Methods for production of platelets from pluripotent stem cells and compositions thereof
AssigneeAstellas Institute for Regenerative Medicine
InventorsQiang Feng, Shi-Jiang Lu, Robert P. Lanza
Life of this patent
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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.
Claims24
A pharmaceutical preparation that is suitable for use in a human patient comprising at least 108 platelets, wherein the preparation is substantially free of leukocytes and wherein substantially all of the platelets are functional, or
- a bioreactor having weakly adherent or non-adherent megakaryocytes that produce functional platelets without feeder cells, or
- a composition comprising at least 109 MLPs, or
- a cryopreserved composition comprising MLPs, optionally comprising 109 to 1014 MLPs, further optionally 109, 1010, 1011, 1012, 1013 or 1014 MLPs, or
- a bank comprising cryopreserved MLPs.
-13. (canceled)
A method for producing platelets from megakaryocytes comprising the steps of:
- (A)
- a) providing a non-adherent culture of megakaryocytes;
- b) contacting the megakaryocytes with
- (i) TPO or a TPO agonist to cause the formation of proplatelets in culture, wherein the proplatelets release platelets;
- 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 of pro-platelets in culture, wherein the pro-platelets release platelets;
- and
- c) isolating the platelets,
- optionally wherein the non-adherent culture of megakaryocytes is a feeder-free, non-adherent culture that includes a cell population comprising megakaryocytes positive for CD41a and CD42b expression, and optionally wherein at least 60% of the released platelets are positive for CD41a and CD42b expression, or
- (B) 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.
-38. (canceled)
-41. (canceled)
-45. (canceled)
A method for producing hemogenic endothelial (PVE-HE) cells comprising differentiating pluripotent stem cells in vitro, thereby forming a cell population comprising hemogenic endothelial cells, wherein the pluripotent stem cells are differentiated without embryoid body formation.
A composition comprising a cell population, wherein at least 70% of cells in the population are CD31/PECAM1+, CD144/VE−Cad+, and CD105/endoglin+ hemogenic endothelial cells, wherein the CD31/PECAM1+, CD144/VE-Cad+, and CD105/endoglin+ hemogenic endothelial cells are produced by in vitro differentiation of pluripotent stem cells.
Dependencies are inferred from the claim text.