Methods for producing enucleated erythroid cells derived from pluripotent stem cells

Abstract

Methods for generating enucleated erythroid cells using pluripotent stem cells are provided. The methods permit the production of large numbers of cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications. Methods for generating megakaryocyte and platelets are also provided.

1 - 83 . (canceled) 
     
     
         84 . A method of producing a human pluripotent stem cell-derived megakaryocyte or a human pluripotent stem cell-derived platelet, the method comprising:
 (a) culturing a human pluripotent stem cell in a culture media comprising bone morphogenic protein 4 (BMP-4) or vascular endothelial growth factor (VEGF), or both, and inducing formation of the pluripotent stem cell into an embryoid body, in the absence of thrombopoietin (TPO), Flt-3L (FL) or stem cell factor (SCF);   (b) culturing the embryoid body in a semi-solid culture media comprising at least two growth factors selected from the group consisting of insulin, transferrin, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), FLT3 (FL), vascular endothelial growth factor (VEGF), and bone morphogenic protein 4 (BMP-4), and generating a hemangioblast, a non-engrafting hemangio cell, or a blast cell from the embryoid body; and   (c) culturing the hemangioblast, the non-engrafting hemangio cell, or the blast cell in a megakaryocyte (MK) culture media comprising TPO, thereby generating a megakaryocyte or a platelet.   
     
     
         85 . The method of  claim 84 , further comprising disaggregating the embryoid body of step (a) to generate a disaggregated embryoid body. 
     
     
         86 . The method of  claim 84 , wherein the human pluripotent stem cell is an embryonic stem cell or an embryo-derived cell. 
     
     
         87 . The method of  claim 84 , wherein the human pluripotent stem cell is an induced pluripotent stem cell. 
     
     
         88 . The method of  claim 84 , wherein the human pluripotent stem cell is cultured in the culture media comprising BMP-4, or VEGF, or both in step (a) for at least the first 48 hours of cell culture. 
     
     
         89 . The method of  claim 84 , wherein the culture media in step (a) further comprises one or more growth factors selected from the group consisting of basic fibroblast growth factor (bFGF), erythropoietin (EPO), and combinations thereof. 
     
     
         90 . The method of  claim 89 , wherein the human pluripotent stem cell is cultured in step (a) in a culture media further comprising the one or more growth factors selected from the group consisting of basic fibroblast growth factor (bFGF), erythropoietin (EPO), and combinations thereof within 48-72 hours of cell culture. 
     
     
         91 . The method of  claim 84 , wherein the embryoid body in step (b) is cultured in the culture media comprising at least two growth factors, for at least 10-13 days. 
     
     
         92 . The method of  claim 84 , wherein the culture media in step (b) further comprises EPO. 
     
     
         93 . The method of  claim 84 , wherein the culture media in step (a) or step (b) further comprises a fusion protein that comprises HOXB4 and a protein transduction domain (PTD). 
     
     
         94 . The method of  claim 93 , wherein the HOXB4 is a mammalian HOXB4. 
     
     
         95 . The method of  claim 84 , wherein the hemangioblast, the non-engrafting hemangio cell, or the blast cell in step (c) are cultured for at least 6 to 8 days. 
     
     
         96 . The method of  claim 84 , wherein the culture media throughout steps (a)-(c) is serum-free. 
     
     
         97 . The method of  claim 84 , wherein the cultures of step (a) and step (b) are cultured under low attachment conditions. 
     
     
         98 . The method of  claim 84 , wherein the culture media of step (b) further comprises methylcellulose. 
     
     
         99 . The method of  claim 84 , wherein the culture media throughout steps (a)-(b) is feeder-free. 
     
     
         100 . The method of  claim 84 , wherein the human pluripotent stem cell is genetically manipulated prior to differentiation. 
     
     
         101 . The method of  claim 84 , wherein the hemangioblast, the non-engrafting hemangio cell, or the blast cell is expanded prior to being differentiated into a megakaryocyte or a platelet. 
     
     
         102 . The method of  claim 101 , wherein the hemangioblast, the non-engrafting hemangio cell, or the blast cell is expanded in a culture media comprising erythropoietin (EPO), interleukin-3 (IL-3), and stem cell factor (SCF). 
     
     
         103 . A pharmaceutical composition comprising at least 1×10 6  megakaryocytes or platelets produced by the method of  claim 84 .