Stimulation of hematopoiesis and stem cell engraftment by senolysis

Abstract

Means, methods, and compositions of matter for accelerating hematopoiesis and / or stem cell engraftment through induction of senolysis and / or deactivation of senescent cells. Senolytic agents can be administered together with hematopoietic growth factors. Resistance to stem cell mobilization can be accomplished by removal of senescent cells through senolytic and / or senescence inactivating approaches.

Description

IMMORTA-SOHASCE-PCTSTIMULATION OF HEMATOPOIESIS AND STEM CELL ENGRAFTMENT BY SENOLYSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from U.S Provisional Application No. 63 / 687,297, entitled STIMULATION OF HEMATOPOIESIS AND STEM CELL ENGRAFTMENT BY SENOLYSIS and filed on August 26, 2024, the entire contents of which are hereby expressly incorporated by reference.FIELD OF INVENTION

[0002] The invention pertains to the field of accelerating hematopoiesis and / or stem cell engraftment through induction of senolysis and / or deactivation of senescent cells.BACKGROUND

[0003] Ablation of the bone marrow (BM) before hematopoietic stem cell transplantation (HSCT) is commonly used to treat a wide array of blood malignancies. Following a BM transplant, hematopoietic stem and progenitor cells (HSPCs) require a stable BM microenvironment to repopulate. Signals from the BM niche are essential for hematopoietic homeostasis.

[0004] Total body irradiation (IR) used to deplete resident HSCs or leukemic cells and make space available in the BM microenvironment destroys not only endogenous HSCs but also non-hematopoietic cells in the vascular niche [1], which mainly comprises endothelial cells (ECs) [2], BM ECs lining the interior of blood vessels produce various factors, such as CXCL12 and SCF, to regulate HSC activities in stable or stressful conditions [3], IR causes endothelial inflammation and hyperplasia. Endothelial dysfunction reduces the cellular capacity to maintain homeostasis [4], While IR preconditioning enables engraftment and creates the available niche for acute regeneration, many hematopoietic conditions due to changes in normal HSC function, such as BM failure and poor graft function (PGF), develop gradually [5], BM fibrosis promotesIMMORTA-SOHASCE-PCT the delay or unsuccessful engraftment [6], and IR causes grade BM fibrosis [7], One serious complication of radiotherapy is fibrotic reactions, which require intense investigation to understand the intimate mechanisms involved in tissue fibrosis and dysfunction.

[0005] There is an urgent need to develop novel ways of stimulating hematopoietic stem cell engraftment and / or reconstitution after injury. Enhanced engraftment / reconstitution would increase efficacy of cord blood transplantation, provide increased successes after classical stem cell transplants.SUMMARY

[0006] Various aspects (embodiments) of the invention are enumerated in the following paragraphs:

[0007] 1 . A method of enhancing engraftment of a hematopoietic stem cell into a recipient comprising the steps of: a) obtaining a recipient in need of a hematopoietic stem cell graft; b) reducing the number of senescent cells in said recipient; c) providing said recipient a conditioning regimen to remove existing hematopoietic stem cells; and d) administering to the recipient exogenous hematopoietic stem cells.

[0008] 2. The method of aspect 1 , wherein said hematopoietic stem cell graft is a purified population of hematopoietic stem cells.

[0009] 3. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses CD34.

[0010] 4. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses CD133.

[0011] 5. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses aldehyde dehydrogenase.

[0012] 6. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses c-kit.

[0013] 7. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses IL-3 receptor.

[0014] 8. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses IL-6 receptor.IMMORTA-SOHASCE-PCT

[0015] 9. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses thrombopoietin receptor.

[0016] 10. The method of aspect 2, wherein said purified population of hematopoietic stem cells expresses c-met.

[0017] 11 . The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of bone marrow derived cells, wherein said population has been treated with G-CSF at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0018] 12. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of peripheral blood derived cells, wherein said population has been treated with G-CSF at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0019] 13. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of mobilized peripheral blood derived cells, wherein said population has been treated with G-CSF at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0020] 14. The method of aspect 13, wherein said mobilization of peripheral blood refers to treating the subject with one or more agents capable of causing stem cells to exit bone marrow niches and enter peripheral circulation.

[0021] 15. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is G-CSF.

[0022] 16. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is GM- CSF.

[0023] 17. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is M-CSF.

[0024] 18. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is beta glucan.

[0025] 19. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is Poly IC.

[0026] 20. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is a tlr2 agonist.IMMORTA-SOHASCE-PCT

[0027] 21 . The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is flt-3 ligand.

[0028] 22. The method of aspect 14, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is detoxified lipopolysaccharide.

[0029] 23. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of bone marrow derived cells, wherein said population has been treated with IL-3 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0030] 24. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of peripheral blood derived cells, wherein said population has been treated with IL-3 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0031] 25. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of mobilized peripheral blood derived cells, wherein said population has been treated with IL-3 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0032] 26. The method of aspect 25, wherein said mobilization of peripheral blood refers to treating the subject with one or more agents capable of causing stem cells to exit bone marrow niches and enter peripheral circulation.

[0033] 27. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is G-CSF.

[0034] 28. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is GM- CSF.

[0035] 29. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is M-CSF.

[0036] 30. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is beta glucan.

[0037] 31 . The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is Poly IC.IMMORTA-SOHASCE-PCT

[0038] 32. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is a tlr2 agonist.

[0039] 33. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is flt-3 ligand.

[0040] 34. The method of aspect 26, wherein said agent capable of causing stem cells to exit bone marrow niches and enter peripheral circulation is detoxified lipopolysaccharide.

[0041] 35. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of bone marrow derived cells, wherein said population has been treated with IL-7 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0042] 36. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of peripheral blood derived cells, wherein said population has been treated with IL-7 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0043] 37. The method of aspect 3, wherein said CD34 cell is selected from a mononuclear population of mobilized peripheral blood derived cells, wherein said population has been treated with IL-7 at a concentration and duration to enhance engraftment ability of said CD34 cell.

[0044] 38. The method of aspect 1 , wherein said hematopoietic stem cells are extracted from a population of bone marrow mononuclear cells that have been treated with one or more senolytic agents and subsequently exposed to an inflammatory stimuli.

[0045] 39. The method of aspect 38, wherein said extraction of said hematopoietic stem cells is performed using magnetic activated cell sorting.

[0046] 40. The method of aspect 39, wherein said extraction is performed using positive selection for the marker CD34.

[0047] 41 . The method of aspect 39, wherein said extraction is performed using positive selection for the marker c-kit.

[0048] 42. The method of aspect 39, wherein said extraction is performed using positive selection for the marker c-mpl.IMMORTA-SOHASCE-PCT

[0049] 43. The method of aspect 39, wherein said extraction is performed using positive selection for the marker c-met.

[0050] 44. The method of aspect 39, wherein said extraction is performed using positive selection for the marker LIF receptor.

[0051] 41 . The method of aspect 38, wherein said inflammatory stimuli is culture with one or more inflammatory cells.

[0052] 42. The method of aspect 41 , wherein said inflammatory cell is a neutrophil.

[0053] 43. The method of aspect 41 , wherein said inflammatory cell is a neutrophil of the type 1 lineage.

[0054] 44. The method of aspect 41 , wherein said inflammatory cell is a monocyte.

[0055] 45. The method of aspect 41 , wherein said inflammatory cell is a macrophage.

[0056] 46. The method of aspect 41 , wherein said inflammatory cell is a T cell.

[0057] 47. The method of aspect 41 , wherein said inflammatory cell is T cytotoxic cell.

[0058] 48. The method of aspect 41 , wherein said inflammatory cell is a T helper cell.

[0059] 49. The method of aspect 41 , wherein said inflammatory cell is a gamma delta T cell.

[0060] 50. The method of aspect 41 , wherein said inflammatory cell is a NK cell.

[0061] 51 . The method of aspect 41 , wherein said inflammatory cell is aNKT cell.

[0062] 52. The method of aspect 41 , wherein said inflammatory cell is a T helper cell.

[0063] 53. The method of aspect 42, wherein said neutrophil is activated by culture with zymosan.

[0064] 54. The method of aspect 42, wherein said neutrophil is activated by culture with bacterial cell wall extract.

[0065] 55. The method of aspect 42, wherein said neutrophil is activated by culture with oxidized LDL.IMMORTA-SOHASCE-PCT

[0066] 56. The method of aspect 42, wherein said neutrophil is activated by culture with necrotic cells.

[0067] 57. The method of aspect 42, wherein said neutrophil is activated by culture with MG.

[0068] 58. The method of aspect 42, wherein said neutrophil is activated by culture with ozone or ozonides.

[0069] 59. The method of aspect 42, wherein said macrophage is activated by culture with one or more allogeneic T cells.

[0070] 60. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-I by 25% compared to baseline prior to culture with said allogeneic T cells.

[0071] 61 . The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-I by 50% compared to baseline prior to culture with said allogeneic T cells.

[0072] 62. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-I by 100% compared to baseline prior to culture with said allogeneic T cells.

[0073] 63. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-II by 25% compared to baseline prior to culture with said allogeneic T cells.

[0074] 64. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-II by 50% compared to baseline prior to culture with said allogeneic T cells.

[0075] 65. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of HLA-II by 100% compared to baseline prior to culture with said allogeneic T cells.

[0076] 66. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient toIMMORTA-SOHASCE-PCT increase expression of CD80 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0077] 67. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of CD80 by 200% compared to baseline prior to culture with said allogeneic T cells.

[0078] 68. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of CD80 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0079] 69. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of CD86 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0080] 70. The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of CD86 by 200% compared to baseline prior to culture with said allogeneic T cells.

[0081] 71 . The method of aspect 59, wherein said macrophages are cultured with interferon gamma at a concentration and duration sufficient to increase expression of CD86 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0082] 72. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-I by 25% compared to baseline prior to culture with said allogeneic T cells.

[0083] 73. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-I by 50% compared to baseline prior to culture with said allogeneic T cells.

[0084] 74. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-I by 100% compared to baseline prior to culture with said allogeneic T cells.IMMORTA-SOHASCE-PCT

[0085] 75. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-II by 25% compared to baseline prior to culture with said allogeneic T cells.

[0086] 76. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-II by 50% compared to baseline prior to culture with said allogeneic T cells.

[0087] 77. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of HLA-II by 100% compared to baseline prior to culture with said allogeneic T cells.

[0088] 78. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of CD80 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0089] 79. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of CD80 by 200% compared to baseline prior to culture with said allogeneic T cells.

[0090] 80. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of CD80 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0091] 81 . The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of CD86 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0092] 82. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increase expression of CD86 by 200% compared to baseline prior to culture with said allogeneic T cells.

[0093] 83. The method of aspect 59, wherein said macrophages are cultured with TNF-alpha at a concentration and duration sufficient to increaseIMMORTA-SOHASCE-PCT expression of CD86 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0094] 84. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-I by 25% compared to baseline prior to culture with said allogeneic T cells.

[0095] 85. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-I by 50% compared to baseline prior to culture with said allogeneic T cells.

[0096] 86. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-I by 100% compared to baseline prior to culture with said allogeneic T cells.

[0097] 87. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-II by 25% compared to baseline prior to culture with said allogeneic T cells.

[0098] 88. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-II by 50% compared to baseline prior to culture with said allogeneic T cells.

[0099] 89. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of HLA-II by 100% compared to baseline prior to culture with said allogeneic T cells.

[0100] 90. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD80 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0101] 91 . The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD80 by 200% compared to baseline prior to culture with said allogeneic T cells.IMMORTA-SOHASCE-PCT

[0102] 92. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD80 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0103] 93. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD86 by 80% compared to baseline prior to culture with said allogeneic T cells.

