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

Patent metadata

Field	Value
Publication number	US-9763984-B2
Application number	US-201314138008-A
Country	US
Kind code	B2
Filing date	Dec 21, 2013
Priority date	Dec 21, 2012
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

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 platelets from a cell population comprising megakaryocytes, comprising:
contacting a feeder-free, non-adherent culture that includes a cell population comprising megakaryocytes positive for CD41a and CD42b expression with
(i) TPO or a TPO agonist or
(ii) hematopoietic expansion medium and optionally
(1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or
(2) TPO or a TPO agonist, SCF, and IL-11

to cause the formation in culture of proplatelets that release platelets,
at least 60% of which are positive for CD41a and CD42b expression.

2. The method of claim 1 , wherein the TPO agonist comprises at least one of: ADP, epinephrine, thrombin, collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoietic agents, romiplostim, eltrombopag (SB497115, Promacta), recombinant human thrombopoietin (TPO), pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, TPO nonpeptide mimetics, AKR-501, monoclonal TPO agonist antibodies, polyclonal TPO agonist antibodies, TPO minibodies, VB22B sc(Fv)2, domain subclass-converted TPO agonist antibodies, MA01G4G344, recombinant human thrombopoietins, recombinant TPO fusion proteins, and TPO nonpeptide mimetics.

3. The method of claim 1 , wherein substantially all the platelets are functional.

4. The method of claim 1 , wherein the cell population comprising megakaryocytes comprises less than 5% CD14 + cells.

5. The method of claim 1 further comprising isolating the platelets.

6. The method of claim 1 , wherein the cell population comprising megakaryocytes is contacted with hematopoietic expansion medium that comprises at least one reagent selected from: Stem Cell Factor (SCF) at a concentration of 0.5-100 ng/ml, thrombopoietin (TPO) at a concentration of 10-100 ng/ml, interleukin-11 (IL-11) at a concentration of 10-100 ng/ml, at least one ROCK inhibitor, and heparin at a concentration of 2.5-25 Units/ml.

7. The method of claim 6 , wherein at least one ROCK inhibitor comprises Y27632 at a concentration of 2-20 μM.

8. The method of claim 1 , wherein the cell population comprising megakaryocytes is contacted with hematopoietic expansion medium that comprises at least one reagent selected from: TPO at a concentration of 10-100 ng/ml, SCF at a concentration of 0.5-100 ng/ml, IL-6 at a concentration of 5-25 ng/ml, IL-9 at a concentration of 5-25 ng/ml, at least one ROCK inhibitor, and Heparin at a concentration of 2.5-25 units/ml.

9. The method of claim 8 , wherein at least one ROCK inhibitor comprises Y27632 at a concentration of 2-20 μM.

10. The method of claim 1 , wherein at least 50 platelets per megakaryocyte are produced.

11. The method of claim 1 , wherein at least 70% of the platelets are positive for CD41a and CD42b expression.

12. The method of claim 1 , wherein the platelets are human platelets.

13. The method of claim 1 , wherein the megakaryocytes are derived from megakaryocyte lineage specific progenitors (MLPs).

14. The method of claim 13 , wherein the MLPs are derived from hemogenic endothelial (PVE-HE) cells.

15. The method of claim 14 , wherein the PVE-HE cells are derived from pluripotent stem cells.

16. The method of claim 15 , wherein the PVE-HE cells are derived without embryoid body formation.

17. The method of claim 15 , wherein the pluripotent stem cells are human pluripotent stem cells.

18. The method of claim 15 , wherein the pluripotent stem cells are induced pluripotent stem cells (iPSC).

19. The method of claim 18 , wherein the iPSCs are human iPSCs.

Assignees

• Advanced Cell Tech 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

• C12N5/0644

  Platelets; Megakaryocytes · CPC title

• C12N2501/2306

  Interleukin-6 (IL-6) · CPC title