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 . The method of  claim 1 , wherein said pluripotent stem cell is an embryonic stem cell or embryo-derived cell. 
     
     
         3 . The method of  claim 1 , wherein said pluripotent stem cell is an induced pluripotent stem cell. 
     
     
         4 . The method of  claim 1 , wherein said pluripotent stem cell is a human cell. 
     
     
         5 . The method of  claim 1 , wherein said pluripotent stem cell is genetically manipulated prior to differentiation. 
     
     
         6 . The method of  claim 1 , wherein differentiating said pluripotent stem cell into an enucleated erythroid cell comprises differentiating said pluripotent stem cell into a hemangioblast, non-engrafting hemangio cell, or blast cell. 
     
     
         7 . The method of  claim 6 , wherein said hemangioblast, non-engrafting hemangio cell, or blast cell is expanded prior to being differentiated into said enucleated erythroid cell. 
     
     
         8 . The method of  claim 7 , wherein said hemangioblasts, non-engrafting hemangio cells, or blast cells are expanded in Stemline II medium with Epo, IL-3, and SCF. 
     
     
         9 . The method of  claim 6 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said human pluripotent stem cell into said hemangioblast is done in vitro by a method comprising:
 (a) culturing a cell culture comprising human pluripotent stem cell in serum-free media 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) adding at least two growth factors to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand said human hemangioblast in said embryoid bodies culture,   wherein said human pluripotent stem cells, embryoid bodies and hemangioblasts are grown in serum-free media throughout steps (a) and (b) of said method, and   wherein said at least two growth factors in step (b) comprise BMP4 and VEGF.   
     
     
         10 . The method of  claim 9 , wherein differentiating said human pluripotent stem cell into said hemangioblast further comprises:
 (c) disaggregating said embryoid bodies into single cells; and   (d) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand human hemangioblasts in said culture comprising said single cells, and   wherein said human pluripotent stem cells, embryoid bodies and hemangio-colony foaming cells are grown in serum-free media throughout steps (a)-(d) of said method.   
     
     
         11 - 18 . (canceled) 
     
     
         19 . The method of  claim 6 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said human pluripotent stem cell into said non-engrafting hemangio cell is done in vitro by a method comprising:
 (a) culturing a cell culture comprising said human pluripotent stem cell in serum-free media 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) adding at least one growth factor to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media,   wherein said growth factor is in an amount sufficient to expand said human non-engrafting hemangio cell in said embryoid bodies culture,   wherein said embryoid bodies are cultured for 10-13 days, and   wherein said human pluripotent stem cell, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a) and (b) of said method.   
     
     
         20 . The method of  claim 19 , wherein differentiating said pluripotent stem cell into said non-engrafting hemangio cell further comprises:
 (c) disaggregating said embryoid bodies into single cells; and   (d) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand said human non-engrafting hemangio cell in said culture comprising said single cells,   wherein said embryo-derived cells, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a)-(d) of said method.   
     
     
         21 - 28 . (canceled) 
     
     
         29 . The method of  claim 1 , wherein differentiating said pluripotent stem cell into said enucleated erythroid cell further comprises culturing said pluripotent stem cell in the culture medium comprising EPO. 
     
     
         30 . The method of  claim 29 , wherein differentiating said pluripotent stem cell into said enucleated erythroid cell further comprises:
 culturing said pluripotent stem cell in a culture medium comprising a supplement selected from the group consisting of inositol, folic acid, monothioglycerol, transferrin, insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine, penicillin-streptomycin and combinations thereof; and   culturing said pluripotent stem cell in said culture medium wherein said culture medium further comprises an agent selected from the group consisting of hydrocortisone, SCF, IL3, Epo and combinations thereof.   
     
     
         31 . An enucleated erythroid cell produced by the method of  claim 1 . 
     
     
         32 . A method of producing a pluripotent stem cell-derived erythroid cell, comprising:
 providing a 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 an embryonic stem cell or embryo-derived cell. 
     
     
         34 . The method of  claim 32 , wherein said pluripotent stem cell is an induced pluripotent stem cell. 
     
     
         35 . The method of  claim 32 , wherein said pluripotent stem cell is a human cell. 
     
     
         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 Stemline II medium with Epo, IL-3, and SCF. 
     
     
         40 . The method of  claim 32 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said human pluripotent stem cell into said hemangioblast is done in vitro by a method comprising:
 (a) culturing a cell culture comprising said human pluripotent stem cell in serum-free media 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) adding at least two growth factors to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand said human hemangioblast in said embryoid bodies culture,   wherein said human pluripotent stem cells, embryoid bodies and hemangioblasts are grown in serum-free media throughout steps (a) and (b) of said method, and   wherein said at least two growth factors in step (b) comprise BMP4 and VEGF.   
     