[0104] 94. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD86 by 200% compared to baseline prior to culture with said allogeneic T cells.

[0105] 95. The method of aspect 59, wherein said macrophages are cultured with HGF at a concentration and duration sufficient to increase expression of CD86 by 400% compared to baseline prior to culture with said allogeneic T cells.

[0106] 96. The method of aspect 1 , wherein said hematopoietic stem cell is administered to said patient after administration of a mesenchymal stem cell.

[0107] 97. The method of aspect 96, wherein one or more senolytic agents are administered prior to administration of mesenchymal stem cells.

[0108] 98. The method of aspect 97, wherein said senolytic agent is an inhibitor of bcl-2.

[0109] 99. The method of aspect 97, wherein said senolytic agent is an inhibitor of bcl-xL.

[0110] 100. The method of aspect 97, wherein said senolytic agent is dasatinib.

[0111] 101. The method of aspect 97, wherein said senolytic agent is imatinib.

[0112] 102. The method of aspect 97, wherein said senolytic agent is rapamycin.

[0113] 103. The method of aspect 97, wherein said senolytic agent is fisutin.

[0114] 104. The method of aspect 97, wherein said senolytic agent is quercetin.IMMORTA-SOHASCE-PCT

[0115] 105. The method of aspect 97, wherein said senolytic agent is dasatinib and quercetin.

[0116] 106. The method of aspect 97, wherein said senolytic agent is fk-506.

[0117] 107. The method of aspect 97, wherein said senolytic agent is natural antibody.

[0118] 108. The method of aspect 97, wherein said senolytic agent is IgM antibody.

[0119] 109. The method of aspect 1 , wherein said hematopoietic stem cell is administered intravenously.

[0120] 110. The method of aspect 1 , wherein said hematopoietic stem cell is administered intra-ossus.

[0121] 111. The method of aspect 96, wherein said mesenchymal stem cell is administered intravenously.

[0122] 112. The method of aspect 96, wherein said mesenchymal stem cell is administered intra-ossus.

[0123] 113. The method of aspect 96, wherein said mesenchymal stem cell is conditioned to possess enhanced hematopoiesis promoting activities.

[0124] 114. The method of aspect 113, wherein said mesenchymal stem cell is gene engeered to express one or more hematopoietic growth factors.

[0125] 115 The method of aspect 114, wherein said hematopoietic growth factor is BMP-2.

[0126] 116. The method of aspect 114, wherein said hematopoietic growth factor is BMP-4.

[0127] 117 The method of aspect 114, wherein said hematopoietic growth factor is IL-4.

[0128] 118 The method of aspect 114, wherein said hematopoietic growth factor is IL-6.

[0129] 119. The method of aspect 114, wherein said hematopoietic growth factor is IL-6-IL-6 receptor complex.

[0130] 120. The method of aspect 114, wherein said hematopoietic growth factor is IL-7.

[0131] 121. The method of aspect 114, wherein said hematopoietic growth factor is IL-9.IMMORTA-SOHASCE-PCT

[0132] 122. The method of aspect 114, wherein said hematopoietic growth factor is IL-10.

[0133] 123. The method of aspect 114, wherein said hematopoietic growth factor is IL-11 .

[0134] 124. The method of aspect 114, wherein said hematopoietic growth factor is thrombopoietin.

[0135] 125. The method of aspect 114, wherein said hematopoietic growth factor is IL-13.

[0136] 126. The method of aspect 114, wherein said hematopoietic growth factor is IL-15.

[0137] 127. The method of aspect 114, wherein said hematopoietic growth factor is IL-20.

[0138] 128. The method of aspect 114, wherein said hematopoietic growth factor is IL-22.

[0139] 129. The method of aspect 114, wherein said hematopoietic growth factor is IL-35.

[0140] 130. The method of aspect 114, wherein said hematopoietic growth factor is IL-37.

[0141] 131. The method of aspect 114, wherein said hematopoietic growth factor is IL-38.

[0142] 132. The method of aspect 114, wherein said hematopoietic growth factor is EGF.

[0143] 133. The method of aspect 114, wherein said hematopoietic growth factor is IGF-1 .

[0144] 134. The method of aspect 114, wherein said hematopoietic growth factor is VEGF.

[0145] 135. The method of aspect 114, wherein said hematopoietic growth factor is KLOTHO.

[0146] 136. The method of aspect 114, wherein said hematopoietic growth factor is PDGF.

[0147] 137. The method of aspect 114, wherein said hematopoietic growth factor is GDF-11.

[0148] 138. The method of aspect 114, wherein said hematopoietic growth factor is GDF-15.IMMORTA-SOHASCE-PCT

[0149] 139. The method of aspect 114, wherein said hematopoietic growth factor is oncostatin.

[0150] 140. The method of aspect 114, wherein said hematopoietic growth factor is HGF.

[0151] 141. The method of aspect 114, wherein said hematopoietic growth factor is stem cell factor.

[0152] 142. The method of aspect 114, wherein said hematopoietic growth factor is M-CSF.

[0153] 143. The method of aspect 114, wherein said hematopoietic growth factor is G-CSF.

[0154] 144. The method of aspect 114, wherein said hematopoietic growth factor is GM-CSF.

[0155] 145. The method of aspect 114, wherein said hematopoietic growth factor is GC-MAF.

[0156] 146. The method of aspect 114, wherein said hematopoietic growth factor is placental growth factor.

[0157] 147. The method of aspect 114, wherein said hematopoietic growth factor is interleukin-1 receptor antagonist.

[0158] 148. The method of aspect 114, wherein said hematopoietic growth factor is ciliary neurotrophic growth factor.

[0159] 149. The method of aspect 114, wherein said hematopoietic growth factor is brain derived growth factor.

[0160] 150. The method of aspect 114, wherein said hematopoietic growth factor is nerve growth factor.

[0161] 151. The method of aspect 96, wherein said mesenchymal stem cell is activated before administration.

[0162] 152. The method of aspect 151 , wherein said activation involved treatment of said mesenchymal stem cell with one or more agents that increase nuclear translocation of NF-kappa B.

[0163] 153. The method of aspect 151 , wherein said activation involved treatment of said mesenchymal stem cell with one or more agents that increase nuclear translocation of HIF-1 alpha.

[0164] 154. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0165] 155. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0166] 156. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0167] 157. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0168] 158. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0169] 159. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0170] 160. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0171] 161. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0172] 162. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0173] 163. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0174] 164. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0175] 165. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0176] 166. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0177] 167. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0178] 168. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0179] 169. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0180] 170. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0181] 171. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with interleukin 1 beta for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0182] 172. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0183] 173. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0184] 174. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0185] 175. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0186] 176. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0187] 177. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.

[0188] 178. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0189] 179. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.IMMORTA-SOHASCE-PCT

[0190] 180. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0191] 181. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0192] 182. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0193] 183. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0194] 184. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0195] 185. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0196] 186. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0197] 187. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0198] 188. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with interleukin 1 beta for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0199] 189. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0200] 190. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0201] 191 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0202] 192. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0203] 193. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0204] 195. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0205] 196. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0206] 197. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0207] 198. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0208] 199. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0209] 200. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0210] 201 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin 1 beta for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0211] 202. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0212] 203. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0213] 204. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0214] 205. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with unmethylated CpG DNA for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0215] 206. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0216] 207. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0217] 208. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0218] 209. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0219] 210. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0220] 211 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0221] 212. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0222] 213. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.IMMORTA-SOHASCE-PCT

[0223] 214. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0224] 215. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0225] 216. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0226] 217. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.

[0227] 218. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0228] 219. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.

[0229] 220. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0230] 221 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0231] 222. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with unmethylated CpG DNA for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0232] 223. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0233] 224. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0234] 225. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0235] 226. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0236] 227. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0237] 228. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0238] 229. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0239] 230. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient toIMMORTA-SOHASCE-PCT soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0240] 231 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0241] 232. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with unmethylated CpG DNA for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0242] 233. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0243] 234. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase LIF expression of said mesenchymal stem cell by 25% or more.

[0244] 235. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0245] 236. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0246] 237. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0247] 238. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with flagellin for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0248] 239. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0249] 240. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0250] 241 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0251] 242. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0252] 243. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0253] 244. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0254] 245. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0255] 246. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0256] 247. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0257] 248. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0258] 249. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0259] 250. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0260] 251 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0261] 252. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0262] 253. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0263] 254. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0264] 255. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with flagellin for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0265] 256. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0266] 257. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.

[0267] 258. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 25% or more.

[0268] 259. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 50% or more.

[0269] 260. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 100% or more.

[0270] 261 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0271] 262. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0272] 263. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0273] 264. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0274] 265. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0275] 266. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0276] 267. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0277] 268. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0278] 269. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0279] 270. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0280] 271 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with flagellin for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0281] 272. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with flagellin for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0282] 273. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0283] 274. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase EPO receptor expression of said mesenchymal stem cell by 25% or more.

[0284] 275. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0285] 276. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0286] 277. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0287] 278. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0288] 279. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0289] 280. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0290] 281 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0291] 282. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0292] 283. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0293] 284. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0294] 285. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0295] 286. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0296] 287. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0297] 288. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0298] 289. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with HMGB1 for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0299] 290. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0300] 291 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0301] 292. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0302] 293. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0303] 294. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0304] 295. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0305] 296. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0306] 297. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0307] 298. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 25% or more.

[0308] 299. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 50% or more.

[0309] 300. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase PDGF- BB expression of said mesenchymal stem cell by 100% or more.

[0310] 301 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0311] 302. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0312] 303. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0313] 304. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0314] 305. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0315] 306. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with HMGB1 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0316] 307. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0317] 308. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0318] 309. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0319] 310. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to soluble TNF- alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0320] 311 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0321] 312. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with HMGB1 for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0322] 313. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0323] 314. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase GDF1 1 expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0324] 315. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0325] 316. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0326] 317. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0327] 318. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0328] 319. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0329] 320. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0330] 321 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0331] 322. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0332] 323. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with interleukin-35 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0333] 324. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0334] 325. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0335] 326. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase M- CSF expression of said mesenchymal stem cell by 25% or more.

[0336] 327. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase M- CSF expression of said mesenchymal stem cell by 50% or more.

[0337] 328. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase M- CSF expression of said mesenchymal stem cell by 100% or more.

[0338] 329. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase G- CSF expression of said mesenchymal stem cell by 25% or more.

[0339] 330. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase G- CSF expression of said mesenchymal stem cell by 50% or more.

[0340] 331 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase G- CSF expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0341] 332. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0342] 333. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0343] 334. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0344] 335. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase IL- 11 expression of said mesenchymal stem cell by 25% or more.

[0345] 336. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase IL- 11 expression of said mesenchymal stem cell by 50% or more.

[0346] 337. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase IL- 11 expression of said mesenchymal stem cell by 100% or more.

[0347] 338. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0348] 339. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.

[0349] 340. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with interleukin-35 for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0350] 341 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0351] 342. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0352] 343. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0353] 344. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0354] 345. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0355] 346. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0356] 347. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0357] 348. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.IMMORTA-SOHASCE-PCT

[0358] 349. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0359] 350. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0360] 351 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0361] 352. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with interleukin-35 for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0362] 353. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0363] 354. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-13 expression of said mesenchymal stem cell by 25% or more.

