Megakaryocyte and platelet production from stem cells

Abstract

Methods for obtaining purified populations of megakaryocytes and platelets by ex vivo culture of stem cells are provided herein.

What is claimed is: 
     
         1 . A platelet production system for the ex vivo production of platelets comprising:
 a bioreactor for expansion of stem cells in the presence of a first growth medium in fluid communication with;   a maturation chamber comprising an artificial bone marrow niche and a second growth medium, wherein the maturation chamber is in fluid communication with;   a cell separation chamber for selecting mature megakaryocytes which is in fluid communication with;   a platelet production module comprising a plurality of platelet production chambers, a three-dimensional matrix, a third growth medium, and a plurality of pumps for moving the third growth medium across the platelet production chambers, wherein the platelet production module is in fluid communication with;   a platelet collection chamber.   
     
     
         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 comprises aryl-hydrocarbon inhibitor/stem regenin-1 and notch-ligand delta-1. 
     
     
         6 . The method of  claim 5 , wherein the first growth medium comprises a plurality of 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, thrombopoietin (TPO), p38 inhibitors, stem cell factor (SCF), dexamethasone, lipids, IGF-1, erythropoietin (EPO), IL-3, IL-6, IL-11, and FLT-3 ligand (FLT-3l). 
     
     
         7 . The method of  claim 5 , wherein the first growth medium further comprises mesenchymal stem cells and/or OP-9 cells. 
     
     
         8 . The method of  claim 1 , wherein the second growth medium comprises TPO, nicotinamide, and a Rho/Rock inhibitor. 
     
     
         9 . The method of  claim 8 , wherein the second growth medium further comprises a plurality of growth factors selected from the group consisting of serotonin, arachidonic acid, Z-VAD-FMK, IL-3, IL-6, FLT-3l. 
     
     
         10 . 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. 
     
     
         11 . The method of  claim 1 , wherein the artificial bone marrow niche further contains mesenchymal stem cells and/or endothelial cells. 
     
     
         12 . The method of  claim 1 , wherein the third growth medium comprises fibrinogen, fibronectin, von Willebrand factor (vWF), an MLCK inhibitor, a Rho/Rock inhibitor, and nicotinamide. 
     
     
         13 . The method of  claim 12 , wherein the third growth medium further 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, Rho/Rock inhibitors, Src inhibitors, Aurora-B inhibitors, Bcr-Abl inhibitors, phorbol 12-myristate 13-acetate (PMA), and blebbistatin. 
     
     
         14 . The method of  claim 1 , wherein the maturation chamber maintains an oxygen concentration between about 10% and about 30% PO 2 . 
     
     
         15 . The method of  claim 1 , wherein the platelet production module maintains an oxygen concentration between about 10% and about 30% PO 2 . 
     
     
         16 . The method of  claim 1 , wherein the platelet production module maintains a shear stress between about 100 μl/min and about 400 μl/min.