Methods and compositions for inducing hematopoietic cell differentiation

Abstract

The invention provides culture platforms, cell media, and methods of differentiating pluriptent cells into hematopoietic cells. The invention further provides pluripotent stem cell-derived hematopoietic cells generated using the culture platforms and methods disclosed herein, which enable feed-free, monolayer culturing and in the absence of ER formation. Specifically, pluripotent stem cell-derived hematopoietic cell of this invention include, and not limited to, iHSC, definitive hemogenic endothelium, hematopoietic multipotent progenitors, T cell progenitors, NK cell progenitors, T cells, and NK cells.

What is claimed is: 
     
         1 . A method for directing differentiation of pluripotent stem cells to hematopoietic lineage cells, comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP pathway activator, and optionally bFGF, to obtain mesodermal cells; and (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator, to obtain mesodermal cells having definitive hemogenic endothelium (HE) potential, wherein the mesodermal cells having definitive hemogenic endothelium (HE) potential are capable of providing hematopoietic lineage cells comprising hematopoietic stem and progenitor cells (HSC), hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells; and wherein mesodermal cells and mesodermal cells having definitive HE potential are obtained in steps (i) and (ii) without forming embryoid bodies. 
     
     
         2 . The method of  claim 1 , further comprising: contacting the mesodermal cells having definitive HE potential with a composition comprising bFGF and a ROCK inhibitor, to obtain definitive HE cells. 
     
     
         3 . The method of  claim 2 , further comprising:
 (i) contacting the definitive HE cells with a composition comprising a BMP activator, and optionally a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11 to obtain hematopoietic multipotent progenitor cells (MPP); or   (ii) contacting the definitive HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; and optionally one or more of a BMP activator, a ROCK inhibitor, VEGF and bFGF to obtain pre-T cell progenitors, T cell progenitors, and/or T cells; or   (iii) contacting the definitive HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15, and optionally one or more of a BMP activator, a ROCK inhibitor, VEGF and bFGF to obtain pre-NK cell progenitors, NK cell progenitors, and/or NK cells.   
     
     
         4 . The method of  claim 1 , further comprising: contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, to seed and expand the cells. 
     
     
         5 . The method of  claim 1 , further comprising: subjecting the pluripotent stem cells, the mesodermal cells, and/or the mesodermal cells having definitive hemogenic endothelium potential under low oxygen tension between about 2% to about 10%. 
     
     
         6 . The method of  claim 1 , wherein the pluripotent stem cell differentiation to hematopoietic lineage cells is (i) free of, or essentially free of, TGFβ receptor/ALK inhibitors; (ii) under feeder-free condition; or (iii) under stromal-free condition. 
     
     
         7 . The method of  claim 1 , wherein the pluripotent stem cells comprise induced pluripotent stem cells (iPSCs). 
     
     
         8 . The method of  claim 7 , wherein the iPSCs are naïve iPSCs. 
     
     
         9 . The method of  claim 1 , wherein the WNT pathway activator is a GSK3 inhibitor; and/or wherein the BMP pathway activator is BMP4. 
     
     
         10 . The method of  claim 9 , wherein the GSK3 inhibitor is CHIR99021. 
     
     
         11 . The method of  claim 2 , wherein the ROCK inhibitor is Thiazovivin or Y-27632. 
     
     
         12 . A method for directing differentiation of pluripotent stem cells to cells of hematopoietic lineage, comprising:
 (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor, and a BMP activator, to obtain mesodermal cells;   (ii) contacting the mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally, a TGFβ receptor/ALK inhibitor, to obtain hemogenic endothelium; and   (iii) contacting the hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and a BMP activator, to obtain definitive HSCs, wherein the composition is optionally free of Wnt pathway activators and TGFβ receptor/ALK inhibitors.   
     
     
         13 . The method of  claim 12 , further comprising:
 (iv) contacting definitive HSCs with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; a BMP activator, and one or more Notch pathway activators, to obtain T cell progenitors, and optionally,   (v) contacting T cell progenitors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; and one or more Notch pathway activators, to obtain T cells;   or   (vi) contacting definitive HSCs with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and a BMP activator, to obtain NK cell progenitors; and optionally,   (vii) contacting NK cell progenitors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, to obtain NK cells, wherein the composition is free of BMP activators.   
     
     
         14 . The method of  claim 12 , wherein the definitive HSCs are CD34+CD45+. 
     
     
         15 . The method of  claim 14 , wherein the definitive HSCs are suitable for long-term engraftment. 
     