[0364] 355. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0365] 356. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0366] 357. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0367] 358. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0368] 359. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0369] 360. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0370] 361 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0371] 362. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0372] 363. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0373] 364. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0374] 365. The metd of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with Klotho for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0375] 366. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0376] 367. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0377] 368. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0378] 369. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0379] 370. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0380] 371 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0381] 372. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0382] 373. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.IMMORTA-SOHASCE-PCT

[0383] 374. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0384] 375. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0385] 376. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0386] 377. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.

[0387] 218. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0388] 379. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.

[0389] 380. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0390] 381 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0391] 382. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with Klotho for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0392] 383. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0393] 384. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0394] 385. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0395] 386. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0396] 387. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0397] 388. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0398] 389. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0399] 390. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0400] 391 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0401] 392. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with Klotho for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0402] 393. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0403] 394. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0404] 395. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0405] 396. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0406] 397. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0407] 398. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0408] 399. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0409] 400. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more.

[0410] 401 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0411] 402. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0412] 403. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0413] 404. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentrationIMMORTA-SOHASCE-PCT sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0414] 405. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0415] 406. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0416] 407. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.

[0417] 408. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0418] 409. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0419] 410. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0420] 411 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0421] 412. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0422] 413. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0423] 414. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0424] 415. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.

[0425] 416. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0426] 417. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.IMMORTA-SOHASCE-PCT

[0427] 418. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0428] 419. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.

[0429] 420. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0430] 421 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0431] 422. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0432] 423. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0433] 424. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentrationIMMORTA-SOHASCE-PCT sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0434] 425. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0435] 426. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0436] 427. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0437] 428. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0438] 429. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0439] 430. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0440] 431 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0441] 432. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.

[0442] 433. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 25% or more.

[0443] 434. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0444] 435. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 50% or more.

[0445] 436. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-3 expression of said mesenchymal stem cell by 100% or more.

[0446] 437. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 25% or more.

[0447] 438. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 50% or more.

[0448] 439. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with oocyte cytoplasm for a period of time and concentration sufficient to increase MMP-9 expression of said mesenchymal stem cell by 100% or more.

[0449] 440. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 25% or more. 4

[0450] 441 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 50% or more.

[0451] 442. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase angiopoietin expression of said mesenchymal stem cell by 100% or more.

[0452] 443. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 25% or more.

[0453] 444. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 50% or more.

[0454] 445. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase VEGF expression of said mesenchymal stem cell by 100% or more.

[0455] 446. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 25% or more.

[0456] 447. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 50% or more.IMMORTA-SOHASCE-PCT

[0457] 448. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase M-CSF expression of said mesenchymal stem cell by 100% or more.

[0458] 449. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 25% or more.

[0459] 450. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 50% or more.

[0460] 451 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with pluripotent stem cell cytoplasm for a period of time and concentration sufficient to increase G-CSF expression of said mesenchymal stem cell by 100% or more.

[0461] 452. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 25% or more.

[0462] 453. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 50% or more.

[0463] 454. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase GM-CSF expression of said mesenchymal stem cell by 100% or more.

[0464] 455. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 25% or more.IMMORTA-SOHASCE-PCT

[0465] 456. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 50% or more.

[0466] 457. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase IL-11 expression of said mesenchymal stem cell by 100% or more.

[0467] 458. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 25% or more.

[0468] 459. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 50% or more.

[0469] 460. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase PDGF-BB expression of said mesenchymal stem cell by 100% or more.

[0470] 461 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 25% or more.

[0471] 462. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 50% or more.

[0472] 463. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase KGF expression of said mesenchymal stem cell by 100% or more.

[0473] 464. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cellIMMORTA-SOHASCE-PCT with oocyte cytoplasm for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 25% or more.

[0474] 465. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 50% or more.

[0475] 466. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase JAGGED expression of said mesenchymal stem cell by 100% or more.

[0476] 467. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 25% or more.

[0477] 468. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 50% or more.

[0478] 469. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase HLA-G expression of said mesenchymal stem cell by 100% or more.

[0479] 470. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to soluble TNF-alpha receptor expression of said mesenchymal stem cell by 25% or more.

[0480] 471 . The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increase soluble TNF-alpha receptor expression of said mesenchymal stem cell by 50% or more.

[0481] 472. The method of aspect 151 , wherein said activation of said mesenchymal stem cell is performed by treatment of said mesenchymal stem cell with oocyte cytoplasm for a period of time and concentration sufficient to increaseIMMORTA-SOHASCE-PCT soluble TNF-alpha receptor expression of said mesenchymal stem cell by 100% or more.BRIEF DESCRIPTION OF THE DRAWINGS

[0482] Figure 1 is a bar graph showing neutrophil counts in mice that were treated with a) saline; b) cyclophosphamide, c) cyclophosphamide + dendritic cells loaded with non-senescent fibroblast lysate, and d) cyclophosphamide + dendritic cells pulsed with senescent fibroblasts (SenoVax) cells.

[0483] Figure 2 is a bar graph showing neutrophil counts in mice that were treated with a) cyclophosphamide, b) G-CSF, c) dendritic cells pulsed with senescent fibroblasts (SenoVax) cells, and d) SenoVax cells with G-CSF.DETAILED DESCRIPTION

[0484] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0485] In one embodiment the invention provides means of generating hematopoietic stem cell grafts by expanding a multicellular population containing hematopoietic stem cells and subsequently extracting said hematopoietic stem cells. In one embodiment bone marrow mononuclear cells are extracted and the whole population is treated with one or more agents, the interaction between these agents and the other cells found in bone marrow mononuclear cells causes expansion and / or stimulation of hematopoietic stem cells. In some embodiments CD34 cells are isolated as a source of pure hematopoietic stem cells. In some embodiments of the invention senolytic compounds are administered into the culture of bone marrow mononuclear cells, said senolytic compounds may include in isolation or in combination quercetin and desatinib.

[0486] In one embodiment the invention teaches the utilization of senolytic agents as a means of stimulating hematopoiesis after injury to the bone marrow compartment. In some embodiments injury is the result of stress, radiation,IMMORTA-SOHASCE-PCT chemotherapy, or immunological insult. In one embodiments the senolytic utilized to stimulate hematopoiesis is a senolytic immunotherapy or senolytic vaccine.

[0487] Embodiments of the invention related to our findings that reducing activity of senescent cells, such as production of senescent associated secretory phenotype, can increase hematopoietic activity and / or hematopoietic reconstitution after a bone marrow insult.

[0488] One aspect of the invention relates to a method for hematopoietic reconstitution in a subject. The method involves administering to the subject hematopoietic stem / progenitor cells (HSPC) and administering to the subject a therapeutically effective amount of an inhibitor senescent cells. Such inhibitor can be a chemical inhibitor, an antisense molecule or RNA interference inducing molecule to bcl-2. Other inhibitors can be immunological effects that target senescent cells such as antibodies, antibody cytoxic conjugates, or immunotherapies. The inhibitor is administered by a route and in a sufficient amount to thereby contact the HSPCs in the subject and thereby promote enhanced proliferation and expansion of the early hematopoietic cells therein. The hematopoietic reconstitution in the subject which would otherwise occur in the absence of the inhibitor is thereby enhanced by the activity of the inhibitor in that the short term reconstitution occurs faster and / or more completely (e.g., with enhanced differentiation into a broader range of cell types) than otherwise would have occurred in the absence of the inhibitor.

[0489] A recipient subject in the methods described herein can be anyone in need of hematopoietic reconstitution or anyone with reduced number of white blood cells in peripheral blood. Such subjects include, without limitation, subjects with hematopoietic cancer such as leukemia and lymphoma, subjects with myelosuppression or myeloablation, such as those who have undergone cytoreductive therapy (e.g., chemotherapy or radiation therapy). The recipient subject may suffer from diseases and disorders including, without limitation, leukopenia of various origins including, congenital leukopenia, childhood or adult cyclic neutropenia, post-infective neutropenia, and myelodysplastic syndrome and aplastic anemia (congenital and acquired). Subjects suitable as recipients include those in which their entire hematopoietic system is ablated, and also those with reduced intensity conditioning. Reduced intensity conditioning doesIMMORTA-SOHASCE-PCT not result in complete myeloablation and is used in patients that are older, in patients who are in complete remission, and in patients with acquired aplastic anemia.

[0490] In one embodiment, the donor has been identified or selected as a candidate for donation of HSPCs prior to administration of the inhibitor of IL-18. In one embodiment, the donor undergoes additional conditioning prior to administration of the HSPCs.

[0491] Administration of the inhibitor to the recipient subject may be prior to, concurrent with, or after administration of the HSPC. It may be advantageous for administration to be ongoing over a period of time, beginning prior to, concurrent with or after administration of the HSPC. Such ongoing administration could be by way of multiple administration time points. In one embodiment, the inhibitor is administered to the subject for a period of from about 1 day to about 5 days (e.g., about 5, 4, 3, 2 or 1 days) beginning on the day of administration of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 5 days to about 10 days (e.g., about 10, 9, 8, 7, or 6 days) beginning on the day of administration of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 10 days to about 20 days (e.g., about 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 days) beginning on the day of administration of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 20 days to about 30 days (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 days) beginning on the day of administration of the HSPC. Benefit may also be obtained from administration on a regular basis up to about day 100 of the HSPC administration.

[0492] Administration of the inhibitor at the time of administration of the HSPC encompasses administration concurrently with the HSPCs, directly prior to (e.g., within an hour prior), and also directly following administration of the HSPC (e.g., within about 1-24 hours). Administration concurrently with the HSPCs may also include combining the HSPCs with the inhibitor and administering the combination to the subject.

[0493] Administration of the inhibitor to the subject prior to administration of the HSPC is expected to have beneficial effect. Administration for a period of from about 1 day up to about 5 days (e.g., about 5, 4, 3, 2 or 1 day) prior to administration of the HSPCs is envisioned. In one embodiment, administration ofIMMORTA-SOHASCE-PCT the inhibitor prior to receipt is combined with administration at the time of receipt and / or ongoing administration for a period of time as described herein. HSPCs are determined suitable for hematopoietic reconstitution by the skilled practitioner, including identification of a suitable donor, appropriate collection and manipulation, prior to administration to the subject.

[0494] The HSPCs can be autologous (where the donor and recipient are the same person) and allogeneic (where the donor and recipient are different individuals). In autologous transplant, HSPCs are removed from the subject before they experience the hematopoietic damaging event (e.g., high-dose chemotherapy or radiation treatment). The cells are stored in a freezer (cryopreservation). After the damaging event, the cells are put back in the subject's body to make (regenerate) normal blood cells. This is referred to as a rescue transplant. In allogeneic transplant, HSPCs are removed from another person, referred to as a donor. Umbilical cord blood transplant is a type of allogeneic or autologous transplant depending on the source of the umbilical cord. Stem cells are removed from a newborn baby's umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Another source of donor cells is placenta.

[0495] Another source of donor cells is alternative sources requiring genetic manipulation such as HSCs obtained through genetic re-programming of more mature cells or induced embryonic stem cells. Donor HSPCs are typically collected in two ways, by bone marrow harvest or leukapheresis. Bone marrow harvest is minor surgery performed under general anesthesia, where the bone marrow is removed from the back of both hip bones. Leukapheresis is the peripheral harvest of HSPCS. The donor receives several (e.g., about 5 days) of treatments to move stem cells from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. HSPCs are separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.

