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 . A method of producing a pluripotent stem cell-derived enucleated erythroid cell, comprising: providing a pluripotent stem cell; and differentiating said pluripotent stem cell into an enucleated erythroid cell by culturing said pluripotent stem cell with OP9 mouse stromal cells or human mesenchymal stem cells (MSCs). 
     
     
         2 - 31 . (canceled) 
     
     
         32 . A method of producing a pluripotent stem cell-derived erythroid cell, comprising: providing a human pluripotent stem cell; and differentiating said pluripotent stem cell into an erythroid cell by culturing said pluripotent stem cell in a medium comprising EPO. 
     
     
         33 . The method of  claim 32 , wherein said pluripotent stem cell is selected from the group consisting of an embryonic stem cell, induced pluripotent stem cell, or embryo-derived cell. 
     
     
         34 - 35 . (canceled) 
     
     
         36 . The method of  claim 32 , wherein said pluripotent stem cell is genetically manipulated prior to differentiation. 
     
     
         37 . The method of  claim 32 , wherein differentiating said pluripotent stem cell into an erythroid cell comprises differentiating said pluripotent stem cell into a hemangioblast, non-engrafting hemangio cell, or blast cell. 
     
     
         38 . The method of  claim 37 , wherein said hemangioblast, non-engrafting hemangio cell, or blast cell is expanded prior being differentiated into said erythroid cell. 
     
     
         39 . The method of  claim 38 , wherein said hemangioblasts, non-engrafting hemangio cells, or blast cells are expanded in the presence of Epo, IL-3, and SCF. 
     
     
         40 . The method of  claim 32 , further comprising:
 (a) culturing a cell culture comprising said human pluripotent stem cell in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said human pluripotent stem cell into embryoid bodies; and   (b) culturing the embryoid bodies in the presence of at least two growth factors in an amount sufficient to produce human hemangioblast, wherein steps (a) and (b) of said method is performed in serum-free media.   
     
     
         41 . The method of  claim 40 , wherein differentiating said human pluripotent stem cell into said hemangioblast further comprises:
 (c) disaggregating said embryoid bodies into single cells; and   (d) culturing said single cells in the presence of at least one additional growth factor in an amount sufficient to produce the human hemangioblasts wherein steps (a)-(d) of said method is performed in serum-free media.   
     
     
         42 . The method of  claim 40 , wherein said at least one growth factor is a fusion protein that comprises HOXB4 and a protein transduction domain (PTD). 
     
     
         43 . The method of  claim 42 , wherein said HOXB4 is mammalian HOXB4. 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 40 , wherein said at least one growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMP), stem cell factor (SCF), Flt-3L (FL) thrombopoietin (TPO) and erythropoietin (EPO). 
     
     
         46 . The method of  claim 45 , wherein the at least one growth factor is vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP), or both, and are present in to step (a) between 0-48 hours of cell culture. 
     
     
         47 . The method of  claim 45 , wherein said at least one growth factor is stem cell factor (SCF), Flt-3L (FL) or thrombopoietin (TPO), or any combination thereof, and are present in step (a) between 48-72 hours from the start of step (a). 
     
     
         48 . The method of  claim 40 , wherein step (a) comprises erythropoietin (EPO). 
     
     
         49 . The method of  claim 41 , wherein step (a) and/or (d) comprises erythropoietin (EPO). 
     
     
         50 . The method of  claim 32 , further comprising:
 (a) culturing a cell culture comprising human pluripotent stem cell in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said human pluripotent stem cell into embryoid bodies; and   (b) culturing the embryoid bodies for at least 10-13 days in the presence of at least one additional growth factor in an amount sufficient to produce said human non-engrafting hemangio cells wherein steps (a) and (b) of said method are performed in serum-free media.   
     
     
         51 - 57 . (canceled) 
     
     
         58 . An erythroid cell produced by the method of  claim 32 . 
     
     
         59 . A method of producing a megakaryocyte or a platelet, comprising: providing a pluripotent stem cell; differentiating said pluripotent stem cell into a hemangioblast, non-engrafting hemangio cell, or blast cell; and differentiating said hemangioblast, non-engrafting hemangio cell, or blast cell into said megakaryocyte or said platelet by culturing in megakaryocyte (MK) culture medium comprising TPO. 
     
     
         60 - 83 . (canceled) 
     
     
         84 . The method of  claim 40 , wherein the at least two growth factors is selected from the group consisting of BMP4 and VEGF.