     
         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) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand human hemangioblasts in said culture comprising said single cells, and   wherein said pluripotent stem cells, embryoid bodies and hemangio-colony forming cells are grown in serum-free media throughout steps (a)-(d) of said method.   
     
     
         42 - 49 . (canceled) 
     
     
         50 . The method of  claim 32 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said pluripotent stem cell into said non-engrafting hemangio cell is done in vitro by a method comprising:
 (a) culturing a cell culture comprising human pluripotent stem cell in serum-free media 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) adding at least one growth factor to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media,   wherein said growth factor is in an amount sufficient to expand said human non-engrafting hemangio cells in said embryoid bodies culture,   wherein said embryoid bodies are cultured for 10-13 days, and   wherein said human pluripotent stem cell, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a) and (b) of said method.   
     
     
         51 . The method of  claim 50 , wherein differentiating said pluripotent stem cell into said non-engrafting hemangio cell further comprises
 (c) disaggregating said embryoid bodies into single cells; and   (d) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand human non-engrafting hemangio cells in said culture comprising said single cells,   wherein said human pluripotent stem cell, embryoid bodies and non-engrafting hemangio cells are gown in scrum-free media throughout steps (a)-(d) of said method.   
     
     
         52 - 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 . The method of  claim 59 , wherein said pluripotent stem cell is an embryonic stem cell or embryo-derived cell. 
     
     
         61 . The method of  claim 59 , wherein said pluripotent stem cell is an induced pluripotent stem cell. 
     
     
         62 . The method of  claim 59 , wherein said pluripotent stern cell is a human cell. 
     
     
         63 . The method of  claim 59 , wherein said pluripotent stem cell is genetically manipulated prior to differentiation. 
     
     
         64 . The method of  claim 59 , wherein said hemangioblast, non-engrafting hemangio cell, or blast cell is expanded prior to being differentiated into said megakaryocyte or said platelet. 
     
     
         65 . The method of  claim 64 , wherein said hemangioblasts, non-engrafting hemangio cells, or blast cells are expanded in Stemline II medium with Epo, IL-3, and SCF. 
     
     
         66 . The method of  claim 59 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said human pluripotent stem cell into said hemangioblast is done in vitro by a method comprising:
 (a) culturing a cell culture comprising human pluripotent stem cell in serum-free media 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) adding at least two growth factors to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand said human hemangioblast in said embryoid bodies culture,   wherein said human pluripotent stem cells, embryoid bodies and hemangioblasts are grown in serum-free media throughout steps (a) and (b) of said method, and   wherein said at least two growth factors in step (b) comprise BMP4 and VEGF.   
     
     
         67 . The method of  claim 66 , wherein differentiating said human pluripotent stem cell into said hemangioblast further comprises:
 (c) disaggregating said embryoid bodies into single cells; and   (d) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand human hemangioblasts in said culture comprising said single cells, and   wherein said human pluripotent stem cells, embryoid bodies and hemangio-colony forming cells are grown in serum-free media throughout steps (a)-(d) of said method.   
     
     
         68 - 73 . (canceled) 
     
     
         74 . The method of  claim 59 , wherein differentiating said hemangioblast, non-engrafting hemangio cell, or blast cell into said megakaryocyte or said platelet is done after about 6 to 8 days of hemangioblast, non-engrafting hemangio cell, or blast cell culture. 
     
     
         75 . The method of  claim 59 , wherein said pluripotent stem cell is a human pluripotent stem cell and differentiating said human pluripotent stem cell into said non-engrafting hemangio cell is done in vitro by a method comprising:
 (a) culturing a cell culture comprising said human pluripotent stem cell in serum-free media 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) adding at least one growth factor to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media,   wherein said growth factor is in an amount sufficient to expand said human non-engrafting hemangio cell in said embryoid bodies culture,   wherein said embryoid bodies are cultured for 10-13 days, and   wherein said human pluripotent stem cell, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a) and (b) of said method.   
     
     
         76 . The method of  claim 75 , wherein differentiating said pluripotent stem cell into said non-engrafting hemangio cell further comprises:
 (c) disaggregating said embryoid bodies into single cells; and   (d) adding at least one growth factor to said culture comprising said single cells and continuing to culture said culture in serum-free media, wherein said growth factor is in an amount sufficient to expand said human non-engrafting hemangio cell in said culture comprising said single cells,   wherein said embryo-derived cells, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a)-(d) of said method.   
     
     
         77 - 82 . (canceled) 
     
     
         83 . A megakaryocyte or a platelet produced by the method of  claim 59 .