[0496] The harvested cells are a mixture of stem cells, progenitors, and white blood cells of various degrees of maturity. The progenitor cells and / or stem cells can reconstitute all of the hematopoietic cells in a subject. These include, but are not limited to, lymphocytes, platelets, erythrocytes and myeloid cells, including, T cells, B cells (plasma cells), natural killer cells, dendritic cells,IMMORTA-SOHASCE-PCT monocytes (macrophages), neutrophils, eosinophils, basophils (mast cells), megakaryocytes (platelets), and erythroblasts (erythrocytes). These cells are also capable, in addition to differentiation, of self-renewal, so as to proliferate the stem-progenitor population that is capable of differentiation.

[0497] Another aspect of the invention relates to treatment of a donor individual with an inhibitor of senescent cells to donation of the HSPC for use in hematopoietic reconstitution in a subject. Hematopoietic reconstitution is achieved in a subject by administering to the subject HSPC obtained from a donor subject that was previously treated with an inhibitor of senescent cells, described herein. The treatment is to thereby induce expansion of early hematopoietic progenitor cells in the donor prior to harvest. The induction occurs by similar mechanism as in the recipient subject. The donor is treated with the inhibitor to thereby contact the HSPCs of the donor with an effective amount of the inhibitor. The inhibitor is administered by a route and in sufficient amount to thereby affect the HSPCs in the donor (e.g., those that express senescent cells at significant levels) and thereby promote enhanced proliferation and expansion of those cells. In one embodiment, administered by a route and in sufficient amount to thereby contact the HSPCs in the donor. As a result of the treatment, the enhanced proliferation and expansion may occur either in the donor prior to harvest, in the recipient following transplant, ex vivo, or any combination thereof. The hematopoietic reconstitution of the recipient subject is enhanced by the activity of the inhibitor in that the short term reconstitution occurs faster and / or more completely (e.g., with a broader cell type populations) than otherwise would have occurred in the absence of administration of the inhibitor to the donor.

[0498] Administration to the donor can be by a variety of methods, examples of which are described herein (e.g., those for the recipient). In one embodiment, the donor is also the recipient of the transplant. In one embodiment, the donor is different from the recipient of the transplant. In one embodiment, the recipient is also administered a therapeutically effective amount of an inhibitor, by the methods discussed herein.

[0499] In one embodiment of the invention, senolytic compounds are administered together rwith stem cells. In one embodiment, adipose tissue derived cells and / or stem cells are utilized. Adipose tissue is an attractive alternative to bone marrow as a source of stem cells for the following reasons: a)IMMORTA-SOHASCE-PCT extraction of adipose derived cells is a simpler procedure that is much less invasive than bone marrow extraction; b) Adipose tissue contains a higher content of mesenchymal stem cells (MSC) as compared to bone marrow; c) MSC from adipose tissue do not decrease in number with aging and can therefore serve as an autologous cell source for all patients; and d) adipose tissue is also a source of unique cell populations in addition to MSC that have therapeutic potential, including endothelial cells and regulatory T cells.

[0500] To date, clinical trials on adipose derived cells have all utilized ex vivo-expanded cells, which share properties with bone marrow derived MSC [8- 13], Preparations of MSC expanded from adipose tissue are equivalent or superior to bone marrow in terms of differentiation ability [14, 15], angiogenesisstimulating potential

[0016] , and immune modulatory effects

[0017] , Given the extra processing steps associated with ex vivo expansion of adipose cells, a simpler and perhaps safer procedure would be the use of primary adipose tissue-derived cells for therapy. SVF comprises the mononuclear cells derived from adipose tissue, which are acquired through a simple isolation procedure whereby fat is lipoaspirated and subjected to enzymatic digestion. In veterinary medicine, over 4,000 horses and 4,000 dogs with various cartilage and bone injuries have been treated with autologous SVF without cellular expansion

[0018] , In double blind studies of canine osteoarthritis, statistically significant improvements in lameness, range of motion, and overall quality of life have been described [19, 20],

[0501] Given the abundance of pre-clinical and veterinary experience with autologous adipose-derived cells, there is great potential for autologous stem cell therapies using adipose-derived cells for a multitude of indications. Indeed, this prospect underlies the interest of commercial entities in devising bench top closed systems for autologous adipose cell therapy, such as Cytori’s Celution™ system

[0021] and Tissue Genesis’ TGI 1000™ platform

[0022] , which are presently entering clinical trials. Although the majority of studies have focused on in vitro expanded adipose derived cells, SVF derived from whole lipoaspirate alleviates the need for extensive processing of the cells, thereby also minimizing the number of steps where contamination could be introduced. The safety of adipose-derived cells is supported by autologus fat grafting, a common practice in cosmetic surgery [23, 24], An important consideration in clinical scenariosIMMORTA-SOHASCE-PCT where bulk SVF is utilized is the potential regenerative, angiogenic and immune regulatory contributions of the numerous cellular populations that are present.Mesenchymal Stem Cells in SVF

[0502] The mononuclear fraction of adipose tissue, referred to as the stromal vascular fraction (SVF), was originally described as the proliferative component of adipose tissue by Hollenberg et al. in 1968

[0025] , The cells comprising SVF morphologically resemble fibroblasts and were demonstrated to differentiate into pre-adipocytes and functional adipose tissue in vitro

[0026] , Although it was suggested that non-adipose differentiation of SVF may occur under specific conditions

[0027] , the notion of “adipose-derived stem cells” was not widely recognized until a seminal paper in 2001 , where Zuk et al demonstrated the SVF contains large numbers of mesenchymal-like stem cells (MSC-like) cells that could be induced to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic lineages

[0028] , Subsequent to the initial description, the same group reported that in vitro expanded SVF derived cells had surface marker expression similar to bone marrow derived MSC, displaying expression of CD29, CD44, CD71 , CD90, CD105 / SH2, and SH3 and lacking CD31 , CD34, and CD45 expression

[0029] , MSC are defined as adherent, non-hematopoietic cells expressing the surface markers CD90, CD105, and CD73, while lacking expression of CD14, CD34, and CD45, and having the ability to differentiate into adipocytes, chondrocytes, and osteocytes in vitro after treatment with the appropriate growth factors

[0030] ,

[0503] Allogeneic bone marrow derived MSC have been administered and conferred clinical benefit for the treatment of diseases such as graft versus host (GVHD) [31-36], osteogenesis imperfecta

[0037] , Hurler syndrome, metachromatic leukodystrophy

[0038] , and acceleration of hematopoietic stem cell engraftment [39- 41], The company Osiris has successfully completed Phase I safety studies using allogeneic bone marrow MSCs and is now conducting efficacy clinical trials (Phase II and Phase III) for Type I Diabetes, Crohn’s Disease, and Graft Versus Host Disease using allogeneic, bone marrow derived MSC. Intravenous administration of allogeneic MSCs by Osiris was also reported to induce aIMMORTA-SOHASCE-PCT statistically significant improvement in cardiac function in a double-blind study

[0042] .

[0504] Other companies have entered clinical trials using allogeneic mesenchymal stem cell based products. Athersys is currently in Phase I trials using its MultiStem technology, which involves administration of ex vivo expanded Multipotent Adult Progenitor Cell (MAPC) for post-infarct heart repair

[0043] , Angioblast Systems has recently announced initiation of Phase II trials using Mesenchymal Precursor Cells for stimulation of cardiac angiogenesis

[0044] , Neuronyx is in Phase I clinical trials using allogeneic, human adult bone marrow- derived somatic cells (h AB MSC) for post infarct healing

[0045] ,

[0505] MSC are poorly immunogenicity and possess immune modulatory activity

[0046] , features that are conserved among MSC from various tissue sources

[0017] , This weak immunogenicity is believed to permit the survival and activity of allogeneic MSC when administered therapeutically.

[0506] Adipose tissue has also been used clinically as a source of regenerative and immune modulatory MSC. Cytori is currently conducting two European clinical trials using autologous, adipose-derived mononuclear cells, of which MSC are believed to be the therapeutic population

[0047] , The PRECISE trial is a 36-patient safety and feasibility study in Europe evaluating adipose- derived stem and regenerative cells as a treatment for chronic cardiac ischemia. The APOLLO trial is a 48-patient safety and feasibility study in Europe to evaluate adipose-derived regenerative cells as a treatment for heart attacks

[0048] , Allogeneic uses of adipose derived MSC included treatment of GVHD associated liver failure

[0012] and steroid refractory GVHD [13, 49],

[0507] Allogeneic placenta and cord blood-derived MSC have also been used for treatment of heart failure

[0050] and Buerger’s Disease

[0051] , respectively. From the above-mentioned clinical trials of allogeneic MSC, graft versus host or pathological immunological reactions have not been reported. Additionally, administration of MSC intravenously, intrathecally, and intramuscularly have not been associated with ectopic tissue formation or teratoma.

[0508] Administration of human MSC has been shown to accelerate hematopoietic reconstitution in animal models [52, 53], Although the in vivo significance of MSC is still highly debated, one theory is that MSC in the bone marrow provide a suitable environment for hematopoiesis. Accordingly, one ofIMMORTA-SOHASCE-PCT the first clinical uses of MSC has been to accelerate hematopoietic recovery. In a 1995 paper, Lazarus et al. reported the use of autologous, in vitro expanded, “mesenchymal progenitor cells” to treat 15 patients suffering from hematological malignancies in remission. The authors demonstrated feasibility of expanding bone marrow derived by MSC in vitro. They showed that a 10 milliliter bone marrow sample was capable of 16,000-fold growth over a four to seven week in vitro culture period. Cell administration was performed in total doses ranging from 1 - 50 x 106cells and was not causative of treatment associated adverse effects

[0054] , In a subsequent study from the same group in 2000, the use of MSC to accelerate hematopoietic reconstitution was performed in a group of 28 breast cancer patients who received high dose chemotherapy. MSC at concentrations of 1 .0 - 2.2 x 106 / kg were administered intravenously. No treatment associated adverse effects where observed, and leukocytic and thrombocytic reconstitution appeared to undergo “rapid recovery”

[0055] , It is interesting that these initial uses were actually in patients with neoplasia and no overt acceleration of cancer progression was noted. Besides feasibility, these studies were important because they established the technique for ex vivo expansion and readministration.

