DIFFERENTIATION OF PLURIPOTENT CELLS

• US 20100279403A1
• Filed: 03/01/2010
• Published: 11/04/2010
• Est. Priority Date: 02/27/2009
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Lewis, Rachel, Rajesh, Deepika
• Granted Patent: US 8,372,642 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• 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 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

Provided herein are methods for the in vitro maintenance, expansion, culture, and/or differentiation of pluripotent cells, such as human embryonic stem cells (hESC) or induced pluripotent cells (iPSC), into hematopoietic precursor cells or endothelial cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the pluripotent cells into hematopoietic precursor cells or endothelial cells. The resulting hematopoietic precursor cells may be further differentiated into various myeloid or lymphoid lineages.

Claims

1. A method of differentiating pluripotent cells into hematopoietic precursor cells or endothelial cells comprising the sequential steps of:

a) culturing or maintaining a plurality of substantially undifferentiated pluripotent cells in a first defined media comprising at least one growth factor;

b) incubating the cells in a second defined media which is free or essentially free of BMP4, VEGF, IL-3, Flt3 ligand, and GMCSF;

c) culturing the cells in a third defined media comprising an amount of BMP4 and VEGF sufficient to expand or promote differentiation in a plurality of the cells; and

d) culturing the cells in a fourth defined media comprising an amount of either (1) IL-3 and Flt3 ligand, or (2) VEGF, FGF-2 or an FGF-2 mimic, and IGF sufficient to expand or promote differentiation in a plurality of the cells;

wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells or endothelial cells.

2. The method of claim 1, wherein the third defined media further comprises FGF-2 or an FGF-2 mimic.

3. The method of claim 1, wherein the fourth defined media comprises IL-3, Flt3 ligand, and GMCSF.

4. The method of claim 1, wherein the fourth defined media comprises IL-3, Flt3 ligand, and at least one of IL-6, SCF, or TPO.

5. The method of claim 4, wherein the fourth defined media further comprises IL-6, SCF, and TPO.

6. The method of claim 5, wherein the fourth defined media further comprises Serum Replacement 3.

7. The method of claim 6, wherein the pluripotent cells are iPSC.

8. The method of claim 1, wherein at least some of the cells are at least partially separated or are substantially individualized prior to step (b).

9. The method of claim 8, wherein the cells are substantially individualized using an enzyme.

10. The method of claim 9, wherein the enzyme is a trypsin.

11. The method of claim 10, wherein the cells are contacted with a ROCK inhibitor and a trypsin inhibitor subsequent to said individualization.

12. The method of claim 11, wherein the ROCK inhibitor is selected from the list consisting of HA-100, H-1152, and Y-27632.

13. The method of claim 11, wherein the trypsin inhibitor is a soybean trypsin inhibitor.

14. The method of claim 1, wherein a plurality of the pluripotent cells form embryoid bodies (EBs).

15. The method of claim 14, wherein from about 200 to about 1000 cells per aggregate are used to generate at least one of said EBs.

16. The method of claim 1, wherein the method comprises culturing the cells at an atmospheric pressure of less than 20% oxygen.

17. The method of claim 16, wherein the method comprises culturing the cells at an atmospheric pressure of about 5% oxygen.

18. The method of claim 1, wherein said cells are partially or substantially reaggregated at least once.

19. The method of claim 18, wherein said cells are reaggregated after culture in the third defined media and prior to or during culture in the fourth defined media.

20. The method of claim 18, wherein said reaggregation comprises exposing said cells to trypsin or TRyPLE.

21. The method of claim 18, wherein said cells are exposed to a ROCK inhibitor subsequent to the reaggregation.

22. The method of claim 18, wherein said cells are cultured in a media essentially free of a ROCK inhibitor subsequent to the reaggregation.

23. The method of claim 18, wherein the method further comprises culturing the cells at an atmospheric pressure of less than about 20% oxygen, and wherein from about 200 to about 1000 cells per aggregate are used to generate a plurality of embryoid bodies (EBs).

24. The method of claim 1, wherein the third defined media or the fourth defined media comprises Serum Replacement 3.

25. The method of claim 1, wherein the first defined media comprises TeSR, mTeSR, or mTeSR1.

26. The method of claim 25, wherein step (a) comprises culturing the cells on a matrix-coated surface.

27. The method of claim 26, wherein the matrix comprises laminin, vitronectin, gelatin, polylysine, thrombospondin or Matrigel™

.

28. The method of claim 1, wherein the second defined media comprises TeSR-GF or X-vivo 15 media

29. The method of claim 28, wherein the second defined media further comprises about 0.1 ng/ml TGF-β

and about 20 ng/ml FGF-2.

30. The method of claim 1, wherein step (b) comprises incubating the cells for a period of from about 12 hours to about 3 days.

31. The method of claim 1, wherein step (c) comprises culturing or differentiating the cells for a period of from about 4 to about 8 days.

32. The method of claim 1, wherein step (d) comprises culturing the cells for a period of at least 4 days.

33. The method of claim 32, wherein a plurality of the pluripotent cells are differentiated into myeloid progenitor cells.

34. The method of claim 33, wherein the myeloid progenitor cells co-express CD31, CD43, and CD45.

35. The method of claim 1, wherein the third defined media comprises about 10-50 ng/ml BMP4 and about 10-50 ng/ml VEGF.

36. The method of claim 35, wherein the third defined media further comprises about 10-50 ng/ml FGF-2.

37. The method of claim 1, wherein the fourth defined media comprises about 5-25 ng/ml IL-3 and about 10-50 ng/ml Flt3 ligand.

