HEMANGIO COLONY FORMING CELLS AND NON-ENGRAFTING HEMANGIO CELLS

• US 20110064705A1
• Filed: 05/06/2009
• Published: 03/17/2011
• Est. Priority Date: 05/06/2008
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: Lu, Shi-Jiang, Lanza, Robert
• Granted Patent: US 9,410,123 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 38/13 Cyclosporins
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61N 5/10 X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy A61N5/01 takes precedence
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 : from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors

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.

Claims

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 growth factor is 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.

3. The method according to claim 1, wherein said pluripotent stem cell is an embryonic stem cell.

4. The method according to claim 1, wherein said growth factor is a protein that comprises a homeobox protein or a functional variant or an active variant thereof.

5. The method according to claim 4, wherein said homeobox protein comprises a HOXB4 protein, or a functional variant or an active variant thereof.

6. The method according to claim 4, wherein said growth factor is added to said culture in step (b) multiple times throughout step (b).

7. The method according to claim 4, wherein said growth factor is added to said cultures in steps (b) and (d) multiple times throughout steps (b) and (d).

12. The method according to claim 1, wherein said growth factor is 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 6, wherein said 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 said 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 4, wherein said growth factor is added to step (b) within 48-72 hours from the start of step (a).

16. The method according to claim 5, wherein said growth factor is added to culturing 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).

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.

23. The method according to claim 4, wherein said growth factor is a fusion protein that comprises HOXB4 and a protein transduction domain (PTD).

24. The method according to claim 23, wherein said PTD and said HOXB4 are conjugated via a linker.

25. The method according to claim 23, wherein said protein transduction domain (PTD) is TAT protein, a functional variant or an active fragment thereof.

27. The method according to claim 5, wherein said HOXB4 is mouse or human HOXB4.

28. The method according to claim 27, wherein said HOXB4 is human HOXB4.

29. The method according to claim 5, wherein said HOXB4 comprises a full-length protein.

30. The method according to claim 29, wherein said HOXB4 comprises the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

3.

31. The method according to claim 23, wherein said PTD comprises one or more copies of a TAT polypeptide with the amino acid sequence of SEQ ID NO:

14.

32. The method according to claim 25, wherein said TAT protein comprises the amino acid sequence of SEQ ID NO:

14.

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 fanning 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.

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.

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.

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.

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.

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.

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.

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.

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.

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-tree 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.

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 said growth factor is 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.

18. The method according to claim 2, further comprising the step of adding erythropoietin (EPO) to steps (b) and (d).

8-11. -11. (canceled)

22. (canceled)

26. (canceled)

37-46. -46. (canceled)

49. (canceled)

52. (canceled)

55. (canceled)

58. (canceled)

61. (canceled)

63. (canceled)

70-71. -71. (canceled)

72. A method for expanding hemangio-colony forming cells comprising growing mammalian hemangio-colony forming cells in serum-free media in the presence of a protein that comprises BMP4 and VEGF or functional equivalents or active fragments thereof in an amount sufficient to support the proliferation of said hemangio-colony forming cells.

73. The method according to claim 72, wherein said hemangio-colony forming cells are enriched, purified, or isolated from cord blood, peripheral blood, or bone marrow.

74. The method according to claim 72, wherein said hemangio-colony forming cells are human hemangio-colony forming cells.

75. The method according to claims 72, wherein said hemangio-colony forming cells are grown in vitro to achieve at least 1×

10⁶ hemangio-colony forming cells.

76. The method according to claim 72, wherein said serum-free media further comprises a homeobox protein, a functional equivalent or an active fragment thereof.

77. The method according to claim 76, wherein the homeobox protein, functional equivalent or active fragment thereof is a HOXB4 protein or a functional equivalent or an active fragment thereof.

79. The method according to claim 77, wherein said HOXB4 is mouse or human HOXB4.

80. The method according to claim 79, wherein said HOXB4 is human HOXB4.

81. The method according to claim 77, wherein said HOXB4 comprises a full-length protein.

82. The method according to claim 77, wherein said HOXB4 comprises the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

3.

83. The method according to claim 72, wherein said serum-free media further comprises a fusion protein comprising HOXB4 and a protein transduction domain (PTD).

