Differentiation of pluripotent cells

• US 10,100,282 B2
• Filed: 02/01/2013
• Issued: 10/16/2018
• Est. Priority Date: 02/27/2009
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Inventors: Rajesh, Deepika, Lewis, Rachel
• Primary Examiner(s): Hill, Kevin Kai
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US13/757,263
• Publication Number:
  US 20130210141A1
• Time in Patent Office: 2,083 Days
• 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 (HPCs) 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, wherein said cells are cultured or maintained in said media on a matrix-coated surface;

(b) culturing individualized pluripotent cells from step a) under aggregate forming conditions in a second defined, feeder-free media comprising a ROCK inhibitor, for a period of time sufficient to form cell aggregates;

(c) culturing the cell aggregates of step (b) in a third defined media comprising an amount of BMP4, VEGF, and FGF-2 or an FGF-2 mimic, for a period of time sufficient to expand or promote differentiation of cells to a mesodermal lineage;

(d) culturing the cells of step (c) in a fourth defined media comprising an amount of IL-3 and Flt3 ligand, for a period of time sufficient to further expand or promote differentiation of the cells to HPCs and endothelial cells;

(e) optionally, where a further enrichment of endothelial cells is desired, the fourth defined media of step (d) further includes VEGF and FGF; and

wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells or endothelial cells, defined as cells that express CD34 and CD3.

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

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

4. The method of claim 3, wherein the fourth defined media of step (d) comprises IL-3, Flt3 ligand, IL-6, SCF, and TPO.

5. The method of claim 1, wherein the pluripotent cells are iPSC.

6. The method of claim 1, wherein the cells employed in step (b) are substantially individualized using an enzyme.

7. The method of claim 6, wherein the enzyme is a trypsin or a trypsin replacement.

8. The method of claim 7, wherein the cells are contacted with a Rho-associated protein kinase (ROCK) inhibitor and a trypsin inhibitor subsequent to said individualization.

9. The method of claim 8, wherein the ROCK inhibitor is selected from the list consisting of HA-100, H-1152, and Y-27632.

10. The method of claim 8, wherein the trypsin inhibitor is a soybean trypsin inhibitor.

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

12. The method of claim 11, wherein from about 200 to about 1000 cells per aggregate are used to generate at least one of said EBs.

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

14. The method of claim 13, wherein the method comprises culturing the cells at an atmospheric pressure of about 5% oxygen.

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

16. The method of claim 15, wherein said cells are reaggregated after culture in the third defined media of step (c) and prior to or during culture in the fourth defined media of step (d).

17. The method of claim 15, wherein said reaggregation comprises exposing said cells to trypsin or trypsin replacement.

18. The method of claim 15, wherein said cells are exposed to a ROCK inhibitor subsequent to the reaggregation.

19. The method of claim 15, wherein said cells are cultured in a media essentially free of a ROCK inhibitor subsequent to the reaggregation.

20. The method of claim 15, 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).

21. The method of claim 1, wherein the matrix-coated surface comprises laminin, vitronectin, gelatin, polylysine, thrombospondin, fibronectin, collagen or CAS Registry 119978-18-6 matrix.

22. 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.

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

24. The method of claim 23, wherein a plurality of the pluripotent cells are differentiated into myeloid progenitor cells.

25. The method of claim 24, wherein the myeloid progenitor cells co-express CD31, CD43, and CD45.

26. The method of claim 1, wherein the third defined media of step (c) comprises about 10-50 ng/ml BMP4 and about 10-50 ng/ml VEGF.

27. The method of claim 26, wherein the third defined media of step (c) further comprises about 10-50 ng/ml FGF-2.

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

29. The method of claim 28, wherein the fourth defined media of step (d) further comprises about 5-25 ng/ml GMCSF.

30. The method of claim 1, wherein the fourth defined media of step (d) further comprises TPO, SCF, IL-6, and IL-3.

31. The method of claim 1, wherein a plurality of the hematopoietic precursor cells express CD34, CD43, CD45 and CD31.

32. 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.

33. The method of claim 32, 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.

34. The method of claim 33, wherein the one or more of the hematopoietic precursor cells are differentiated into a eosinophil, basophil, neutrophil, monocyte, or macrophage.

35. 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 group 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.

36. 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 group 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.

37. 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 group consisting of IL-7, SCF, and IL-2, in an amount sufficient to promote differentiation of the cells into NK cells.

38. The method of claim 33, 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 group consisting of IL-7, SCF, and IL-2 in an amount sufficient to promote differentiation of the cells into T cells.

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

40. The method of claim 39, wherein said mammalian pluripotent cells are human pluripotent cells.

41. The method of claim 40, wherein said human pluripotent cells are human embryonic stem cells (hESC).

42. The method of claim 41, 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.

43. The method of claim 40, wherein said human pluripotent cells are induced pluripotent cells (iPSC).

44. The method of claim 1, wherein the defined, feeder-free media of step (b) comprises polyvinyl alcohol (PVA).

45. The method of claim 1, wherein the fourth defined media of the second culturing step (d) further includes VEGF and FGF to promote enrichment of endothelial cells, and wherein steps b) through d) are carried out in a defined media that is xeno-free, serum-free, feeder cell-free, free of feeder-cell conditioned media and free of bovine serum albumin and there are no intervening steps carried out using a media that is not comprising xeno-free, serum-free, feeder cell-free, free of feeder-cell conditioned media and free of bovine serum albumin.

46. The method of claim 1, wherein steps b) through d) are carried out in a defined media that is xeno-free, serum-free, feeder cell-free, free of feeder-cell conditioned media and free of bovine serum albumin and there are no intervening steps carried out using a media that is not comprising xeno-free, serum-free, feeder cell-free, free of feeder-cell conditioned media and free of bovine serum albumin serum, feeder cells, feeder-cell conditioned media or bovine serum albumin.

47. The method of claim 30, wherein the fourth defined media of step (d) 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.

48. The method of claim 1, wherein the fourth defined media of step (d) further comprises VEGF and FGF.