Methods for production of platelets from pluripotent stem cells and compositions thereof

Patent metadata

Field	Value
Publication number	US-10426799-B2
Application number	US-201314653969-A
Country	US
Kind code	B2
Filing date	Dec 21, 2013
Priority date	Dec 21, 2012
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

Methods for production of platelets from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are provided. These methods may be performed without forming embryoid bodies or clusters of pluripotent stem cells, and may be performed without the use of stromal inducer cells. Additionally, the yield and/or purity can be greater than has been reported for prior methods of producing platelets from pluripotent stem cells. Also provided are compositions and pharmaceutical preparations comprising platelets, preferably produced from pluripotent stem cells.

First claim

What is claimed is:

1. A method for producing human megakaryocytes comprising:
(a) culturing human pluripotent stem cells under an adherent condition in culture medium that comprises bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor to form human hemogenic endothelial cells;
(b) culturing the human hemogenic endothelial cells in culture medium that comprises two or more factors selected from thrombopoietin, interleukin-3, and interleukin-6 to form human megakaryocyte progenitors; and
(c) culturing the human megakaryocyte progenitors under a non-adherent culture condition in culture medium that comprises thrombopoietin and one or more factor(s) selected from stem cell factor, interleukin-6, interleukin-9, and a ROCK inhibitor to form human megakaryocytes.

2. The method of claim 1 , wherein the human pluripotent stem cells are human induced pluripotent stem cells.

3. The method of claim 1 , wherein the human hemogenic endothelial cells are cultured in the presence of a BET inhibitor.

4. The method of claim 3 , wherein the BET inhibitor is IBET151.

5. The method of claim 3 , wherein the concentration of BET inhibitor is within a micromolar or submicromolar range.

6. The method of claim 5 , wherein the concentration of BET inhibitor is 0.25 μM.

7. The method of claim 1 , wherein the culture medium of step (b) comprises thrombopoietin, interleukin-3, and interleukin-6.

8. The method of claim 1 , wherein the culture medium of step (c) comprises stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.

9. The method of claim 8 , wherein the ROCK inhibitor is Y27632.

10. The method of claim 1 , wherein the human hemogenic endothelial cells are differentiated from the human pluripotent stem cells under low oxygen conditions comprising 1% to 10% oxygen.

11. The method of claim 10 , wherein the human hemogenic endothelial cells are differentiated from the human pluripotent stem cells under low oxygen conditions comprising 4% to 6% oxygen.

12. The method of claim 1 , wherein the human megakaryocyte progenitors are cultured to form human megakaryocytes at a temperature between 38-40° C.

13. The method of claim 1 , wherein the human hemogenic endothelial cells express CD31.

14. The method of claim 1 , wherein the human megakaryocyte progenitors express CD41a.

15. The method of claim 1 , wherein the step of culturing human pluripotent stem cells under an adherent condition comprises culturing human pluripotent stem cells on an extracellular matrix.

16. The method of claim 1 , wherein the human pluripotent stem cells of step (a) are cultured under a feeder-free culture condition.

17. A method for producing human megakaryocytes comprising:
(a) culturing human pluripotent stem cells under an adherent condition in culture medium that comprises bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor to form human hemogenic endothelial cells;
(b) culturing the human hemogenic endothelial cells in culture medium that comprises stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-3, and interleukin-6 to form human megakaryocyte progenitors; and
(c) culturing the human megakaryocyte progenitors under a non-adherent culture condition in culture medium that comprises stem cell factor, thrombopoietin, interleukin-6, and interleukin-9 to form human megakaryocytes.

18. The method of claim 17 , wherein the human hemogenic endothelial cells are cultured in the presence of a ROCK inhibitor and/or a BET inhibitor.

19. A method for producing human megakaryocytes comprising:
(a) culturing human pluripotent stem cells under an adherent condition in culture medium that comprises bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor to form human hemogenic endothelial cells;
(b) culturing the human hemogenic endothelial cells in culture medium that comprises two or more factors selected from thrombopoietin, interleukin-3, interleukin-6, and Fms-related tyrosine kinase 3 to form human megakaryocyte progenitors; and
(c) culturing the human megakaryocyte progenitors under a non-adherent culture condition in culture medium that comprises thrombopoietin and one or more factor(s) selected from stem cell factor, interleukin-6, interleukin-9, and a ROCK inhibitor to form human megakaryocytes.

20. The method of claim 19 , wherein the human pluripotent stem cells are human induced pluripotent stem cells.

21. The method of claim 19 , wherein the human hemogenic endothelial cells are cultured in the presence of a BET inhibitor.

22. The method of claim 19 , wherein the culture medium of step (b) comprises thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-3, and interleukin-6.

23. The method of claim 19 , wherein the culture medium of step (c) comprises stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.

24. The method of claim 19 , wherein the step of culturing human pluripotent stem cells under an adherent condition comprises culturing human pluripotent stem cells on an extracellular matrix.

25. The method of claim 19 , wherein the human pluripotent stem cells of step (a) are cultured under a feeder-free culture condition.

26. A method for producing human megakaryocytes comprising:
(a) culturing human pluripotent stem cells under an adherent condition in culture medium that comprises bone morphogenetic protein 4, basic fibroblast growth factor, and vascular endothelial growth factor to form human hemogenic endothelial cells;
(b) culturing the human hemogenic endothelial cells in culture medium that comprises two or more factors selected from thrombopoietin, interleukin-3, interleukin-6, and stem cell factor to form human megakaryocyte progenitors; and
(c) culturing the human megakaryocyte progenitors under a non-adherent culture condition in culture medium that comprises thrombopoietin and one or more factor(s) selected from stem cell factor, interleukin-6, interleukin-9, and a ROCK inhibitor to form human megakaryocytes.

27. The method of claim 26 , wherein the human pluripotent stem cells are human induced pluripotent stem cells.

28. The method of claim 26 , wherein the human hemogenic endothelial cells are cultured in the presence of a BET inhibitor.

29. The method of claim 26 , wherein the culture medium of step (b) comprises thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.

30. The method of claim 26 , wherein the culture medium of step (c) comprises stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.

31. The method of claim 26 , wherein the step of culturing human pluripotent stem cells under an adherent condition comprises culturing human pluripotent stem cells on an extracellular matrix.

32. The method of claim 26 , wherein the human pluripotent stem cells of step (a) are cultured under a feeder-free culture condition.

Assignees

• Ocata Therapeutics Inc
• Astellas Inst For Regenerative Medicine

Inventors

• Feng Qiang
• Lu Shi-Jiang
• Lanza Robert P

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• A61P33/06

  Antimalarials · CPC title

• A61P17/02

  for treating wounds, ulcers, burns, scars, keloids, or the like · CPC title

• C12N2501/2306

  Interleukin-6 (IL-6) · CPC title

• A61K35/19 Primary

  Platelets; Megacaryocytes · CPC title