differentiation medium comprising a basic cell culture medium, 5-10% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines.

18. A kit of parts for expanding and differentiating stem cells into NK progenitor cells, comprising an expansion medium comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines; and an initial differentiation medium comprising a basic cell culture medium, 5-10% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines comprising TPO, FLT-3L, SCF, IL-7, VEGF, IL-2, GM-CSF, G-CSF, LIF, MIP-I-α and IL-6.

19. A method for expanding and differentiating stem cells into NK progenitor cells, comprising expanding stem cells in a medium comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium and a combination of cytokines; and differentiating the expanded cells in a differentiation medium comprising a basic cell culture medium, 5-10% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines.

20. A method for expanding and differentiating stem cells into NK progenitor cells, comprising expanding stem cells in a medium comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium and a combination of cytokines; and differentiating the expanded cells in an initial differentiation medium comprising a basic cell culture medium, 5-10% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines comprising TPO, FLT-3L, SCF, IL-7, VEGF, IL-2, GM-CSF, G-CSF, LIF, MIP-I-α and IL-6.

21. A method for expanding stem cells comprising culturing said stem cells in a medium according to claim 4 .

22. A method for differentiating stem cells comprising culturing stem cells into NK progenitor cells in a differentiation medium according to claim 10 .

23. A method for differentiating stem cells comprising culturing stem cells into NK progenitor cells in an initial differentiation medium according to claim 15 .

24. A medium according to claim 1 , for differentiating stem cells into vascular progenitor cells, said medium comprising about 10% human serum in relation to the volume of culture medium.

25. A differentiation medium according to claim 24 , wherein the basic medium is M199 basal medium.

26. An initial differentiation medium according to claim 24 , wherein the combination of cytokines is SCGF, VEGF, Angiopoietin-1, angiopoietin-2, bFGF, IGF, TPO, FLT-3L, IL-1β, GM-CSF, G-CSF, LIF, MIP-I-α and 11-6.

27. A secondary differentiation medium according to claim 1 , wherein the combination of cytokines comprises SCGF, VEGF, bFGF, IGF, TPO, FLT-3L and IL-IB and the amount of human serum is 1-4% in relation to the volume of culture medium.

28. A method for expanding and differentiating stem cells into vascular progenitor cells, comprising expanding stem cells in a medium for expanding stem cells comprising a basic cell culture medium, 10% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of the culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of the culture medium and a combination of cytokines, differentiating the expanded cells in a differentiation medium according to claim 24 .

29. A method for expanding and differentiating stem cells into vascular progenitor cells, comprising expanding stem cells in a medium for expanding stem cells comprising a basic cell, culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of the culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of the culture medium and a combination of cytokines, differentiating the expanded cells in an initial differentiation medium according to claim 26 .

30. A method for differentiating stem cells comprising culturing stem cells, into vascular progenitor cells in a differentiation medium according to claim 24 .

31. A method for differentiating stem cells comprising culturing stem cells, into vascular progenitor cells in an initial differentiation medium according to claim 26 .

32. A kit of parts for expanding and differentiating stem cells into vascular progenitor cells, comprising an expansion medium for expanding stem cells comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation, to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of the culture medium and a combination of cytokines, and a differentiation medium according to claim 24 .

33. A kit of parts for expanding and differentiating stem cells into vascular progenitor cells, comprising an expansion medium for expanding stem cells comprising a basic cell culture medium, 10-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of the culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of the culture medium and a combination of cytokines, an initial differentiation medium according to claim 26 .

34. A kit of parts according to claim 18 and a secondary differentiation medium for differentiating stem cells into Natural Killer cell progenitors comprising a basic cell culture medium, 1-20% human serum in relation to the volume of culture medium, 2-10 mmol/l O-acetyl-L-carnitine in relation to the volume of culture medium, 40-80 mg/l N-desulphated-N-acetylated heparin in relation to the volume of culture medium, and a combination of cytokines, wherein the combination of cytokines is TPO, FLT-3 ligand, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

35. A method according to claim 20 and further differentiating the resulting cells in a secondary differentiation medium comprising thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7 wherein the combination of cytokines is TPO, FLT-3 ligand, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

36. A method according to claim 23 and further differentiating the resulting cells in a secondary differentiation medium comprising thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7 wherein the combination of cytokines is TPO, FLT-3 ligand, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

37. A method according to claim 29 and further differentiating the resulting cells in a secondary differentiation medium comprising thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7 wherein the combination of cytokines is TPO, FLT-3, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

38. A method according to claim 31 and further differentiating the resulting cells in a secondary differentiation medium comprising thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7 wherein the combination of cytokines is TPO, FLT-3, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, IL-6, and MIP-I-α.

39. A kit of parts according to claim 33 and further differentiating the resulting cells in a secondary differentiation medium comprising thrombopoietin, fit-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, LIF, VEGF, bFGF, IL-3 and IL-7 wherein the combination of cytokines is TPO, FLT-3, SCF, IL-7, IL-15, IL-2, GM-CSF, G-CSF, LIF, LIF, IL-6, and MIP-I-α.

40. A medium according to claim 1 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

41. A medium according to claim 4 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

42. A medium according to claim 17 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

43. A medium according to claim 18 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

44. A medium according to claim 19 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6,MIP-I-α, and LIF.

45. A medium according to claim 20 , wherein said cytokines encompass three or more of thrornbopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

46. A medium according to claim 28 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

47. A medium according to claim 29 , wherein said cytokines encompass three or more of thrombopoictin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6 MIP-I-α, and LIF.

48. A medium according to claim 32 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

49. A medium according to claim 33 , wherein said cytokines encompass three or more of thrombopoietin, flt-3 ligand, stem cell factor, G-CSF, GM-CSF, IL-6, MIP-I-α, and LIF.

50. A medium according to claim 5 , wherein said serum is AB.

51. A medium according to claim 24 , wherein said serum is AB.

52. The medium of claim 2 wherein the medium comprises about 5 mmol/l of O-acetyl-L-carnitine in relation to the volume of culture medium.

53. The medium of claim 3 wherein the medium comprises about 60 mg/l of N-desulphated-N-acetylated heparin in relation to the volume of culture medium.

54. The stem cells of claim 22 wherein the stem cells are expanded.

55. The stem cells of claim 23 wherein the stem cells are expanded.