[0509] Studies along these lines continued which reaffirmed the feasibility of the approach of “repairing bone marrow stroma” with expanded MSC cells. In 2005, Lazarus et al treated 46 patients suffering from hematological malignancies with HLA-matched allografts comprising bone marrow and donor-derived expanded MSC. The numbers of MSC administered were 1 - 5 million I kg. On average, the time to neutrophil reconstitution (as defined by absolute neutrophil count > or = 0.500 x 109 / L) and platelet reconstitution (as defined by platelet count > or = 20 x 109 / L was 14.0 days (range 11 .0 - 26.0 days) and 20 days (range 15.0 - 36.0 days). Incidence of acute, Grade ll-IV GVHD was 13 / 46 and chronic was 22 / 36 patients that survived for at least 90 days. Relapse of malignancy occurred in 11 patients with a median time to progression of 213.5 days (range 14 - 688 days). The authors concluded that cotransplantation of HLA-identical sibling culture-expanded MSCs with an HLA-identical sibling HSC transplant was feasible and safe, without immediate infusional or late MSC- associated toxicities

[0056] , These data were of importance since one of the concerns regarding MSC treatment is associated with growth factor production.IMMORTA-SOHASCE-PCTLeukemic patients have minimally residual disease, which seems to be at least in part controlled by recipient immune function [57, 58], The demonstration that the recipient did not have an overtly higher incidence of relapse suggests that MSC do not endow a preferential advantage to leukemic cells. This is interesting given that MSC are generally considered immune suppressive cells [59, 60], In addition to its stem / progenitor cell content, the SVF is known to contain monocytes / macrophages. Although pluripotency of monocytic populations have previously been described [61 , 62], we will focus our discussion to immunological properties, specifically, the apparent anti-inflammatory / angiogenic activities of these cells. Initial experiments suggested that macrophage content of adipose tissue was associated with the chronic low-grade inflammation found in obese patients. This was suggested by co-culture experiments in which adipocytes were capable of inducing TNF-alpha secretion from macrophage cell lines in vitro

[0063] , Clinical studies demonstrated that adipocytes also directly release a constitutive amount of TNF-alpha and leptin, which are capable of inducing macrophage secretion of inflammatory mediators

[0064] , Interestingly, it appears from several studies in mice and humans that when monocytes / macrophages are isolated from adipose tissue, they in fact possess anti-inflammatory functions characterized by high expression of IL-10 and IL-1 receptor antagonist [65-67], These adipose derived macrophages have an “M2” phenotype, which physiologically is seen in conditions of immune suppression such as in tumors

[0068] , post-sepsis compensatory anti-inflammatory syndrome [69, 70], or pregnancy associated decidual macrophages

[0071] , It is estimated that the monocytic / macrophage compartment of the SVF is approximately 10% based on CD14 expression

[0072] , Interestingly, administrations of ex vivo generated M2 macrophages have been demonstrated to inhibit kidney injury in an adriamycin- induced model

[0073] , In the context of multiple sclerosis, alternatively activated, M2-like microglial cells are believed to inhibit progression in the EAE model

[0074] , Thus the anti-inflammatory activities of M2 cells are a potential mechanism of therapeutic effect of SVF cells when isolated from primary sources and not expanded. It has been reported by us and others, that activation of T cells in the absence of costimulatory signals leads to generation of immune suppressive CD4+ CD25+ T regulatory (Treg) cells [75, 76], Thus local activation of immunity in adipose tissue would theoretically be associated with reduced costimulatoryIMMORTA-SOHASCE-PCT molecule expression by the M2 macrophages, which may predispose to Treg generation. Conversely, it is known that Tregs are involved in maintaining macrophages in the M2 phenotype

[0077] , Supporting the possibility of Treg in adipose tissue also comes from the high concentration of local MSC which are known to secrete TGF-beta

[0078] and IL-10

[0079] , both involved in Treg generation

[0080] , Indeed numerous studies have demonstrated the ability of MSC to induce Treg cells [79, 81-83], In AA, Treg are functionally abnormal and their numbers are significantly reduced, whereas pro-inflammatory Th1 and Th17 cells are numerically increased. It is believed that the clonally restricted T cell populations that are pro-inflammatory preferentially expand in AA patients, thereby further suppressing the activity of Treg. Therefore, Treg contained within the SVF fraction could serve as a source of immune regulatory cells to counter the pro- inflammatory responses that underlie AA pathophysiology. Over the past two decades the endothelium has received significant attention as adynamic surface cell that acts as an adaptable, anti-coagulated barrier between the blood stream and interior of the blood vessel. This allows for selective transmigration of cells in and out of the blood stream, regulates blood flow through controlling smooth muscle contraction, and participates in tissue remodeling and angiogenesis [84- 88], Endothelial cells are believed to originate from a primitive stem cell, the hemangioblast, which is capable of giving rise to both hematopoietic and endothelial cells

[0089] , During adulthood, the endothelium is continually selfrenewed by a population of bone marrow-derived cells termed endothelial progenitor cells (EPC). This progenitor population has previously been characterized as expressing the CD34 HSC marker as well as VEGF-receptor 2 and AC133

[0090] , These cells have been demonstrated using in vivo chimeric models to repair damaged blood vessels in non-diseased

[0091] as well as in pathological settings [92, 93],

[0510] Given the importance of endothelial cells in so many aspects of biological systems, it would be reasonable to explore the ways in which endothelial cells provide support for hematopoietic processes. Specifically, the observation that hematopoietic and endothelial cells originate developmentally from a common precursor may suggest that in adulthood these cells are interrelated. The original experiments highlighting the interaction between hematopoietic cells and endothelial cells were studies which attempted toIMMORTA-SOHASCE-PCT recapitulate hematopoiesis in vitro. Early experiments demonstrated that an endothelial cell layer was essential as part of the “stroma” for in vitro hematopoiesis [94, 95], Interestingly, soluble factors generated by endothelial cells that supported in vitro hematopoiesis were not only identified

[0096] , but it was demonstrated that their production was inducible by various agents such as lipopolysaccharide

[0097] , This suggests that the endothelium was an inducible source of factors stimulating hematopoiesis when physiologically necessary, such as during infection

[0098] , Detailed characterization of the role of endothelium in hematopoiesis was performed initially by anatomical studies, which defined the sinusoidal aspects of the bone marrow hematopoietic endothelium [99, 100], Morphological changes of this specialized endothelium have been identified during times of excessive production of various blood cells

[0101] , hinting at a possible involvement in the process of hematopoiesis

[0102] , During times of inflammation, structural changes occur in the bone marrow endothelium, in part to support release of granulocytes

[0103] , Such changes also occur in response to administration of bone marrow stem cell mobilizing agents, such as Neupogen, which causes stimulation of localized complement activation through activation of proteases that expose neoepitopes in the bone marrow microenvironment [104, 105], Because of the physical proximity of HSC and endothelial cells, as well as the ability of endothelial cells to support hematopoiesis in vitro, investigators have sought to identify molecular means by which endothelial cells may control hematopoiesis.

[0511] One of the major goals of hematology research has been the development of methods of expanding hematopoietic stem cells outside of the body. This would hypothetically allow for various approaches to purging leukemic cells out of autologous grafts while expanding non-malignant hematopoietic cells, as well as expanding cord blood stem cells in order to accelerate engraftment after transplantation [106-108], As previously discussed, early studies have used bone marrow stromal cells for expanding hematopoietic stem cells

[0109] , Components of the bone marrow stroma include monocytes, adipocytes, and mesenchymal stem cells

[0110] , An interesting finding was that endothelial cells, whether originating from bone marrow, brain, or fat, all possessed the ability to stimulate hematopoietic cell expansion. Specifically, Davis et al

[0111] examined the ability of porcine microvascular endothelial cells (PMVECs) together withIMMORTA-SOHASCE-PCT combinations of cytokines (GM-CSF, IL-3, SCF, IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In seven-day cultures, the greatest HSC expansion was observed when the HSC were in direct contact with PMVEC monolayers, followed by PMVEC noncontact and liquid suspension cultures. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM- CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocytemacrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. These experiments demonstrated the efficacy of endothelial cells to stimulate HSC proliferation. Also, such factors are not species-specific, thus enabling animal experiments with human cells. These studies were also confirmed in another paper

[0112] ,

[0512] Other studies have associated endothelial cell production of IL-6, SCF, G-CSF and GM-CSF ex vivo HSC expansion

[0113] , The expansion of HSC by endothelial cells has been shown to accelerate bone marrow engraftment in vivo

[0114] , Acceleration of hematopoietic recovery has been demonstrated not only with endothelial cells but also with conditioned media of these cells, suggesting both contact dependent and contact independent effects

[0115] , Ex vivo expansion of HSC using endothelial cells was demonstrated to generate HSC that were functional in a large animal model

[0116] , specifically, a control baboon received no transplant and two animals that received a suboptimal number of marrow mononuclear cells died 37, 43, and 59 days post-irradiation. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in porcine microvascular endothelial cells (PMVEC) co-cultured with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte- macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone areaforming cells (CAFC) over input. Both baboons receiving the treatment became transfusion independent by day 23 post-transplant and achieved absoluteIMMORTA-SOHASCE-PCT neutrophil count ANC >5001 microL by day 25 + 1 and platelets > 20,0001 microL by day 29 + 2.

[0513] In addition to expanding functional HSC ex vivo, the direct injection of exogenous endothelial cells has demonstrated hematopoietic stimulatory effects. Salter et al treated BALB / c mice with total body irradiation (TBI) followed by infusion with C57BI6-derived endothelial progenitor cells (EPCs). TBI caused pronounced disruption of the BM vasculature, BM hypocel lularity , ablation of HSCs, and pancytopenia in control mice. Irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti-VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non-EPC- treated mice compared with irradiated controls

[0117] , Such hematopoietic stimulatory effects were observed with endothelial cells from a variety of sources. For example, it has been shown that both brain and fetal blood derived endothelial cells have the ability to endow radioprotection, as well as to accelerate reconstitution in C57BI6 mice treated with 1050 cGy of radiation

[0118] , In another series of experiments, Fleming’s group demonstrated that transplantation of segments of adult thoracic aorta or inferior vena cava under the kidney capsule of lethally irradiated recipients (1100 cGy) resulted in significant radioprotection. Specifically, 10 mg of transplanted vascular tissue would protect 80% of recipients from lethality. Furthermore, this procedure gave rise to similar numbers of colony forming units as rescue using 105bone marrow cells and prevented the development of severe anemia. Labeling of proliferating cells using BRDll revealed that the cells within the intima of donor vascular tissue would begin proliferation within 48 hours of transplantation. It was also demonstrated in cell tracking studies that donor-derived vascular cells migrated to the recipient spleen; hematopoietic colony forming units were of host origin. Although donor-derived cells were readily detected in the peripheral blood two to three weeks after transplant, they rapidly declined in frequency to approximately 1 .0% by four weeks and persisted at these levels for more than one year. Bone marrow from rescued primary recipients provided radioprotection after transplantation into secondary recipients, but only CD3(+) donor-derived cells were detected

[0119] , The question remained from these experiment aboutIMMORTA-SOHASCE-PCT whether similar hematopoietic effects could be observed by endothelial cells alone or if they require other vascular cells that mediate therapeutic effects. This was addressed in another paper by the same group which assessed vascular endothelial cells administered in suspension. The investigators found that as little as 104endothelial cells either isolated from the murine lung or brain were capable of radioprotecting mice

[0120] , Furthermore, endothelial cell administration was associated with reconstitution of host hematopoiesis, with the expanded cells being able to transfer hematopoiesis to secondary recipients.

