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 human megakaryocytes comprising:
(a) culturing 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
(b) 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 , further comprising culturing human pluripotent stem cells under adherent culture conditions and/or feeder free culture conditions to form the human hemogenic endothelial 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 1 , wherein the culture medium of step (a) comprises thrombopoietin, interleukin-3, and interleukin-6.
6. The method of claim 1 , wherein the culture medium of step (b) comprises stem cell factor, thrombopoietin, interleukin-6, interleukin-9, and a ROCK inhibitor.
7. The method of claim 6 , wherein the ROCK inhibitor is Y27632.
8. The method of claim 1 , wherein the human megakaryocyte progenitors are cultured to form human megakaryocytes at a temperature between 38-40° C.
9. The method of claim 1 , wherein the human hemogenic endothelial cells express CD31.
10. The method of claim 1 , wherein the human megakaryocyte progenitors express CD41a.
11. The method of claim 1 , wherein the culture medium of step (a) comprises stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-3, and interleukin-6, and the culture medium of step (b) comprises stem cell factor, thrombopoietin, interleukin-6, and interleukin-9.
12. A method for producing human megakaryocytes comprising:
(a) culturing human hemogenic endothelial cells in culture medium that comprises two or more factors selected from (i) thrombopoietin, (ii) interleukin-3, (iii) interleukin-6, and (iv) Fms-related tyrosine kinase 3 or stem cell factor to form human megakaryocyte progenitors; and
(b) 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.