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

• US 10,894,065 B2
• Filed: 08/15/2017
• Issued: 01/19/2021
• Est. Priority Date: 12/21/2012
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Feng, Qiang, Lu, Shi-Jiang, Lanza, Robert P.
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): Pyla, Evelyn Y
• Application Number:
  US15/677,770
• Publication Number:
  US 20180008640A1
• Time in Patent Office: 1,253 Days
• Field of Search: None
• CPC Class Codes: A61K 35/19 Platelets; Megacaryocytes
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 2501/04 Immunosuppressors, e.g. cyclosporin, tacrolimus
• CPC Class Codes: C12N 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2309 Interleukin-9 (IL-9)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/30 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C12N2501/85
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/734 Proteases (EC 3.4.)
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 5/0644 Platelets; Megakaryocytes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A method for producing a pharmaceutical preparation comprising:

differentiating megakaryocytes positive for CD41a and CD42b expression in a feeder-free, non-adherent culture to cause formation in the culture of platelets, at least 60% of which are positive for CD41a and CD42b;

isolating the platelets from the culture; and

concentrating the platelets to form a pharmaceutical preparation comprising at least 10⁸ platelets, wherein the preparation is substantially free of leukocytes and wherein substantially all of the platelets are functional, and wherein the step of differentiating megakaryocytes comprises contacting the megakaryocytes with;

(i) Thrombopoietin (TPO) or a TPO agonist;

or

(ii) a hematopoietic expansion medium, wherein the hematopoietic expansion medium comprises TPO or a TPO agonist and one or more of Stem Cell Factor (SCF), Interleukin (IL)-6, IL-9, and IL-11.

2. The method of claim 1, wherein the pharmaceutical preparation comprises 10⁹-10¹⁴ platelets.

3. The method of claim 1, wherein the platelets have one or more of the following attributes:

a mean platelet volume range of 9.7-12.8 fL;

a unimodal distribution of size in the preparation; and

/or a log normal platelet volume distribution wherein one standard deviation is less than 2 μ

m³, less than 1.5 μ

m³, less than 1 μ

m³ or less than 0.5 μ

m³.

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

5. The method of claim 1, wherein at least 50%, 60%, 70%, 80% or 90% of the platelets are functional for at least 2, 3 or 4 days after storage at room temperature.

6. The method of claim 1, wherein the TPO agonist comprises one or more of:

ADP, epinephrine, thrombin, collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoietic agents, romiplostim, eltrombopag, recombinant human 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 TPO fusion proteins, and/or TPO nonpeptide mimetics.

7. The method of claim 1, wherein the hematopoietic expansion medium comprises one or more of:

SCF at 0.5-100 ng/ml, TPO at 10-100 ng/ml, IL 11 at 10-100 ng/ml, at least one Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and/or Heparin at 2.5-25 Units/ml.

8. The method of claim 1, wherein the hematopoietic expansion medium comprises one or more of:

TPO at 10-100 ng/ml, SCF at 0.5-100 ng/ml, IL-6 at 5-25 ng/ml, IL-9 at 5-25 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25 units/ml.