Hemangio-colony forming cells

Abstract

Methods of generating and expanding human hemangio-colony forming 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 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.

1 - 232 . (canceled) 
     
     
         233 . A method, comprising:
 (a) culturing human embryonic stem cells in serum free media in the presence of vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP-4) in an amount sufficient to induce the differentiation of said embryonic stem cells into embryoid bodies; and   (b) adding at least one first growth factor to said culture comprising the embryoid bodies to generate CD34 −  CD31 −  human hemangio-colony forming cells,   wherein the embryonic stem cells, embryoid bodies, and CD34 −  CD31 −  hemangio-colony forming cells are continuously grown in serum-free media to generate CD34 −  CD31 −  human hemangio-colony forming cells.   
     
     
         234 . The method of  claim 233 , wherein the VEGF and BMP-4 are added to step (a) within 0-48 hours of cell culture, and optionally, multiple times throughout step (a). 
     
     
         235 . The method of  claim 233 , wherein the at least one first growth factor is selected from the group consisting of: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), stem cell factor (SCF), thrombopoietin (TPO), Flt-3L (FL), HOX protein, erythropoietin (EPO) and combinations thereof. 
     
     
         236 . The method of  claim 233 , wherein the at least one first growth factor is added to step (b) within 48-72 hours of cell culture, and optionally, added multiple times throughout step (b). 
     
     
         237 . The method of  claim 235 , wherein the HOX protein is HOXB4, a functional equivalent or an active fragment thereof, and the functional equivalent or an active fragment thereof possess the same or substantially similar properties as wild-type HOXB4. 
     
     
         238 . The method of  claim 233 , further comprising:
 (c) disaggregating the embryoid bodies into single cells; and   (d) adding at least one second growth factor to said culture in an amount sufficient to expand CD34 −  CD31 −  human hemangio-colony forming cells,   wherein the embryonic stem cells, embryoid bodies, and CD34 −  CD31 −  hemangio-colony forming cells are continuously grown in serum-free media to expand CD34 −  CD31 −  human hemangio-colony forming cells.   
     
     
         239 . The method of  claim 238 , wherein the at least one second growth factor is 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), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), HOX protein, thrombopoietin (TPO), Flt-3L (FL), and combinations thereof. 
     
     
         240 . The method of  claim 238 , wherein the at least one second growth factor is added to step (d) after 72 hours of cell culture and optionally, added multiple times throughout step (d). 
     
     
         241 . The method of  claim 239 , wherein the HOX protein is HOXB4, a functional equivalent or an active fragment thereof, and the functional equivalent or an active fragment thereof possess the same or substantially similar properties as wild-type HOXB4. 
     
     
         242 . The method of  claim 241 , wherein the functional equivalent or an active fragment thereof, comprises a fusion protein with a protein transduction domain (PTD). 
     
     
         243 . The method of  claim 242 , wherein the fusion protein comprises one or more copies of SEQ ID NO:14. 
     
     
         244 . The method of  claim 238 , further comprising purifying the CD34 −  CD31 −  human hemangio-colony forming cells. 
     
     
         245 . The method of  claim 244 , further comprising the use of an anti-CD71 antibody to purify the CD34 −  CD31 −  human hemangio-colony forming cells. 
     
     
         246 . The method of  claim 238 , further comprising isolating the CD34 −  CD31 −  human hemangio-colony forming cells. 
     
     
         247 . The method of  claim 233 , wherein the embryonic stem cells are from a library of embryonic stem cells that are hemizygous or homozygous for at least one majorhistocompatibility (MHC) allele present in a human population, and each member of the library is hemizygous or homozygous for a unique set of MHC alleles compared to other members of the library of embryonic stem cells, thereby generating a library of CD34 −  CD31 −  human hemangio-colony forming cells that are hemizygous or homozygous for at least one majorhistocompatibility (MHC) allele present in a human population, and each member of the library of CD34 −  CD31 −  human hemangio-colony forming cells is hemizygous or homozygous for a unique set of MHC alleles compared to other members of the library of human hemangio-colony forming cells. 
     
     
         248 . The method of  claim 247 , wherein the embryonic stem cells are hemizygous or homozygous all majorhistocompatibility (MHC) alleles present in a human population, thereby generating a library of CD34 −  CD31 −  human hemangio-colony forming cells that are hemizygous or homozygous all majorhistocompatibility (MHC) alleles present in a human population. 
     
     
         249 . The method of  claim 238 , further comprising differentiating CD34 −  CD31 −  human hemangio-colony forming cells into human hematopoietic cells. 
     
     
         250 . The method of  claim 238 , further comprising differentiating CD34 −  CD31 −  human hemangio-colony forming cells into human endothelial cells. 
     
     
         251 . A method of administering to a patient in need thereof, the human hematopoietic cells of  claim 249 , comprising:
 selecting the patient;   providing a quantity of hematopoietic cells; and   administering the quantity of hematopoietic cells to the patient.   
     
     
         252 . The method of  claim 251 , wherein the patient is in need of a blood transfusion. 
     
     
         253 . A method of administering to a patient in need thereof, the human endothelial cells of  claim 250 , comprising:
 selecting the patient;   providing a quantity of endothelial cells; and   administering the quantity of endothelial cells to the patient.   
     
     
         254 . A method of expanding CD34 −  CD31 −  human hemangio-colony forming cells, comprising:
 providing a quantity of CD34 −  CD31 −  human hemangio-colony forming cells; and   adding at least one growth factor in an amount sufficient to expand CD34 −  CD31 −  human hemangio-colony forming cells, wherein the CD34 −  CD31 −  hemangio-colony forming cells are continuously grown in serum-free media.   
     
     
         255 . The method of  claim 254 , wherein the at least one growth factor is 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), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), HOX protein, thrombopoietin (TPO), Flt-3L (FL), and combinations thereof. 
     
     
         256 . The method of  claim 254 , further comprising differentiating CD34 −  CD31 −  human hemangio-colony forming cells into human hematopoietic cells cells. 
     
     
         257 . The method of  claim 254 , further comprising differentiating CD34 −  CD31 −  human hemangio-colony forming cells into human endothelial cells. 
     
     
         258 . A method of administering to a patient in need thereof, the human hematopoietic cells of  claim 256 , comprising:
 selecting the patient;   providing a quantity of hematopoietic cells; and   administering the quantity of hematopoietic cells to the patient.   
     
     
         259 . The method of  claim 258 , wherein the patient is in need of a blood transfusion. 
     
     
         260 . A method of administering to a patient in need thereof, the human endothelial cells of  claim 257 , comprising:
 selecting the patient;   providing a quantity of endothelial cells; and   administering the quantity of endothelial cells to the patient.   
     
     
         261 . A method of inducing tolerance in a human subject receiving a donor allograft, comprising:
 (a) creating thymic space in the human subject;   (b) depleting or inactivating donor-reactive T cells in the human subject;   (c) introducing into the human subject, the human hemangio-colony forming cells generated by the method of claim  1 , wherein said hemangio-colony forming cells are match with respect to the donor, and   (d) implanting the allograft into the human subject, wherein said human hemangio-colony forming cells induce tolerance in the human subject to the allograft.