Methods for production of platelets from pluripotent stem cells and compositions thereof
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.
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 10 8 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 10 9 -10 14 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 μm 3 , less than 1.5 μm 3 , less than 1 μm 3 or less than 0.5 μm 3 .
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.