[0514] The most practical embodiment of the concept that endothelial cells stimulate hematopoiesis in vivo would be the utilization of autologous, adipose derived, endothelial cells. In this system, it may be useful to utilize the whole stromal vascular fraction (SVF) as a heterogenous cellular product. SVF is comprised of the mononuclear cells derived from adipose tissue and are known to contain not only endothelial cells, but also T regulatory cells, monocytes, and hematopoietic stem cells. This term is more than four decades old and is used to describe the mitotically active source of adipocyte precursors [25, 26], SVF as a source of stem cells was first described by Zuk et al who identified mesenchymal- like stem cells cells in SVF that could be induced to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic lineages

[0028] , Subsequent to the initial description, the same group reported after in vitro expansion the SVF derived cells had surface marker expression similar to bone marrow derived MSC, comprising of positive for CD29, CD44, CD71 , CD90, CD105 / SH2, and SH3 and lacking CD31 , CD34, and CD45 expression [29, 121],

[0515] Liposuction is performed routinely and numerous protocols exist for autologous collection and administration of SVF, which contains all of the cellular elements mentioned. In fact, safety of this procedure has been previously published for rheumatoid arthritis in a 13 patient study in which patients were monitored for over one year

[0122] , Several other studies have demonstrated a high content of EPC in adipose tissue [123, 124], Functional demonstration of adipose EPC was performed in experiments in which SVF was purified for CD34 positive cells. This fraction was demonstrated to induce angiogenesis in immune compromised mice that were subjected to hindlimb ischemia. Mechanistically, the cells were identified as EPC based on ability to form endothelial colonies when cultured in vitro

[0125] , Numerous groups have reported SVF containsIMMORTA-SOHASCE-PCT cellular activity stimulatory of angiogenesis. For example, Sumi et al showed that administration of SVF but not adipocytes led to revascularization in the hindlimb ischemia model

[0126] , Other studies have shown that EPC-like activities are found in SVF

[0127] , and also that conditioned media from SVF is capable of stimulating host angiogenesis [128, 129], It is reported that EPC in the SVF capable of stimulating angiogenesis directly or through release of growth factors such as IGF-1 , HGF-1 and VEGF [127, 129-131],

[0516] Use of non-expanded SVF is commonplace in veterinary medicine and has been shown to be safe and effective. Pioneered by the commercial company Vet-Stem, double blind studies of SVF administration to treat canine osteoarthritis have shown statistically significant improvements in lameness, range of motion, and overall quality of life after treatment with autologous SVF [19, 20], To date Vet-Stem has treated over 5,000 canines using local, intraarticular administration as well as systemic intravenous administration of these cells. Additionally, it is reported that over 3,000 horses with various cartilage and bone injuries have been treated with autologous lipoaspirate fractions without cellular expansion

[0018] , These studies have not only demonstrated safety and in some cases efficacy, but also have established the practical foundation of commercialized stem cell therapeutics

[0132] , For clinical use closed system point-of-care devices have been developed by the companies Cytori and Tissue Genesis to allow for rapid processing of adipose tissue, without need for a Good Manufacturing Practices compliant laboratory [21 , 22], In the published literature, the clinical use of intravaneously administered SVF cells has been reported in three studies. The first study was a description of 3 patients suffering from multiple sclerosis who received intravenous administration of autologous adipose SVF. All 3 patients reported significant improvement neurologically and demonstrated a good safety profile

[0133] , In another paper, a single patient case report described a remission of rheumatoid arthritis

[0134] , More recently, a paper demonstrating one year safety of rheumatoid arthritis patients treated with autologous SVF was published

[0122] , The general safety of fat grafting is widely accepted, as this is a common procedure in cosmetic surgery [23, 24],

[0517] Administration of the inhibitor of senescent cells to the donor subject is prior to harvest of the HSPC. Administration may be in a single dose, or by way of multiple separate administrations over a period of time, beginning at a definedIMMORTA-SOHASCE-PCT time point prior to harvest. In one embodiment, the inhibitor is administered to the subject for a period of from about 1 day to about 5 days (e.g., about 5, 4, 3, 2 or 1 days) prior to harvest of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 5 days to about 10 days (e.g., about 10, 9, 8, 7, or 6 days) prior to harvest of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 10 days to about 20 days (e.g., about 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 days) prior to harvest of the HSPC. In one embodiment, the inhibitor is administered to the subject for a period of from about 20 days to about 30 days (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 days) prior to harvest of the HSPC. Donor cells may be obtained from any suitable source from the donor, examples of which are described herein.

[0518] The results presented herein also indicate that treatment of the HSPC after harvest but prior to administration (ex vivo) with the inhibitor of senescent cells will also enhance expansion of the early hematopoietic progenitor cells. Such expansion is beneficial to the recipient subject and will accelerate posttransplant recovery, as described herein. In one embodiment of the invention, senolytic agents can be used that include: a) kinase inhibitors such as dasatinib

[0135] , nintedanib [136-139], Terreic acid

[0140] ; b) bcl-2 family protein inhibitors; c) naturally occurring polyphenols; d) heat shock protein inhibitors; e) BET family protein inhibitors; f) p53 stabilizers; g) repurposed anticancer drugs; h) cardiac steroids; and i) antibiotics.EXAMPLESAcceleration of Neutrophil Recovery After SenoVax Administration

[0519] BALB / c mice (female, 10 per group) were treated with a) saline (control); b) 1 mg / kg cyclophosphamide by intraperitoneal administration of the substance diluted in phosphate buffered saline (Cyc); c) 1 million dendritic cells loaded with non-senescent fibroblast lyaste; and d) 1 million dendritic cells pulsed with senescent fibroblasts (SenoVax) cells. Neutrophil counts were measured at days 7, 14, and 21 by Coulter Counter.

[0520] SenoVax (Dendritic Cells pulsed with Senescent Fibroblast Lysate) was prepared as follows: Bone marrow mononuclear cells were isolated fromIMMORTA-SOHASCE-PCTBALB / c mice. Cells were extracted from femurs and tibia by isolation of bone marrow cords through flushing bones with saline. Cords were dissociated over a sterile mesh to remove debris or aggregates and single cells were diluted in 12 ml of phosphate buffered saline. Cells were spun for 15 minutes at 1000g. The supernatant was decanted and the pellet was resuspended by gentle tapping. 2 ml of lysis buffer (ThermoFisher, Carlsbad, CA) was added to the pellet for 3 minutes after which 10 ml of phosphate buffered saline was added. Cells were washed twice and subsequently mononuclear cells were plated in 20 ml of DMEM media with 10% fetal calf serum and GM-CSF (100 lU / ml) and IL-4 (100 lll / ml). Cells were incubated in a fully humidified atmosphere with 5 % carbon dioxide. Media was changed every second day. This generates CD80 and CD86 expressing dendritic cells at 7 days of culture. Concentration of cells was 100,000 per ml. Lysate of senescent dermal fibroblasts of BALB / c origin were used. The concentration added was 10 ug of lysate per million cells.

[0521] BALB / c dermal fibroblasts were obtained by shaving a 1 x 1 centimeter portion of the mouse dermis and surgically removing the skin. The excised skin was subsequently cut into 1 x 1 millimeter pieces using surgical scissors and incubated in RPMI containing 100 microgram per ml collagenase for 20 minutes. Cells were first isolated by passing through the sterile mesh and subsequently washed in phosphate buffered saline. Adherent cells were growth for 2 weeks in the presence of 5 ug / ml of doxorubicin. Cells expressing more than 80% beta galactosidase were considered “senescent” and lyzed by 10 freeze-thaw cycles in liquid nitrogen. Lysate was passed through a 2 micron filtrate to removed debris and used to pulse dendritic cells.

[0522] A mouse dendritic cell (DC) vaccine using senescent fibroblasts as antigens, begin by inducing senescence in mouse fibroblasts (e.g., NIH / 3T3 or primary dermal fibroblasts). Seed fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1% penicillin / streptomycin) and incubate overnight at 37°C, 5% CO2 to allow attachment. Treat the cells with 0.5 pM doxorubicin (from a 1 mM stock in DMSO) for 24 hours, then wash three times with sterile PBS to remove the drug. Replace with fresh complete DMEM and culture for 7 days, refreshing the medium every 2-3 days. Confirm senescence by observing >70% cells positive for SA-[3-gal staining (blue under a microscope) or by detecting increased p16 / p21 expression via qPCR or WesternIMMORTA-SOHASCE-PCT blot, if desired. Next, prepare the senescent fibroblast lysate by harvesting the cells with trypsin-EDTA, washing with PBS, and resuspending at 1 x 1 OA7 cells / ml in PBS. Subject the cells to six freeze-thaw cycles (10 minutes in liquid nitrogen or -80°C, followed by 5 minutes in a 37°C water bath) to lyse them. Centrifuge the lysate at 12,000 x g for 10 minutes at 4°C to remove debris, collect the supernatant, quantify the protein content (target 1 -5 mg / ml using a BCA assay), and store aliquots at -80°C.For DC generation, harvest bone marrow from the femurs and tibias of 6-8 week old mice (e.g., C57BL / 6) euthanized per institutional guidelines. Flush the bones with 10 ml complete RPMI (RPMI-1640 with 10% heat-inactivated FBS and 1 % penicillin / streptomycin) using a 25G needle and syringe, filter through a 70-pm cell strainer, and centrifuge at 300 x g for 5 minutes. Lyse red blood cells by resuspending the pellet in 5 ml ACK buffer for 5 minutes at room temperature, quench with 10 ml RPMI, and centrifuge again. Resuspend the cells at 2-4 x 10A6 cells / ml in 10 ml complete RPMI supplemented with 20 ng / ml recombinant mouse GM-CSF and 20 ng / ml IL-4, and plate in 10-cm non-tissue culture dishes. Incubate at 37°C, 5% CO2. On day 3, add 10 ml fresh medium with 40 ng / ml each of GM-CSF and IL-4. On day 5, remove half the medium, centrifuge floating cells, resuspend in fresh medium with cytokines, and return to the dish. On day 7, harvest loosely adherent immature DCs, which should yield approximately 1 -2 x 10A7 cells per mouse, with >80% CD11 c+ by flow cytometry.To pulse the DCs, resuspend the immature DCs at 1 -5 x 1 OA6 cells / ml in complete RPMI and add senescent fibroblast lysate at 100-500 pg protein per 10A6 DCs. Incubate for 18- 24 hours at 37°C to allow antigen uptake and processing. Optionally, add 100 ng / ml LPS for the last 6 hours to mature the DCs for enhanced immunogenicity. Wash the pulsed DCs three times with PBS, resuspend at 1 x 1 OA7 cells / ml in PBS, and the vaccine is ready for in vivo administration (e.g., 1 -5 x 1 OA6 DCs per mouse, subcutaneous or intravenous). Ensure all procedures are performed under sterile conditions in a biosafety cabinet, and obtain IACUC approval for animal work. Validate vaccine efficacy through T-cell activation assays or tumor challenge models, and scale up as needed based on mouse strain and experimental requirements.

[0523] To generate the mouse dendritic cell (DC) vaccine using senescent fibroblasts as antigens (SenoVax), mouse fibroblasts (e.g., NIH / 3T3 or primaryIMMORTA-SOHASCE-PCT dermal fibroblasts) were seeded at 5 x 1 OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1 % penicillin / streptomycin) and incubated overnight at 37°C, 5% CO2 to allow attachment. The cells were treated with 0.5 pM doxorubicin (from a 1 mM stock in DMSO) for 24 hours, then washed three times with sterile PBS to remove the drug. Fresh complete DMEM was added, and the cells were cultured for 7 days, with the medium refreshed every 2-3 days. Senescence was confirmed by observing >70% cells positive for SA-[3-gal staining (blue under a microscope) or by detecting increased p16 / p21 expression via qPCR or Western blot, if performed. The senescent fibroblast lysate was prepared by harvesting the cells with trypsin-EDTA, washing with PBS, and resuspending at 1 x 1OA7 cells / ml in PBS. The cells were subjected to six freezethaw cycles (10 minutes in liquid nitrogen or -80°C, followed by 5 minutes in a 37°C water bath) to lyse them. The lysate was centrifuged at 12,000 x g for 10 minutes at 4°C to remove debris, the supernatant was collected, the protein content was quantified (targeting 1 -5 mg / ml using a BCA assay), and aliquots were stored at -80°C.For DC generation, bone marrow was harvested from the femurs and tibias of 6-8 week old mice (e.g., C57BL / 6) euthanized per institutional guidelines. The bones were flushed with 10 ml complete RPMI (RPMI-1640 with 10% heat-inactivated FBS and 1 % penicillin / streptomycin) using a 25G needle and syringe, filtered through a 70-pm cell strainer, and centrifuged at 300 x g for 5 minutes. Red blood cells were lysed by resuspending the pellet in 5 ml ACK buffer for 5 minutes at room temperature, quenched with 10 ml RPMI, and centrifuged again. The cells were resuspended at 2-4 x 1 OA6 cells / ml in 10 ml complete RPMI supplemented with 20 ng / ml recombinant mouse GM-CSF and 20 ng / ml IL-4 and plated in 10-cm non-tissue culture dishes. The cells were incubated at 37°C, 5% CO2. On day 3, 10 ml of fresh medium with 40 ng / ml each of GM-CSF and IL-4 was added. On day 5, half the medium was removed, floating cells were centrifuged, resuspended in fresh medium with cytokines, and returned to the dish. On day 7, loosely adherent immature DCs were harvested, yielding approximately 1 -2 x 1 OA7 cells per mouse, with >80% CD11c+ by flow cytometry.To pulse the DCs, the immature DCs were resuspended at 1 -5 x 1 OA6 cells / ml in complete RPMI, and senescent fibroblast lysate was added at 100-500 pg protein per 10A6 DCs. The cells were incubated for 18-24 hours at 37°C to allow antigen uptake and processing. In some cases, 100 ng / ml LPS was addedIMMORTA-SOHASCE-PCT for the last 6 hours to mature the DCs for enhanced immunogenicity. The pulsed DCs were washed three times with PBS, resuspended at 1 x 1OA7 cells / ml in PBS, and the vaccine was prepared for in vivo administration (e.g., 1-5 x 1OA6 DCs per mouse, subcutaneous or intravenous). All procedures were performed under sterile conditions in a biosafety cabinet, and IACUC approval was obtained for animal work. Vaccine efficacy was validated through T-cell activation assays or tumor challenge models, with scaling adjusted based on mouse strain and experimental requirements. Results are shown in FIG. 1 .Acceleration of Neutrophil Recovery After SenoVax Administration