84. The method according to claim 83, wherein said protein transduction domain (PTD) is TAT protein, a functional variant or an active fragment thereof.

85. The method according to claim 84, wherein said TAT protein comprises the amino acid sequence of SEQ ID NO:

14.

86. The method according to claim 83, wherein said PTD comprises one or more copies of a TAT polypeptide with the amino acid sequence of SEQ ID NO:

14.

78. (canceled)

103. A human non-engrafting hemangio cell, which cell can differentiate to produce at least one or more hematopoietic cell types, wherein the hemangio cell does not have the capacity of engrafting and repopulating the bone marrow in a host.

104. The non-engrafting hemangio cell according to claim 103, wherein the hemangio cell expresses some but not all of the proteins which characterize human hemangio-colony forming cells that have the capacity of engrafting and repopulating the bone marrow in a host.

105. The non-engrafting hemangio cell according to claim 103, wherein the hemangio cell has one or more characteristics of human hemangio-colony forming cells that have the capacity of engrafting and repopulating the bone marrow in a host.

106. The non-engrafting hemangio cell according to claim 103, wherein the hemangio cell is loosely adherent to other hemangio cells.

107. The non-engrafting hemangio cell according to claim 104, wherein the human hemangio cell does not express one or more proteins selected from CD34, CD31, KDR and CD133.

108. The non-engrafting hemangio cell according to claim 104, wherein the human hemangio cell expresses LMO2 and GATA2 proteins.

109. The non-engrafting hemangio cell according to claim 103, wherein the host is a mammal.

110. The non-engrafting hemangio cell according to claim 109, wherein the mammal is a human.

118. A cell culture comprising a hematopoietic cell differentiated from the non-engrafting hemangio cells according to claim 103.

111. A cell culture comprising a substantially purified population of human non-engrafting hemangio cells, which cells can differentiate to produce at least one or more hematopoietic cell types, wherein the hemangio cells do not have the capacity of engrafting and repopulating the bone marrow in a host.

113. The cell culture according to claim 111, wherein the non-engrafting hemangio cells express some but not all of the proteins which characterize human hemangio-colony forming cells that have the capacity of engrafting and repopulating the bone marrow in a host.

114. The cell culture according to claim 111, wherein the non-engrafting hemangio cells has one or more of the characteristics of human hemangio-colony forming cells that have the capacity of engrafting and repopulating the bone marrow in a host.

115. The cell culture according to claim 111, wherein the hemangio cell is loosely adherent to other hemangio cells.

116. The cell culture according to claim 113, wherein the human hemangio cell does not express one or more proteins selected from CD34, CD31, KDR and CD133.

117. The cell culture according to claim 113, wherein the human hemangio cell expresses LMO2 and GATA2 proteins.

112. A cell culture comprising human non-engrafting hemangio cells differentiated from embryo-derived cells, wherein the non-engrafting hemangio cells can differentiate to produce at least one or more hematopoietic cell types, and wherein the hemangio cells do not have the capacity of engrafting and repopulating the bone marrow in a host.

119-137. -137. (canceled)

138. A method for generating and expanding human non-engrafting hemangio cell in vitro, said method comprising the steps of:

(a) culturing a cell culture comprising human embryo-derived cells in serum-free media in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said embryo-derived cells 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 human non-engrafting hemangio cells in said embryoid bodies culture,

wherein said embryoid bodies are cultured for 10-13 days, and

wherein said embryo-derived cells, embryoid bodies and non-engrafting hemangio cells are grown in serum-free media throughout steps (a) and (b) of said method.

140. The method according to claim 138, wherein said embryo-derived cell is an embryonic stem cell.

141. The method according to claim 138, wherein said growth factor is a protein that comprises a homeobox protein or a functional variant or an active variant thereof.

142. The method according to claim 141, wherein said homeobox protein comprises a HOXB4 protein, or a functional variant or an active variant thereof.

143. The method according to claim 138, wherein said growth factor is 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).

144. The method according to claim 143, wherein said vascular endothelial growth factor (VEGF) or bone morphogenic protein (BMP), or both, are added to step (a) within 0-48 hours of cell culture.

145. The method according to claim 143, wherein said 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).

146. The method according to claim 141, wherein said growth factor is added to step (b) within 48-72 hours from the start of step (a).

