Compositions and methods for the generation of platelets and methods of use thereof
Compositions and methods for generating platelets and methods of use thereof are disclosed.
1. A method of treating thrombocytopenia in a subject, said method comprising:
a) decreasing the expression of GATA-1 in human pluripotent stem cells, thereby generating GATA-1 knockout or knockdown stem cells;
b) culturing the GATA-1 knockout or knockdown stem cells in the presence of thrombopoietin, thereby generating a self-replicating, megakaryocyte-erythroid progenitor (MEP)-like cell line (G1ME);
c) expressing GATA-1 in said G1ME cells, thereby differentiating the cells into terminal erythrocytes and megakaryocytes;
d) isolating megakaryocytes from the cells of step c); and
e) administering the isolated megakaryocytes to said subject, thereby treating said thrombocytopenia,
wherein step a) comprises delivering GATA-1 antisense, GATA-1 siRNA, GATA-1 shRNA, or a nucleic acid molecule encoding said antisense, siRNA or shRNA to the human pluripotent stem cells.
2. The method of claim 1 , wherein said human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem (iPS) cells.
3. The method of claim 1 , further comprising the delivery of a nucleic acid molecule encoding a protein of interest to the cells prior to step e).
4. The method of claim 3 , further comprising the delivery of a nucleic acid molecule encoding a protein of interest to said G1ME cells.
5. The method of claim 1 , wherein step c) comprises removal of the suppression of GATA-1 expression by said GATA-1 antisense, GATA-1 siRNA, GATA-1 shRNA, or by the nucleic acid molecule encoding said antisense, siRNA or shRNA.
6. The method of claim 1 , wherein step c) comprises expressing GATA-1 to native levels.
7. The method of claim 1 , wherein step b) further comprises culturing the GATA-1 knockout or knockdown stem cells with stromal cells.
8. The method of claim 1 , wherein step d) comprises isolating cells having a diameter of at least about 50 μm.
9. The method of claim 3 , wherein said protein of interest is a procoagulant protein.