     
         16 . The method of  claim 13 , wherein the T cell progenitors are CD34+CD7+. 
     
     
         17 . The method of  claim 13 , wherein the NK cell progenitors are CD56+CD7+CD161+. 
     
     
         18 . The method of  claim 12 , wherein directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage is (i) void of generation of embryoid bodies; (ii) under monolayer culturing; (iii) under feeder-free condition; or (iv) under stromal-free condition. 
     
     
         19 . The method of  claim 12 , wherein the pluripotent stem cells are iPSCs. 
     
     
         20 . The method of  claim 19 , where in the iPSCs are naïve iPSCs. 
     
     
         21 . A composition comprising:
 (a) a culture medium, optionally, free of TGFβ receptor/ALK inhibitors; and   (b) one or more cell populations generated from the method of  claim 1 , wherein the one or more cell populations are selected from:
 (i) pluripotent stem cell-derived CD34+ definitive hemogenic endothelium (iCD34 HE), wherein the iCD34 cells have capacity to differentiate into multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells and NK cells, and wherein the iCD34 cells are CD34+CD43−; 
   (ii) pluripotent stem cell-derived definitive hemogenic endothelium (iHE), wherein the iHE cells are CD34+;   (iii) pluripotent stem cell-derived definitive HSCs (iHSCs), wherein the iHSCs are CD34+CD45+;   (iv) pluripotent stem cell-derived multipotent progenitor (iMPP) cells, wherein the iMPP cells are CD34+CD45+;   (v) pluripotent stem cell-derived T cell progenitors, wherein the T cell progenitors are CD34+CD7+;   (vi) pluripotent stem cell-derived T cells, wherein the T cells are CD4+or CD8+;   (vii) pluripotent stem cell-derived NK cell progenitors, wherein the NK cell progenitors are CD56+CD7+CD161+; and   (viii) pluripotent stem cell-derived NK cells, wherein the NK cells are CD56+CD57+CD16+CD94−.   
     
     
         22 . A composition comprising pluripotent stem cell-derived hematopoietic cells and one or more culture medium selected from the group consisting of:
 (i) CD34+ HE cells (iCD34), and one or more culture medium selected from iCD34-C, iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-Al, iNK-A2, iNK-B1 and iNK-B2;   (ii) definitive hemogenic endothelium (iHE), and one or more culture medium selected from iCD34-C, iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-Al, iNK-A2, iNK-B1 and iNK-B2;   (iii) definitive HSCs, and one or more culture medium selected from iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-Al, iNK-A2, iNK-B1 and iNK-B2;   (iv) multipotent progenitor cells (iMPP), and iMPP-A;   (v) T cell progenitors (iproT), and one or more culture medium selected from iTC-A1, iTC-A2, iTC-B1, and iTC-B2;   (vi) T cells (iTC), and iTC-B1 or iTC-B2;   (vii) NK cell progenitors (iproNK), and one or more culture medium selected from iNK-A1, iNK-A2, iNK-B1, and iNK-B2;   (viii) NK cells (iNK), and iNK-B1 or iNK-B2; and   (ix) HSC (iHSC), and iHSC-A, iHSC-B, and iHSC-C;   wherein iHSC-A comprises a Wnt pathway activator, and a BMP activator;   wherein iHSC-B comprises a Wnt pathway activator, a BMP activator, and optionally, a TGFβ receptor/ALK inhibitor;   wherein iHSC-C comprises a BMP activator, and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO;   wherein iTC-A1 comprises a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators selected from the group consisting of Jag1, Jag2, DLL-1, DLL-3 and DLL-4;   wherein iTC-B1 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators selected from the group consisting of Jag1, Jag2, DLL-1, DLL-3 and DLL-4;   wherein iNK-A1 comprises a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15;   wherein iNK-B1 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15;   wherein iCD34-C comprises a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11;   wherein iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11;   wherein iTC-A2 comprises a BMP activator, a ROCK inhibitor, VEGF, bFGF, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7;   wherein iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7;   wherein iNK-A2 comprises a BMP activator, a ROCK inhibitor, VEGF, bFGF, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL15; and   wherein iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.   
     
     
         23 . The composition of  claim 22 , wherein iHSC-C is free of Wnt pathway activators and TGFβ receptor/ALK inhibitors; wherein iTC-A1 is free of VEGF and/or IL15; wherein iCD34-C is free of TGFβ receptor/ALK inhibitor; wherein iTC-B1 is free of BMP activator; and/or wherein iTC-B2 is free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors.