Expansion of renewable stem cell populations using modulators of PI 3-kinase

• US 20050054103A1
• Filed: 03/04/2004
• Published: 03/10/2005
• Est. Priority Date: 03/07/2003
• Status: Abandoned Application

• Current Assignee: Gamida Cell, Ltd.
• Original Assignee: Gamida Cell, Ltd.
• Inventors: Peled, Tony, Grynspan, Frida
• Application Number:
  US10/795,215
• Publication Number:
  US 20050054103A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/515 Animal cells
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: G01N 2333/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Abstract

Provided are ex vivo and in vivo methods of expanding renewable stem cells using modulators of PI 3-kinase activity, expanded populations of renewable stem cells, and uses thereof.

Claims

1. A method of ex vivo expanding and inhibiting differentiation of a population of stem cells, the method comprising:

(a) providing the cells ex vivo with conditions for cell proliferation;

(b) ex vivo providing the cells with an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase;

thereby ex vivo expanding and inhibiting differentiation of the population of stem cells.

2. The method of claim 1, wherein said stem cells are early hematopoietic and/or hematopoietic progenitor cells.

3. The method of claim 1, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

4. The method of claim 3, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

5. The method of claim 1, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

6. The method of claim 5, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

7. The method of claim 3, wherein said providing is effected by transiently expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme within a stem cell.

8. The method of claim 3, wherein said providing is effected by

(a) providing an expressible polynucleotide capable of expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme, and

(b) stably integrating said expressible polynucleotide into a genome of a cell,

thereby providing a modulator capable of downregulating a PI 3-kinase activity or PI 3-kinase gene expression.

9. The method of claim 3, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

10. The method of claim 1, wherein said providing said conditions for cell proliferation is effected by providing the cells with nutrients and cytokines.

11. The method of claim 10, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

12. The method of claim 11, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

13. The method of claim 11, wherein said early acting cytokine is FLT3 ligand.

14. The method of claim 11, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

15. The method of claim 11, wherein said late acting cytokine is granulocyte colony stimulating factor.

16. The method of claim 1, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

17. The method of claim 16, wherein said stem cells are derived from bone marrow or peripheral blood.

18. The method of claim 16, wherein said stem cells are derived from neonatal umbilical cord blood.

19. The method of claim 1, further comprising the step of selecting a population of stem cells enriched for hematopoietic stem cells.

20. The method of claim 19, wherein said selection is affected via CD34.

21. The method of claim 1, further comprising the step of selecting a population of stem cells enriched for early hematopoietic stem/progenitor cells.

22. The method of claim 21, wherein said selection is affected via CD133.

46. A therapeutic ex vivo cultured stem cell population comprising undifferentiated hematopoietic cells expanded according to the methods of any of claims 1-45.

47. The cell population of claim 46, in a culture medium comprising a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase.

48. The cell population of claim 47, isolated from said medium.

49. A pharmaceutical composition comprising the cell population of claim 46 and a pharmaceutically acceptable carrier.

50. A pharmaceutical composition comprising the cell population of claim 48 and a pharmaceutically acceptable carrier.

23. A method of transducing expanded, undifferentiated stem cells with an exogene, the method comprising:

(a) obtaining a population of stem cells;

(b) expanding and inhibiting differentiation of said stem cells by;

(i) providing said stem cells with conditions for cell proliferation;

(ii) providing said stem cells with an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase;

wherein steps (i) and (ii) are effected in vitro or ex vivo, thereby expanding and inhibiting differentiation of said stem cells; and

(c) transducing said expanded, undifferentiated stem cells with the exogene.

24. The method of claim 23, wherein said transducing is effected by a vector including the exogene.

25. The method of claim 23, wherein said stem cells are early hematopoietic and/or hematopoietic progenitor cells.

26. The method of claim 23, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

27. The method of claim 26, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

28. The method of claim 23, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

29. The method of claim 28, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

30. The method of claim 26, wherein said providing is effected by transiently expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme within a stem cell.

31. The method of claim 26, wherein said providing is effected by

(a) providing an expressible polynucleotide capable of expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme, and

(b) stably integrating said expressible polynucleotide into a genome of a cell,

thereby providing a modulator capable of downregulating a PI 3-kinase activity or PI 3-kinase gene expression.

32. The method of claim 26, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

33. The method of claim 23, wherein said providing said conditions for cell proliferation is effected by providing the cells with nutrients and cytokines.

34. The method of claim 33, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

35. The method of claim 34, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

36. The method of claim 34, wherein said early acting cytokine is FLT3 ligand.

37. The method of claim 34, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

38. The method of claim 34, wherein said late acting cytokine is granulocyte colony stimulating factor.

39. The method of claim 23, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

40. The method of claim 39, wherein said stem cells are derived from bone marrow or peripheral blood.

41. The method of claim 39, wherein said stem cells are derived from neonatal umbilical cord blood.

42. The method of claim 23, further comprising the step of selecting a population of stem cells enriched for hematopoietic stem cells.

43. The method of claim 42, wherein said selection is affected via CD34.

44. The method of claim 23, further comprising the step of selecting a population of stem cells enriched for early hematopoietic stem/progenitor cells.

45. The method of claim 44, wherein said selection is affected via CD133.

51. A method of hematopoietic stem cells transplantation into a recipient, the method comprising:

(a) obtaining a population of hematopoietic stem cells;

(b) ex vivo expanding and inhibiting differentiation of said hematopoietic stem cells by;

(i) ex vivo providing said hematopoietic stem cells with conditions for cell proliferation;

(ii) providing said hematopoietic stem cells ex vivo with an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase;

thereby expanding and inhibiting differentiation of said stem hematopoietic cells; and

(c) transplanting said hematopoietic stem cells into the recipient.

52. The method of claim 51, wherein said hematopoietic stem cells are early hematopoietic and/or hematopoietic progenitor cells.

53. The method of claim 51, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

54. The method of claim 53, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

55. The method of claim 51, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

56. The method of claim 55, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

57. The method of claim 53, wherein said providing is effected by transiently expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme within a stem cell.

58. The method of claim 53, wherein said providing is effected by

(a) providing an expressible polynucleotide capable of expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme, and

(b) stably integrating said expressible polynucleotide into a genome of a cell,

thereby providing a modulator capable of downregulating a PI 3-kinase activity or PI 3-kinase gene expression.

59. The method of claim 53, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

60. The method of claim 51, wherein said providing said conditions for cell proliferation is effected by providing the cells with nutrients and cytokines.

61. The method of claim 60, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

62. The method of claim 61, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

63. The method of claim 61, wherein said early acting cytokine is FLT3 ligand.

64. The method of claim 61, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

65. The method of claim 61, wherein said late acting cytokine is granulocyte colony stimulating factor.

66. The method of claim 51, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

67. The method of claim 66, wherein said stem cells are derived from bone marrow or peripheral blood.

68. The method of claim 66, wherein said stem cells are derived from neonatal umbilical cord blood.

69. The method of claim 51, further comprising the step of selecting a population of stem cells enriched for hematopoietic stem cells.

70. The method of claim 69, wherein said selection is affected via CD34.

71. The method of claim 51, further comprising the step of selecting a population of stem cells enriched for early hematopoietic stem/progenitor cells.

72. The method of claim 71, wherein said selection is affected via CD133.

73. A method of adoptive immunotherapy comprising:

(a) obtaining progenitor hematopoietic cells from a patient;

(b) ex vivo expanding and inhibiting differentiation of said hematopoietic cells by;

(i) providing said progenitor hematopoietic cells ex vivo with conditions for cell proliferation;

(ii) providing said progenitor hematopoietic cells with an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase;

thereby expanding and inhibiting differentiation of said progenitor hematopoietic cells; and

(c) transplanting said progenitor hematopoietic cells into a recipient.

74. The method of claim 73, wherein said hematopoietic stem cells are early hematopoietic and/or hematopoietic progenitor cells.

75. The method of claim 73, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

76. The method of claim 75, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

77. The method of claim 73, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

78. The method of claim 77, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

79. The method of claim 75, wherein said providing is effected by transiently expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme within a stem cell.

80. The method of claim 75, wherein said providing is effected by

(a) providing an expressible polynucleotide capable of expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme, and

(b) stably integrating said expressible polynucleotide into a genome of a cell,

thereby providing a modulator capable of downregulating a PI 3-kinase activity or PI 3-kinase gene expression.

81. The method of claim 75, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

82. The method of claim 73, wherein said providing said conditions for cell proliferation is effected by providing the cells with nutrients and cytokines.

83. The method of claim 82, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

84. The method of claim 83, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

85. The method of claim 83, wherein said early acting cytokine is FLT3 ligand.

86. The method of claim 83, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

87. The method of claim 83, wherein said late acting cytokine is granulocyte colony stimulating factor.

88. The method of claim 73, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

89. The method of claim 88, wherein said stem cells are derived from bone marrow or peripheral blood.

90. The method of claim 88, wherein said stem cells are derived from neonatal umbilical cord blood.

91. The method of claim 73, further comprising the step of selecting a population of stem cells enriched for hematopoietic stem cells.

92. The method of claim 91, wherein said selection is affected via CD34.

93. The method of claim 73, further comprising the step of selecting a population of stem cells enriched for early hematopoietic stem/progenitor cells.

94. The method of claim 93, wherein said selection is affected via CD133.

95. A method of mobilization of bone marrow stem cells into the peripheral blood of a donor for harvesting the cells comprising:

(a) administering to the donor an effective concentration of a a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase, thereby expanding and inhibiting differentiation of a population of bone marrow stem cells; and

(b) harvesting the cells by leukopheresis.

96. A method of inhibiting maturation/differentiation of erythroid precursor cells for treatment of a β

-hemoglobinopathic patient comprising administering to said patient an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase, thereby expanding and inhibiting differentiation of a population of stem cells of said patient such that upon removal of said modulator of PI 3-kinse from said patient, said stem cells undergo accelerated maturation resulting in elevated fetal hemoglobin production,

thereby ameliorating symptoms of β

-hemoglobinopathy in said patient.

98. The method of claim 96, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

99. The method of claim 96, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

100. The method of claim 96, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

101. The method of claim 100, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

102. The method of claim 96, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

97. The method of 96, further comprising the step of administering to said patient a cytokine.

103. The method of claim 97, wherein said cytokine is selected from the group consisting of early acting cytokines and late acting cytokines.

104. The method of claim 103, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

105. The method of claim 103, wherein said early acting cytokine is FLT3 ligand.

106. The method of claim 103, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

107. The method of claim 103, wherein said late acting cytokine is granulocyte colony stimulating factor.

108. A method of preservation of undifferentiated stem cells comprising providing the undifferentiated stem cells with an effective concentration of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase of said undifferentiated stem cells, wherein said providing is performed in at least one of the steps of harvesting, isolating and storage of the undifferentiated hematopoietic cells.

109. The method of claim 108, wherein said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase is selected from the group consisting of:

(a) an inhibitor of PI 3-kinase catalytic activity;

(b) an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding a PI 3-kinase;

(c) a ribozyme which specifically cleaves PI 3-kinase transcripts, coding sequences and/or promoter elements;

(d) an siRNA molecule capable of inducing degradation of PI 3-kinase transcripts; and

(e) a DNAzyme which specifically cleaves PI 3-kinase transcripts or DNA.

110. The method of claim 109, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

111. The method of claim 109, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

112. The method of claim 111, wherein said anti-PI 3-kinase antibody is ScFV or Fab.

113. The method of claim 109, wherein said providing is effected by transiently expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme within a stem cell.

114. The method of claim 109, wherein said providing is effected by

(a) providing an expressible polynucleotide capable of expressing said antisense polynucleotide, said ribozyme, said siRNA molecule or said DNAzyme, and

(b) stably integrating said expressible polynucleotide into a genome of a cell,

thereby providing a modulator capable of downregulating a PI 3-kinase activity or PI 3-kinase gene expression.

115. The method of claim 109, wherein said inhibitor of PI 3-kinase activity is an expressible polynucleotide encoding an anti-PI 3-kinase ScFv or Fab.