[0524] BALB / c mice (female, 10 per group) were treated with a) 1 mg / kg cyclophosphamide by intraperitoneal administration of the substance diluted in phosphate buffered saline (Cyc); b) G-CSF every second day 100 ng / mouse); c) 1 million SenoVax cells and d) SenoVax cells with G-CSF. Neutrophil counts were measured at days 7, 14, and 21 by Coulter Counter. Results are shown in FIG. 2.Example 3: Enhanced Platelet Recovery After Cyclophosphamide using Senolytic ImmunotherapyTo assess the recovery of platelets following cyclophosphamide-induced thrombocytopenia and the potential acceleration by senolytic immunotherapy using a dendritic cell vaccine (Senovax), an experimental protocol was implemented using 6-8 week old C57BL / 6 mice (n=10 per group, balanced for sex). Mice were housed under specific pathogen-free conditions with ad libitum access to food and water, and all procedures were approved by the institutional animal care and use committee (IACUC). Senescent fibroblasts were induced by seeding NIH / 3T3 mouse fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1 % penicillin / streptomycin) and incubating overnight at 37°C, 5% CO2 for attachment. Cells were treated with 0.5 pM doxorubicin for 24 hours, washed three times with PBS, and cultured in fresh complete DMEM for 7 days with medium changes every 2-3 days; senescence was confirmed by >70% SA-[3-gal positivity. Lysates from senescent and nonsenescent (control) fibroblasts were prepared by harvesting cells with trypsin-IMMORTA-SOHASCE-PCTEDTA, resuspending at 1 x 1OA7 cells / ml in PBS, subjecting to six freeze-thaw cycles, centrifuging at 12,000 x g for 10 minutes at 4°C, quantifying protein (1-5 mg / ml via BCA assay), and storing at -80°C. Bone marrow-derived dendritic cells (BMDCs) were generated by flushing femurs and tibias with complete RPMI, lysing red blood cells with ACK buffer, and plating 2-4 x 1OA6 cells / ml in complete RPMI with 20 ng / ml GM-CSF and IL-4. Medium with cytokines was added on day 3, half-replaced on day 5, and immature DCs were harvested on day 7. DCs were pulsed with 100-500 pg lysate protein per 10A6 cells for 18-24 hours (with optional LPS maturation for the last 6 hours), washed, and resuspended at 1 x 10A7 cells / ml in PBS. Four groups were established: Group 1 (no intervention) received saline only; Group 2 (cyclophosphamide only) received a single intraperitoneal injection of cyclophosphamide (200 mg / kg) on day 0; Group 3 (cyclophosphamide plus DCs loaded with fibroblast lysate) received cyclophosphamide on day 0 followed by intravenous injection of 2 x 1OA6 pulsed DCs (normal fibroblast lysate) on days 1 , 3, and 5; Group 4 (cyclophosphamide plus DCs loaded with senescent fibroblast lysate, i.e., Senovax) received cyclophosphamide on day 0 followed by the same DC dosing schedule with senescent lysate-pulsed DCs. Blood was collected via tail vein at baseline and weeks 2, 4, and 6 post-cyclophosphamide; platelet counts were measured using an automated hematology analyzer (e.g., Sysmex XN-1000) in duplicate, with technicians blinded to group assignments. Data were analyzed for statistical significance using one-way ANOVA with Tukey's post-hoc test (p<0.05 considered significant). Results indicated baseline platelet counts were similar across groups (approximately 950 ± 50 xA3 / pL). Cyclophosphamide induced thrombocytopenia in Groups 2-4, with nadir counts around day 7 (not shown). Platelet recovery was markedly accelerated in Group 4 (Senovax) compared to Groups 2 and 3, achieving near-baseline levels by week 4, while Groups 2 and 3 showed delayed recovery. Group 1 remained stable. Table 1 below summarizes platelet counts (x 10A3 / pL) at weeks 2, 4, and 6.IMMORTA-SOHASCE-PCT

[0525] Table 1Example 4: Enhanced Platelet Recovery After Adriamycin using Senolytic Immunotherapy

[0526] To assess the recovery of platelets following adriamycin (doxorubicin)-induced thrombocytopenia and the potential acceleration by senolytic immunotherapy using a dendritic cell vaccine (Senovax), an experimental protocol was implemented using 6-8 week old C57BL / 6 mice (n=10 per group, balanced for sex). Mice were housed under specific pathogen-free conditions with ad libitum access to food and water, and all procedures were approved by the institutional animal care and use committee (IACUC). Senescent fibroblasts were induced by seeding NIH / 3T3 mouse fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1 % penicillin / streptomycin) and incubating overnight at 37°C, 5% CO2 for attachment. Cells were treated with 0.5 pM doxorubicin for 24 hours, washed three times with PBS, and cultured in fresh complete DMEM for 7 days with medium changes every 2-3 days; senescence was confirmed by >70% SA-[3-gal positivity. Lysates from senescent and non-senescent (control) fibroblasts were prepared by harvesting cells with trypsin-EDTA, resuspending at 1 x 1OA7 cells / ml in PBS, subjecting to six freeze-thaw cycles, centrifuging at 12,000 x g for 10 minutes at 4°C, quantifying protein (1-5 mg / ml via BCA assay), and storing at -80°C. Bone marrow-derived dendritic cells (BMDCs) were generated by flushing femurs and tibias with complete RPMI, lysing red blood cells with ACK buffer, and plating 2-4 x 1OA6 cells / ml in complete RPMI with 20 ng / ml GM-CSF and IL-4. Medium with cytokines was added on day 3, half-replaced on day 5,IMMORTA-SOHASCE-PCT and immature DCs were harvested on day 7. DCs were pulsed with 100-500 pg lysate protein per 10A6 cells for 18-24 hours (with optional LPS maturation for the last 6 hours), washed, and resuspended at 1 x 1 OA7 cells / ml in PBS. Four groups were established: Group 1 (no intervention) received saline only; Group 2 (adriamycin only) received a single intraperitoneal injection of adriamycin (doxorubicin, 20 mg / kg) on day 0; Group 3 (adriamycin plus DCs loaded with fibroblast lysate) received adriamycin on day 0 followed by intravenous injection of 2 x 1 OA6 pulsed DCs (normal fibroblast lysate) on days 1 , 3, and 5; Group 4 (adriamycin plus DCs loaded with senescent fibroblast lysate, i.e., Senovax) received adriamycin on day 0 followed by the same DC dosing schedule with senescent lysate-pulsed DCs. Blood was collected via tail vein at baseline and weeks 2, 4, and 6 post-adriamycin; platelet counts were measured using an automated hematology analyzer (e.g., Sysmex XN-1000) in duplicate, with technicians blinded to group assignments. Data were analyzed for statistical significance using one-way ANOVA with Tukey's post-hoc test (p<0.05 considered significant). Results indicated baseline platelet counts were similar across groups (approximately 950 ± 50 xA3 / pL). Adriamycin induced thrombocytopenia in Groups 2-4, with nadir counts around day 7 (not shown). Platelet recovery was markedly accelerated in Group 4 (Senovax) compared to Groups 2 and 3, achieving near-baseline levels by week 4, while Groups 2 and 3 showed delayed recovery. Group 1 remained stable. Table 2 below summarizes platelet counts (x 10A3 / pL) at weeks 2, 4, and 6.

[0527] Table 2IMMORTA-SOHASCE-PCTExample 5: Protection from Adriamycin Induced Cardiotoxicity

[0528] To assess the protective effect of senolytic immunotherapy using a dendritic cell vaccine (Senovax) on heart function following adriamycin (doxorubicin)-induced cardiotoxicity, an experimental protocol was implemented using 6-8 week old C57BL / 6 mice (n=10 per group, balanced for sex). Mice were housed under specific pathogen-free conditions with ad libitum access to food and water, and all procedures were approved by the institutional animal care and use committee (IACUC). Senescent fibroblasts were induced by seeding NIH / 3T3 mouse fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1 % penicillin / streptomycin) and incubating overnight at 37°C, 5% CO2 for attachment. Cells were treated with 0.5 pM doxorubicin for 24 hours, washed three times with PBS, and cultured in fresh complete DMEM for 7 days with medium changes every 2-3 days; senescence was confirmed by >70% SA- [3-gal positivity. Lysates from senescent and non-senescent (control) fibroblasts were prepared by harvesting cells with trypsin-EDTA, resuspending at 1 x 1OA7 cells / ml in PBS, subjecting to six freeze-thaw cycles, centrifuging at 12,000 x g for 10 minutes at 4°C, quantifying protein (1-5 mg / ml via BCA assay), and storing at -80°C. Bone marrow-derived dendritic cells (BMDCs) were generated by flushing femurs and tibias with complete RPMI, lysing red blood cells with ACK buffer, and plating 2-4 x 1OA6 cells / ml in complete RPMI with 20 ng / ml GM-CSF and IL-4. Medium with cytokines was added on day 3, half-replaced on day 5, and immature DCs were harvested on day 7. DCs were pulsed with 100-500 pg lysate protein per 10A6 cells for 18-24 hours (with optional LPS maturation for the last 6 hours), washed, and resuspended at 1 x 1OA7 cells / ml in PBS. Four groups were established: Group 1 (no intervention) received saline only; Group 2 (adriamycin only) received a single intraperitoneal injection of adriamycin (doxorubicin, 20 mg / kg) on day 0; Group 3 (adriamycin plus DCs loaded with fibroblast lysate) received adriamycin on day 0 followed by intravenous injection of 2 x 1OA6 pulsed DCs (normal fibroblast lysate) on days 1 , 3, and 5; Group 4 (adriamycin plus DCs loaded with senescent fibroblast lysate, i.e., Senovax) received adriamycin on day 0 followed by the same DC dosing schedule with senescent lysate-pulsed DCs. Heart function was evaluated via transthoracic echocardiography (e.g., using a Vevo 2100 system) under isoflurane anesthesiaIMMORTA-SOHASCE-PCT at baseline and weeks 2, 4, and 6 post-adriamycin; left ventricular ejection fraction (LVEF) was measured from M-mode images in the parasternal short-axis view, with operators blinded to group assignments. Data were analyzed for statistical significance using one-way ANOVA with Tukey's post-hoc test (p<0.05 considered significant). Results indicated baseline LVEF values were similar across groups (approximately 70 ± 3%). Adriamycin induced cardiotoxicity in Groups 2-4, manifesting as progressive decline in LVEF, but Group 4 (Senovax) showed significant protection, maintaining LVEF near baseline levels throughout, while Groups 2 and 3 exhibited marked heart failure with reduced systolic function. Group 1 remained stable. Table 3 below summarizes LVEF (%) at weeks 2, 4, and 6.

