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 . A method for generating and expanding human hemangio-colony forming cells in vitro, said method comprising the steps of:
 (a) culturing a cell culture comprising human pluripotent stem cells in serum-free media in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said pluripotent stem cells 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 at least two growth factors are in an amount sufficient to expand human hemangio-colony forming cells in said embryoid bodies culture,   wherein said pluripotent stem cells, embryoid bodies and hemangio-colony forming cells 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.   
     
     
         2 . A method for generating and expanding human hemangio-colony forming cells in vitro, said method comprising the steps of:
 (a) culturing a cell culture comprising human pluripotent stem cells in serum-free media in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said pluripotent stem cells 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 at least two growth factors are in an amount sufficient to expand human hemangio-colony forming cells in said embryoid bodies culture,   (c) said embryoid bodies are disaggregated into single cells;   (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 hemangio-colony forming cells in said culture comprising said single cells,   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,   and wherein said at least two growth factors in step (b) comprise BMP4 and VEGF.   
     
     
         3 . The method according to  claim 1 , wherein said pluripotent stem cell is an embryonic stem cell. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method according to  claim 1 , wherein said at least two growth factors are added to said culture in step (b) multiple times throughout step (b). 
     
     
         7 . The method according to  claim 2 , wherein said at least two growth factors are added to said cultures in steps (b), and said at least one growth factor is added in step (d) multiple times throughout steps (b) and (d). 
     
     
         8 - 11 . (canceled) 
     
     
         12 . The method according to  claim 1 , wherein said at least two growth factors are 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). 
     
     
         13 . The method according to  claim 1 , wherein vascular endothelial growth factor (VEGF) or bone morphogenic protein (BMP), or both, are added to step (a) within 0-48 hours of cell culture. 
     
     
         14 . The method according to  claim 6 , wherein stem cell factor (SCF), Flt-3L (FL) or thrombopoietin (TPO), or any combination thereof, are added to said culture within 48-72 hours from the start of step (a). 
     
     
         15 . The method according to  claim 1 , wherein said at least two growth factors are added to step (b) within 48-72 hours from the start of step (a). 
     
     
         16 . The method according to  claim 2 , wherein said at least two growth factors are added to step (b) within 48-72 hours from the start of step (a). 
     
     
         17 . The method according to  claim 1 , further comprising the step of adding erythropoietin (EPO) to step (b). 
     
     
         18 . The method according to  claim 2 , further comprising the step of adding erythropoietin (EPO) to steps (b) and (d). 
     
     
         19 . The method according to  claim 1 , further comprising the step of purifying said human hemangio-colony forming cells from said culture. 
     
     
         20 . The method according to  claim 19 , wherein said step of purifying said hemangio-colony forming cells comprises the step of using immunoaffinity column chromatography with an anti-CD71 antibody. 
     
     
         21 . The method according to  claim 1 , further comprising the step of isolating said human hemangio-colony forming cells from said culture. 
     
     
         22 .- 32 . (canceled) 
     
     
         33 . The method according to  claim 1 , wherein said cell culture comprising human pluripotent stem cells is derived from a library of human embryonic stem cells, wherein said library of human embryonic stem cells comprises stem cells, each of which is hemizygous or homozygous for at least one MHC allele present in a human population, wherein each member of said library of stem cells is hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library, wherein said method generates a library of hemangio-colony forming cells, each of which is hemizygous or homozygous for at least one MHC allele present in a human population, wherein each member of said library of stem cells is hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library. 
     
     
         34 . The method according to  claim 33 , wherein said library of human embryonic stem cells comprises stem cells that are hemizygous or homozygous for all MHC alleles present in a human population, wherein said method generates a library of hemangio-colony forming cells comprising hemangio-colony forming cells that are hemizygous or homozygous for all MHC alleles present in a human population. 
     
     
         35 . The method according to  claim 1 , further comprising the step of culturing said human hemangio-colony forming cells under conditions suitable to induce the differentiation of said human hemangio-colony forming cells into human hematopoietic stem cells. 
     
     
         36 . The method according to  claim 1 , further comprising the step of culturing said human hemangio-colony forming cells under conditions suitable to induce the differentiation of said human hemangio-colony forming cells into human endothelial cells. 
     
     
         37 - 46 . (canceled) 
     
     
         47 . A method for administering human hemangio-colony forming cells to a patient in need thereof, comprising the steps of:
 (a) selecting the patient in need thereof;   (b) supplying human hemangio-colony forming cells made by the method of  claim 1 ;   (c) administering some or all of said human hemangio-colony forming cells to said patient.   
     
     
         48 . The method according to  claim 47 , wherein said hemangio-colony forming cells are made by culturing without feeder cells. 
     
     
         49 . (canceled) 
     
     
         50 . A method for administering human hematopoietic stem cells to a patient in need thereof, comprising the steps of:
 (a) selecting the patient in need thereof;   (b) supplying human hemangio-colony forming cells made by the method of  claim 1 ;   (c) differentiating said human hemangio-colony forming cells into human hematopoietic stem cells; and   (d) administering some or all of said human hematopoietic stem cells to said patient.   
     
     
         51 . The method according to  claim 50 , wherein said hemangio-colony forming cells are made by culturing without feeder cells. 
     
     
         52 . (canceled) 
     
     
         53 . A method for administering human endothelial cells into a patient in need thereof, comprising the steps of:
 (a) selecting the patient in need thereof;   (b) supplying human hemangio-colony forming cells made by the method of  claim 1 ;   (c) differentiating said human hemangio-colony forming cells into human endothelial cells; and   (d) administering some or all of said human endothelial cells to said patient.   
     
     
         54 . The method according to  claim 53 , wherein said hemangio-colony forming cells are made by culturing without feeder cells. 
     
     
         55 . (canceled) 
     
     
         56 . A method of treating a hematopoietic stem cell disorder in a patient in need thereof, comprising the steps of:
 (a) selecting the patient in need thereof;   (b) supplying human hemangio-colony forming cells made by the method of  claim 1 ;   (c) differentiating said human hemangio-colony forming cells into human hematopoietic stem cells; and   (d) administering some or all of said human hematopoietic stem cells to said patient.   
     
     
         57 . The method according to  claim 56 , wherein said hemangio-colony forming cells are made by culturing without feeder cells. 
     
     
         58 . (canceled) 
     
     
         59 . A method of treating an endothelial cell disorder in a patient in need thereof, comprising the steps of:
 (a) selecting the patient in need thereof;   (b) supplying human hemangio-colony forming cells made by the method of  claim 1 ;   (c) differentiating said human hemangio-colony forming cells into human endothelial cells; and   (d) administering some or all of said human endothelial cells to said patient.   
     
     
         60 . The method according to  claim 59 , wherein said hemangio-colony forming cells are made by culturing without feeder cells. 
     
     
         61 . (canceled) 
     
     
         62 . The method according to  claim 56 , wherein said endothelial cell disorder is selected from the group consisting of myocardium infarction, stroke, atherosclerosis and ischemia. 
     
     
         63 . (canceled) 
     
     
         64 . A method for producing human hematopoietic stem cells in vitro, comprising the steps of:
 (a) supplying human hemangio-colony forming cells made by the method of  claim 1 ; and   (b) growing said human hemangio-colony forming cells under conditions suitable to induce the differentiation of said human hemangio-colony forming cells into human hematopoietic stem cells.   
     
     
         65 . A human hematopoietic stem cell produced by the method of  claim 64 . 
     
     
         66 . A method for producing human endothelial cells in vitro, comprising the steps of:
 (a) supplying human hemangio-colony forming cells made by the method of  claim 1 ; and   (b) growing said human hemangio-colony forming cells under conditions suitable to induce the differentiation of said human hemangio-colony forming cells into human endothelial cells.   
     
     
         67 . A human endothelial cell produced by the method of  claim 66 . 
     
     
         68 . A library of human hemangio-colony forming cells made by a method according to  claim 33 . 
     
     
         69 . A method to treat a human patient in need of treatment involving administering human hematopoietic stem cells or human endothelial cells to said patient, comprising the steps of:
 (a) selecting said patient;   (b) identifying MHC proteins expressed on the surface of said patient's cells;   (c) providing a library of human hemangio-colony forming cells according to  claim 68 ;   (d) selecting the human hemangio-colony forming cells that match said patient's MHC proteins on said patient's cells;   (e) differentiating said human hemangio-colony forming cells identified in step (d) into human hematopoietic stem cells, endothelial cells or both, depending on need;   (f) administering some or all of said human hematopoietic stem cells, endothelial cells or both from step (e) to said patient.   
     