38. The method of claim 37, wherein the fourth media further comprises about 5-25 ng/ml GMCSF.

39. The method of claim 37, wherein the fourth media further comprises about 10-100 ng/ml TPO, 10-100 ng/ml SCF, about 5-25 ng/ml IL-6, and about 5-25 ng/ml IL-3

40. The method of claim 1, wherein a plurality of the hematopoietic precursor cells express at least two cell markers selected from the list comprising CD43, CD34, CD31 and CD45.

41. The method of claim 40, wherein a plurality of the hematopoietic precursor cells express CD34, CD43, CD45 and CD31.

42. The method of claim 1, wherein one or more of the hematopoietic precursor cells are differentiated into an erythroid cell, a myeloid cell, or a lymphoid cell.

43. The method of claim 42, wherein one or more of the hematopoietic precursor cells are differentiated into a myeloid cell, wherein the myeloid cell is selected from the group consisting of macrophage, mast cell, erythrocyte, megakaryocyte/platelet, dendritic cell, and polymorph nuclear granulocyte.

44. The method of claim 43, wherein the one or more of the hematopoietic precursor cells are differentiated into a granulocyte selected from the list consisting of eosinophil, basophil, neutrophil, monocyte, and macrophage.

45. The method of claim 1, wherein method comprises culturing a plurality of said cells in a fifth defined media comprising one or more growth factor selected from the list consisting of IL-3, IL-6, SCF, EPO, and TPO, in an amount sufficient to promote differentiation of a plurality of the cells into erythroblasts.

46. The method of claim 1, wherein said method further comprises culturing a plurality of said cells in a fifth defined media, wherein the fifth defined media comprises one or more growth factor selected from the list consisting of SCF, IL-6, G-CSF, EPO, TPO, FGF2, IL-7, IL-11, IL-9, IL-13, IL-2, or M-CSF in an amount sufficient to promote expansion or further differentiation of the cells.

47. The method of claim 1, wherein a plurality of the cells are cultured in a fifth defined media comprising one or more growth factor selected from the list consisting of IL-7, SCF, and IL-2, in an amount sufficient to promote differentiation of the cells into NK cells.

48. The method of claim 43, wherein the method further comprises culturing a plurality of the cells in a fifth defined media comprising Fc chimeric Notch DLL-1 ligand and one or more growth factor selected from the list consisting of IL-7, SCF, and IL-2 in an amount sufficient to promote differentiation of the cells into T cells.

49. The method of claim 1, wherein said pluripotent cells are mammalian pluripotent cells.

50. The method of claim 49, wherein said mammalian pluripotent cells are human pluripotent cells.

51. The method of claim 50, wherein said human pluripotent cells are human embryonic stem cells (hESC).

52. The method of claim 51, wherein the hESC comprise cells selected from the list consisting of H1, H9, hES2, hES3, hES4, hES5, hES6, BG01, BG02, BG03, HSF1, HSF6, H1, H7, H9, H₁₃B, and H14.

53. The method of claim 50, wherein said human pluripotent cells are induced pluripotent cells (iPSC).

54. The method of claim 53, wherein said iPSC are selected from the list consisting of iPS6.1, iPS 6.6, iPS, iPS 5.6, iPS iPS 5.12, iPS 5.2.15, iPS iPS 5.2.24, iPS 5.2.20, iPS 6.2.1, iPS-Bl-SONL, iPS-B1-SOCK, iPS-TIPS lEE, iPS-TiPS IB, iPS-KIPS-5, and iPS 5/3-4.3.

55. A hematopoietic precursor cell differentiated according to the method of claim 1 or derived from a separate hematopoietic precursor cell differentiated according to the method of claim 1.

56. The hematopoietic precursor cells of claim 55, wherein the hematopoietic precursor cell expresses at least two cell markers selected from the list comprising CD34, CD43, CD45, and CD31.

57. The method of claim 56, wherein the hematopoietic precursor cell expresses CD34, CD43, CD45, and CD31

58. A cell derived from a hematopoietic precursor cell differentiated according to the method of claim 1, wherein the cell is a myeloid cell or a common myeloid progenitor.

59. The cell of claim 58, wherein the cell is selected from the list consisting of monocyte, macrophage, mast cell, neutrophil, basophil, eosinophil, erythrocyte, megakaryocyte, platelet, and dendritic cell.

60. The cell of claim 59, wherein the cell is an erythrocyte.

61. The cell of claim 58, wherein the myeloid cell is comprised in a pharmaceutical preparation.

62. A lymphoid cell derived from a hematopoietic precursor cell differentiated according to the method of claim 1.

63. The lymphoid cell of claim 62, wherein the lymphoid cell is a T-cell, B-cell, or a natural killer cell.

64. The lymphoid cell of claim 62, wherein the lymphoid cell is comprised in a pharmaceutical preparation.

65. An endothelial cell differentiated according to the method of claim 1.

66. The endothelial cell of claim 65, wherein the endothelial cell is a lymphatic endothelial cell, a vascular endothelial cell, a vascular arterial endothelial cell, a vascular venous endothelial cell, or an organ-specific endothelial cell.