147. The method according to claim 138, further comprising the step of adding erythropoietin (EPO) to step (b).

149. The method according to claim 138, further comprising the step of purifying the human non-engrafting hemangio cells from said culture.

150. The method according to claim 138, further comprising the step of isolating the human non-engrafting hemangio cells from said culture.

151. The method according to claim 141, wherein said growth factor is a fusion protein that comprises HOXB4 and a protein transduction domain (PTD).

152. The method according to claim 151, wherein said PTD and said HOXB4 are conjugated via a linker.

153. The method according to claim 151, wherein said protein transduction domain (PTD) is TAT protein, a functional variant or an active fragment thereof.

155. The method according to claim 153, wherein said mammalian HOXB4 is mouse or human HOXB4.

156. The method according to claim 142, wherein said HOXB4 comprises a full-length protein.

157. The method according to claim 138, further comprising the step of culturing said non-engrafting hemangio cells under conditions suitable to induce the differentiation of said non-engrafting hemangio cells into human hematopoietic stem cells.

160. A method for producing human hematopoietic stem cells in vitro, comprising the steps of:

(a) supplying human non-engrafting hemangio cells made by the method of claim 138; and

(b) growing said human non-engrafting hemangio cells under conditions suitable to induce the differentiation of said human non-engrafting hemangio cells into human hematopoietic stem cells.

162. A method for administering human non-engrafting hemangio cells to a patient in need thereof, comprising the steps of:

(a) selecting the patient in need thereof;

(b) supplying human non-engrafting hemangio cells made by the method of claim 138;

(c) administering some or all of said human non-engrafting hemangio cells to said patient.

165. 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 non-engrafting hemangio cells made by the method of claim 138;

(c) differentiating said human non-engrafting hemangio cells into human hematopoietic stem cells; and

(d) administering some or all of said human hematopoietic stem cells to said patient.

168. 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 non-engrafting hemangio cells made by the method of claim 138;

(c) differentiating said human non-engrafting hemangio cells into human hematopoietic stem cells; and

(d) administering some or all of said human hematopoietic stem cells to said patient.

174. A method for producing differentiated hematopoietic cells from non-engrafting hemangio cells in vitro, said method comprising the steps of:

(a) providing human non-engrafting hemangio cells made by the method of claim 138; and

(b) differentiating said non-engrafting hemangio cells into differentiated hematopoietic cells.

176. A method for performing blood transfusions using hematopoietic cells differentiated in vitro from human non-engrafting hemangio cells, said method comprising the steps of:

(a) providing human non-engrafting hemangio cells made by the method of claim 138;

(b) differentiating said non-engrafting hemangio cells into differentiated hematopoietic cells; and

(c) performing blood transfusions with said differentiated hematopoietic cells.

180. The method of claim 138, 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.

181. The method of claim 160, 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.

183. The method of claim 174, 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.

139. A method for generating and expanding human non-engrafting hemangio cell in vitro, said method comprising the steps of:

(a) culturing a cell culture comprising human embryo-derived cells in serum-free media in the presence of at least one growth factor in an amount sufficient to induce the differentiation of said embryo-derived cells 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 human non-engrafting hemangio 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 non-engrafting hemangio cells in said culture comprising said single cells,

wherein said culture of single cells are cultured for 10-13 days, and

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.

148. The method according to claim 139, further comprising the step of adding erythropoietin (EPO) to steps (b) and (d).

154. (canceled)

158-159. -159. (canceled)

161. (canceled)

163-164. -164. (canceled)

166-167. -167. (canceled)

169-170. -170. (canceled)

171. A method for producing differentiated hematopoietic cells from non-engrafting hemangio cells in vitro, said method comprising the steps of:

(a) providing human non-engrafting hemangio cells; and

(b) differentiating said non-engrafting hemangio cells into differentiated hematopoietic cells.

182. The method of claim 171, 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.

172. (canceled)

173. A method for performing blood transfusions using hematopoietic cells differentiated in vitro from human non-engrafting hemangio cells, said method comprising the steps of:

(a) providing human non-engrafting hemangio cells;

(b) differentiating said non-engrafting hemangio cells into differentiated hematopoietic cells; and

(c) performing blood transfusions with said differentiated hematopoietic cells.

175. (canceled)