Hemangio colony forming cells and non-engrafting hemangio cells

Abstract

Methods of generating and expanding human hemangio-colony forming cells and non-engrafting hemangio cells in vitro and methods of expanding and using such cells are disclosed. The methods permit the production of large numbers of hemangio-colony forming cells, non-engrafting hemangio cells as well as derivative cells, such as hematopoietic and endothelial cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications. Human non-engrafting hemangio cells are a novel progenitor cell population that is related to but distinct from the hemangioblast and human hemangio-colony forming cells. The invention also provides compositions, preparations, and solutions comprising hemangio-colony forming cells, non-engrafting hemangio cells or cells differentiated therefrom. The compositions, preparations, and solutions include cryopreserved preparations and substantially purified preparations, as well as mixed compositions formulated in combination with related hemangioblast progenitor cell types that can engraft into the bone marrow.

1 - 183 . (canceled) 
     
     
         184 . A method for generating and expanding human pluripotent stem cell-derived human hemangio-colony forming cells in vitro, the method comprising the steps of:
 (a) culturing human pluripotent stem cells in a first serum-free media comprising bone morphogenic protein 4 (BMP-4) or vascular endothelial growth factor (VEGF), or both, and inducing the differentiation of the pluripotent stem cells into embryoid bodies; and   (b) culturing the embryoid bodies in a second serum-free media comprising at least thrombopoietin (TPO) and Flt-3L (FL), and generating and expanding human hemangio-colony forming cells from the embryoid bodies,   wherein the pluripotent stem cells, the embryoid bodies and the hemangio-colony forming cells are grown in serum-free media throughout steps (a) and (b) of the method.   
     
     
         185 . A method for generating and expanding human pluripotent stem cell-derived human hemangio-colony forming cells in vitro, the method comprising the steps of:
 (a) culturing human pluripotent stem cells in a first serum-free media comprising bone morphogenic protein 4 (BMP-4) or vascular endothelial growth factor (VEGF), or both, and inducing the differentiation of the pluripotent stem cells into embryoid bodies;   (b) disaggregating the embryoid bodies; and   (c) culturing the disaggregated embryoid bodies in a second serum-free media comprising at least thrombopoietin (TPO) and Flt-3L (FL), and generating and expanding human hemangio-colony forming cells from the disaggregated embryoid bodies;   wherein the pluripotent stem cells, the embryoid bodies and the hemangio-colony forming cells are grown in serum-free media throughout steps (a)-(c) of the method.   
     
     
         186 . The method of  claim 184 , wherein the pluripotent stem cells are embryonic stem cells. 
     
     
         187 . The method of  claim 184 , wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs). 
     
     
         188 . The method of  claim 184 , wherein the human pluripotent stem cells are cultured in the first serum-free media comprising vascular endothelial growth factor (VEGF) or bone morphogenic protein 4 (BMP-4), or both, for at least the first 48 hours of inducing the differentiation of the pluripotent stem cells into embryoid bodies. 
     
     
         189 . The method of  claim 184 , wherein the first serum-free media further comprises at least one growth factor selected from the group consisting of stem cell factor (SCF), Flt-3L (FL), thrombopoietin (TPO), basic fibroblast growth factor (bFGF), homeobox protein and erythropoietin (EPO). 
     
     
         190 . The method according to  claim 189 , wherein the stem cell factor (SCF), the Flt-3L (FL) or the thrombopoietin (TPO), or any combination thereof, are added to step (a) within 48-72 hours of cell culture. 
     
     
         191 . The method of  claim 184 , wherein the second serum-free media further comprises one or more additional 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 (GCSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMPs), basic fibroblast growth factor (bFGF), HOX protein, and combinations thereof. 
     
     
         192 . The method of  claim 184 , wherein the second serum-free media further comprises erythropoietin (EPO). 
     
     
         193 . The method of  claim 184 , wherein the first serum-free media, second serum-free media, or both further comprise a rho-kinase (ROCK) inhibitor. 
     
     
         194 . The method of  claim 184 , wherein the pluripotent stem cells in step (a) and the embryoid bodies in step (b) are cultured under low attachment conditions. 
     
     
         195 . The method of  claim 184 , wherein the embryoid bodies in step (b) are cultured in a semi-solid culture media. 
     
     
         196 . The method of  claim 184 , wherein the second serum-free media further comprises methylcellulose. 
     
     
         197 . The method of  claim 184 , wherein the pluripotent stem cells, the embryoid bodies and the hemangio colony forming cells are cultured in feeder-free media throughout steps (a) and (b) of the method. 
     
     
         198 . The method of  claim 184 , wherein the first serum-free media does not comprise one or more of stem cell factor (SCF), Flt-3L (FL), or thrombopoietin (TPO). 
     
     
         199 . The method of  claim 184 , further comprising culturing the human hemangio-colony forming cells under conditions suitable to induce the differentiation of the human hemangio-colony forming cells into human hematopoietic stem cells. 
     
     
         200 . The method of  claim 184 , further comprising culturing the human hemangio-colony forming cells under conditions suitable to induce the differentiation of the human hemangio-colony forming cells into human endothelial cells. 
     
     
         201 . A human hemangio-colony forming cell prepared by the method of  claim 184 . 
     
     
         202 . A method of treating a hematopoietic stem cell disorder in a patient in need thereof, the method comprising the steps of:
 (a) providing human hemangio-colony forming cells generated by the method of  claim 184 ;   (b) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; and   (c) administering the human hematopoietic stem cells to the patient, thereby treating the hematopoietic stem cell disorder in the patient.   
     
     
         203 . A method of treating an endothelial cell disorder in a patient in need thereof, the method comprising the steps of:
 (a) providing human hemangio-colony forming cells generated by the method of  claim 184 ;   (b) differentiating the human hemangio-colony forming cells into human endothelial cells; and   (c) administering the human endothelial cells to the patient, thereby treating the endothelial cell disorder in the patient.