116. The method of claim 108, further comprising providing the cells with nutrients and cytokines.

117. The method of claim 116, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

118. The method of claim 117, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

119. The method of claim 117, wherein said early acting cytokine is FLT3 ligand.

120. The method of claim 117, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

121. The method of claim 117, wherein said late acting cytokine is granulocyte colony stimulating factor.

122. The method of claim 108, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

123. The method of claim 122, wherein said stem cells are derived from bone marrow or peripheral blood.

124. The method of claim 122, wherein said stem cells are derived from neonatal umbilical cord blood.

125. The method of claim 108, further comprising the step of selecting a population of stem cells enriched for hematopoietic stem cells.

126. The method of claim 125, wherein said selection is affected via CD34.

127. The method of claim 108, further comprising the step of selecting a population of stem cells enriched for early hematopoietic stem/progenitor cells.

128. The method of claim 127, wherein said selection is affected via CD133.

129. Stem cell collection bags, stem cell separation and stem cell washing buffers supplemented with an amount of a modulator of PI 3-kinase activity, said modulator selected capable of downregulating a PI 3-kinase activity or an expression of a gene encoding a PI 3-kinase, said amount sufficient to inhibit differentiation of a population of undifferentiated hematopoietic cells.

130. The stem cell collection bags and buffers of claim 129, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an inhibitor of PI 3-kinase activity.

131. The stem cell collection bags and buffers of claim 130, wherein said inhibitor of PI 3-kinase activity is Wortmannin or LY294002.

132. The stem cell collection bags and buffers of claim 129, wherein said modulator capable of downregulating PI 3-kinase activity or expression of a gene encoding PI 3-kinase is an anti-PI 3-kinase antibody.

133. The stem cell collection bags and buffers of claim 129, further supplemented with nutrients and cytokines.

134. The stem cell collection bags and buffers of claim 133, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

135. The stem cell collection bags and buffers of claim 134, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin-6, thrombopoietin and interleukin-3.

136. The stem cell collection bags and buffers of claim 134, wherein said early acting cytokine is FLT3 ligand.

137. The stem cell collection bags and buffers of claim 134, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor and erythropoietin.

138. The stem cell collection bags and buffers of claim 134, wherein said late acting cytokine is granulocyte colony stimulating factor.

139. An assay for determining whether a modulator of PI 3-kinase activity is capable of inhibiting differentiation of cells, the assay comprising:

(a) culturing a population of cells capable of differentiating, in the presence or absence of said modulator of PI 3-kinase activity; and

(b) assessing changes in differentiation of said cells,

wherein an increase in differentiation as compared to untreated cells indicates a modulator of PI 3-kinase activity incapable of inhibiting differentiation, and whereas a lack of or decrease in differentiation as compared to untreated cells, indicates a modulator of PI 3-kinase activity capable of inhibiting differentiation.

140. The assay of claim 139, wherein said cells capable of differentiating are stem or progenitor cells, or substantially undifferentiated cells of a cell line.

141. The assay of claim 140, wherein said stem or progenitor cells are early hematopoietic and/or hematopoietic progenitor cells.

142. The assay of claim 139, further comprising providing the cells with nutrients and cytokines.

143. The assay of claim 142, wherein said cytokines are selected from the group consisting of early acting cytokines and late acting cytokines.

144. The assay of claim 140, wherein said stem cells are derived from a source selected from the group consisting of hematopoietic cells, neural cells, oligodendrocyte cells, skin cells, hepatic cells, embryonal stem cells, muscle cells, bone cells, mesenchymal cells, pancreatic cells, chondrocytes and stroma cells.

145. The assay of claim 139, wherein said assessing changes in differentiation is effected via differentiation markers.

146. The assay of claim 145, wherein said differentiation markers are selected from the group consisting of CD133, CD34, CD38, CD33, CD14, CD15, CD3, CD61 and CD19.

147. A method of ex vivo expanding and inhibiting differentiation of a population of stem cells, the method comprising:

(a) providing the cells ex vivo with conditions for cell proliferation;

(b) ex vivo reducing a capacity of said stem cells in responding to signaling pathways involving a PI 3-kinase activity;

thereby ex vivo expanding and inhibiting differentiation of the population of stem cells.