Megakaryocyte and platelet production from stem cells

1. A method for producing platelets for clinical transfusion in vitro comprising:

(1) culturing stem cells in a first growth medium to produce a megakaryocyte progenitor cell population, wherein the first growth medium comprises aryl-hydrocarbon inhibitor/stem regenin-1 and notch-ligand delta-1;

(2) maturing the expanded megakaryocyte progenitor cells in an artificial bone marrow niche environment comprising a second growth medium in the presence of an oxygen concentration between about 10% and about 30% PO 2 to differentiate the megakaryocyte progenitor cells into megakaryocytes, wherein the second growth medium comprises thrombopoietin (TPO), stem cell factor (SCF), nicotinamide, and a Rho/Rock inhibitor;

(3) isolating the mature megakaryocytes;

(4) culturing the mature megakaryocytes in a three-dimensional matrix and a third growth medium and in the presence of an oxygen concentration between about 10% and about 30% PO 2 and a shear stress between about 100 and 400 μl/min to produce platelets, wherein the third growth medium comprises fibrinogen, fibronectin, von Willebrand factor (vWF), an MLCK inhibitor, a Rho/Rock inhibitor, and nicotinamide; and

(5) collecting the platelets in a number suitable for transfusion.

2. The method of claim 1 , wherein the stem cells are selected from the group consisting of hematopoietic stem cells, induced pluripotent stem cells, embryonic stem cells, and fibroblasts.

3. The method of claim 2 , wherein the hematopoietic stem cells are obtained from the bone marrow, peripheral blood, or cord blood.

4. The method of claim 1 , wherein the stem cells are enriched for CD34 + cells prior to culture-expansion.

5. The method of claim 1 , wherein the first growth medium further comprises additional growth factors selected from the group consisting of prostaglandin-E2, SALL4 gene activators, Hoxb4 activators, stromal cell-derived factor-1 (SDF-1α), histone acetyl transferase inhibitors, valproic acid, tropoelastin, copper chelation, Z-VAD-FMK, banana lectin, garlic lectin, interferon-α, TPO, p38 inhibitors, SCF, dexamethasone, lipids, IGF-1, erythropoietin (EPO), IL-3, IL-6, IL-11, and FLT-3 ligand (FLT-3l).

6. The method of claim 5 , wherein the growth factors are IL-3, IL-6, and IL-11.

7. The method of claim 1 , wherein the second growth medium further comprises additional growth factors selected from the group consisting of serotonin, arachidonic acid, Z-VAD-FMK, IL-3, IL-6, and FLT-3l.

8. The method of claim 1 , wherein the cell growth matrix is selected from the group consisting of extracellular matrix extracts, extracellular matrix gels, gelatin, fibrinogen, collagen, methylcellulose, and combinations thereof.

9. The method of claim 1 , wherein the artificial bone marrow niche further contains mesenchymal stem cells and/or endothelial cells.

10. The method of claim 1 , wherein the third growth medium comprises a plurality of growth factors selected from the group consisting of Fas-ligand, PMA, nitric oxide, Src inhibitors, SDF-1α, folic acid, vitamin B12, Aurora-B inhibitors, Bcr-Abl inhibitors, phorbol 12-myristate 13-acetate (PMA), and blebbistatin.

11. The method of claim 1 , wherein the first growth medium further comprises mesenchymal stem cells and/or OP-9 cells.