     
         70 - 86 . (canceled) 
     
     
         87 . A method of inducing tolerance in a human recipient to a donor allograft comprising the steps of:
 (a) administering to the recipient an agent that inhibits T cell co-stimulation;   (b) introducing into the recipient human hemangio-colony forming cells generated by the method of  claim 1 , wherein said hemangio-colony forming cells are matched with respect to the donor, and   (c) implanting the allograft into the recipient, wherein said human hemangio-colony forming cells induce tolerance in the recipient to the allograft.   
     
     
         88 . The method according to  claim 87 , wherein the agent that inhibits T cell co-stimulation is an agent that inhibits the CD40 ligand-CD40 interaction. 
     
     
         89 . The method according to  claim 87 , wherein the method further comprises the step of administering an agent that inhibits the CD28-B7 interaction. 
     
     
         90 . The method according to  claim 87 , wherein the method does not comprise the step of thymic irradiation or T cell depletion or inactivation. 
     
     
         91 . The method according to  claim 87 , wherein the method further comprises the step of administering thymic irradiation to the recipient. 
     
     
         92 . The method according to  claim 87 , wherein the method further comprises the step of administering to the recipient a treatment that depletes or inactivates T cells. 
     
     
         93 . The method according to  claim 87 , wherein the method further comprises the steps of administering to the recipient thymic irradiation and a treatment that depletes or inactivates T cells. 
     
     
         94 . A method of inducing tolerance in a human recipient to a donor allograft comprising the steps of:
 (a) creating thymic space in the recipient;   (b) depleting or inactivating donor-reactive T cells in the recipient;   (c) introducing into the recipient human hemangio-colony forming cells generated by the method of  claim 1 , wherein said hemangio-colony forming cells are matched with respect to the donor, and   (d) implanting the allograft into the recipient,   wherein said human hemangio-colony forming cells induce tolerance in the recipient to the allograft.   
     
     
         95 . The method according to  claim 94 , wherein said thymic space is created by administering to the recipient at least one treatment selected from the group consisting of thymic irradiation, steroids, corticosteroids, brequinar, and an immune suppressant chemical or drug. 
     
     
         96 . The method according to  claim 94 , wherein said thymic space is created by administering thymic irradiation to the recipient. 
     
     
         97 . The method according to  claim 94 , wherein said thymic space is created by administering an immune suppressant chemical or drug to the recipient. 
     
     
         98 . The method according to  claim 94 , wherein said thymic space is created by administering cyclosporin to the recipient. 
     
     
         99 . The method according to  claim 87 , wherein multiple hemangio-colony forming cell administrations are provided to the recipient. 
     
     
         100 . The method according to  claim 87 , wherein said tolerance is induced in the absence of hematopoietic space created by whole body irradiation. 
     
     
         101 . The method according to  claim 87 , wherein said human hemangio-colony forming cells and said allograft are obtained from the same donor. 
     
     
         102 . The method according to  claim 87 , wherein said human hemangio-colony forming cells and said allograft are obtained from different donors. 
     
     
         103 - 176 . (canceled) 
     
     
         177 . The method of  claim 1 , wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC), and
 said iPSC is passaged with trypsin,   said iPSC is maintained with mouse embryonic fibroblasts (MEFs) or is maintained feeder-free, and/or   Y-27632 is added to serum-free embryoid body media and/or differentiation media.   
     
     
         178 . The method of  claim 64 , wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC), and
 said iPSC is passaged with trypsin,   said iPSC is maintained with mouse embryonic fibroblasts (MEFs) or is maintained feeder-free, and/or   Y-27632 is added to serum-free embryoid body media and/or differentiation media.   
     
     
         179 . The method of  claim 66 , wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC), and
 said iPSC is passaged with trypsin,   said iPSC is maintained with mouse embryonic fibroblasts (MEFs) or is maintained feeder-free, and/or   Y-27632 is added to serum-free embryoid body media and/or differentiation media.   
     
     
         180 - 183 . (canceled)