[0529] Table 3Example 6: Protection from Doxorubicin Induced Cardiotoxicity

[0530] To assess the protective effect of senolytic immunotherapy using a dendritic cell vaccine (Senovax) on heart function following doxorubicin-mediated cardiac toxicity, an experimental protocol was implemented using 6-8 week old C57BL / 6 mice (n=10 per group, balanced for sex). Mice were housed under specific pathogen-free conditions with ad libitum access to food and water, and all procedures were approved by the institutional animal care and use committee (IACUC). Senescent fibroblasts were induced by seeding NIH / 3T3 mouse fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1% penicillin / streptomycin) and incubating overnight at 37°C, 5% CO2 for attachment. Cells were treated with 0.5 pM doxorubicin for 24 hours, washed three times with PBS, and cultured in fresh complete DMEM for 7 days with medium changes every 2-3 days; senescence was confirmed by >70% SA-[3-galIMMORTA-SOHASCE-PCT positivity. Lysates from senescent and non-senescent (control) fibroblasts were prepared by harvesting cells with trypsin-EDTA, resuspending at 1 x 1 OA7 cells / ml in PBS, subjecting to six freeze-thaw cycles, centrifuging at 12,000 x g for 10 minutes at 4°C, quantifying protein (1 -5 mg / ml via BCA assay), and storing at -80°C. Bone marrow-derived dendritic cells (BMDCs) were generated by flushing femurs and tibias with complete RPMI, lysing red blood cells with ACK buffer, and plating 2-4 x 1 OA6 cells / ml in complete RPMI with 20 ng / ml GM-CSF and IL-4. Medium with cytokines was added on day 3, half-replaced on day 5, and immature DCs were harvested on day 7. DCs were pulsed with 100-500 pg lysate protein per 10A6 cells for 18-24 hours (with optional LPS maturation for the last 6 hours), washed, and resuspended at 1 x 1 OA7 cells / ml in PBS. Four groups were established: Group 1 (no intervention) received saline only; Group 2 (doxorubicin only) received a single intraperitoneal injection of doxorubicin (20 mg / kg) on day 0; Group 3 (doxorubicin plus DCs loaded with fibroblast lysate) received doxorubicin on day 0 followed by intravenous injection of 2 x 1 OA6 pulsed DCs (normal fibroblast lysate) on days 1 , 3, and 5; Group 4 (doxorubicin plus DCs loaded with senescent fibroblast lysate, i.e., Senovax) received doxorubicin on day 0 followed by the same DC dosing schedule with senescent lysate-pulsed DCs. Heart function was evaluated via transthoracic echocardiography (e.g., using a Vevo 2100 system) under isoflurane anesthesia at baseline and weeks 2, 4, and 6 post-doxorubicin; left ventricular ejection fraction (LVEF) was measured from M-mode images in the parasternal short-axis view, with operators blinded to group assignments. Data were analyzed for statistical significance using one-way ANOVA with Tukey's post-hoc test (p<0.05 considered significant). Results indicated baseline LVEF values were similar across groups (approximately 70 ± 3%). Doxorubicin induced cardiac toxicity in Groups 2-4, manifesting as progressive decline in LVEF, but Group 4 (Senovax) showed significant protection, maintaining LVEF near baseline levels throughout, while Groups 2 and 3 exhibited marked heart failure with reduced systolic function. Group 1 remained stable. Table 4 below summarizes LVEF (%) at weeks 2, 4, and 6.IMMORTA-SOHASCE-PCT

[0531] Table 4Example ?: Protection from Doxorubicin Induced Neurotoxicity

[0532] To evaluate the protective effect of senolytic immunotherapy using a dendritic cell vaccine (Senovax) against doxorubicin-induced neurotoxicity, an experimental protocol was conducted using 6-8 week old C57BL / 6 mice (n=10 per group, balanced for sex). Mice were housed under specific pathogen-free conditions with ad libitum access to food and water, and all procedures were approved by the institutional animal care and use committee (IACUC). Senescent fibroblasts were induced by seeding NIH / 3T3 mouse fibroblasts at 5 x 1OA5 cells per 10-cm dish in complete DMEM (DMEM with 10% FBS and 1 % penicillin / streptomycin) and incubating overnight at 37°C, 5% CO2 for attachment. The fibroblasts were treated with 0.5 pM doxorubicin for 24 hours, washed three times with PBS, and cultured in fresh complete DMEM for 7 days with medium changes every 2-3 days; senescence was confirmed by >70% SA- [3-gal positivity. Lysates from senescent and non-senescent (control) fibroblasts were prepared by harvesting cells with trypsin-EDTA, resuspending at 1 x 1OA7 cells / ml in PBS, subjecting to six freeze-thaw cycles, centrifuging at 12,000 x g for 10 minutes at 4°C, quantifying protein (1-5 mg / ml via BCA assay), and storing at -80°C. Bone marrow-derived dendritic cells (BMDCs) were generated by flushing femurs and tibias with complete RPMI, lysing red blood cells with ACK buffer, and plating 2-4 x 1OA6 cells / ml in complete RPMI with 20 ng / ml GM-CSF and IL-4. Medium with cytokines was added on day 3, half-replaced on day 5, and immature DCs were harvested on day 7. DCs were pulsed with 100-500 pg lysate protein per 10A6 cells for 18-24 hours (with optional LPS maturation for the last 6 hours), washed, and resuspended at 1 x 1OA7 cells / ml in PBS. Four groupsIMMORTA-SOHASCE-PCT were established: Group 1 (no intervention) received saline only; Group 2 (doxorubicin only) received a single intraperitoneal injection of doxorubicin (20 mg / kg) on day 0; Group 3 (doxorubicin plus DCs loaded with fibroblast lysate) received doxorubicin on day 0 followed by intravenous injection of 2 x 1OA6 pulsed DCs (normal fibroblast lysate) on days 1 , 3, and 5; Group 4 (doxorubicin plus DCs loaded with senescent fibroblast lysate, i.e., Senovax) received doxorubicin on day 0 followed by the same DC dosing schedule with senescent lysate-pulsed DCs. Neurotoxicity was assessed via the novel object recognition test (NOR), a standard measure of cognitive function, at baseline and weeks 2, 4, and 6 post-doxorubicin. The NOR test involved a 5-m inute familiarization phase with two identical objects, followed by a 5-minute test phase 24 hours later with one familiar and one novel object; the discrimination index (DI) was calculated as (time spent exploring novel object - time spent exploring familiar object) I total exploration time, with higher DI indicating better cognitive function. Tests were conducted in a dimly lit arena with video tracking, and operators were blinded to group assignments. Data were analyzed using one-way ANOVA with Tukey's post-hoc test (p<0.05 considered significant). Results showed baseline DI values were similar across groups (approximately 0.65 ± 0.05). Doxorubicin induced neurotoxicity in Groups 2-4, evidenced by reduced DI scores indicating impaired cognitive function, but Group 4 (Senovax) exhibited significant protection, maintaining DI near baseline levels throughout, while Groups 2 and 3 showed persistent cognitive deficits. Group 1 remained stable. Table 5 below summarizes DI scores at weeks 2, 4, and 6.

[0533] Table 5IMMORTA-SOHASCE-PCT

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Claims

1. IMMORTA-SOHASCE-PCTWHAT IS CLAIMED IS:1 . A method of enhancing engraftment of a hematopoietic stem cell into a recipient comprising the steps of: a) obtaining a recipient in need of a hematopoietic stem cell graft; b) reducing the number of senescent cells in said recipient; c) providing said recipient a conditioning regimen to remove existing hematopoietic stem cells; and d) administering to the recipient exogenous hematopoietic stem cells.

2. The method of claim 1 , wherein said hematopoietic stem cell graft is a purified population of hematopoietic stem cells expressing one or more markers selected from the group comprising: CD34, CD133, aldehyde dehydrogenase, c-kit, IL-3 receptor, IL-6 receptor, thrombopoietin receptor, and c-met, and combinations thereof.

3. The method of claim 2, wherein said purified population of hematopoietic stem cells expressing CD34 is selected from a mononuclear population derived from bone marrow, peripheral blood, or mobilized peripheral blood, and said population has been treated with G-CSF at a concentration and duration to enhance engraftment ability of said CD34 cell.

4. The method of claim 3, wherein said mobilization of peripheral blood comprises treating the subject with one or more agents capable of causing stem cells to exit bone marrow niches and enter peripheral circulation, selected from the group consisting of G-CSF, GM-CSF, M-CSF, beta glucan, Poly IC, a TLR2 agonist, FLT-3 ligand, and detoxified lipopolysaccharide, and combinations thereof.

5. The method of claim 2, wherein said purified population of hematopoietic stem cells expressing CD34 is selected from a mononuclear population derived from bone marrow, peripheral blood, or mobilized peripheral blood, and said population has been treated with IL-3 at a concentration and duration to enhance engraftment ability of said CD34 cell.IMMORTA-SOHASCE-PCT6. The method of claim 2, wherein said purified population of hematopoietic stem cells expressing CD34 is selected from a mononuclear population derived from bone marrow, peripheral blood, or mobilized peripheral blood, and said population has been treated with IL-7 at a concentration and duration to enhance engraftment ability of said CD34 cell.

7. The method of claim 1 , wherein said hematopoietic stem cells are extracted from a population of bone marrow mononuclear cells that have been treated with one or more senolytic agents and subsequently exposed to an inflammatory stimuli.

8. The method of claim 7, wherein said extraction of said hematopoietic stem cells is performed using magnetic activated cell sorting with positive selection for a marker selected from the group consisting of CD34, c-kit, c-mpl, c-met, and LIF receptor, and combinations thereof.

9. The method of claim 7, wherein said inflammatory stimuli comprises culture with one or more inflammatory cells selected from the group consisting of neutrophils, type 1 lineage neutrophils, monocytes, macrophages, T cells, T cytotoxic cells, T helper cells, gamma delta T cells, NK cells, and NKT cells, and combinations thereof.

10. The method of claim 9, wherein said inflammatory cell is a neutrophil activated by culture with an agent selected from the group consisting of zymosan, bacterial cell wall extract, oxidized LDL, necrotic cells, IVIG, ozone, and ozonides, and combinations thereof.11 . The method of claim 9, wherein said inflammatory cell is a macrophage activated by culture with one or more allogeneic T cells, and optionally cultured with interferon gamma, TNF-alpha, or HGF at a concentration and duration sufficient to increase expression of one or more markers selected from HLA-I, HLA-II, CD80, and CD86 and combinations thereof by at least 25%, 50%, 80%, 100%, 200%, or 400% compared to baseline.IMMORTA-SOHASCE-PCT12. The method of claim 1 , wherein said hematopoietic stem cell is administered to said recipient after administration of a mesenchymal stem cell, and optionally one or more senolytic agents are administered prior to administration of said mesenchymal stem cell.

13. The method of claim 12, wherein said senolytic agent is selected from the group consisting of a bcl-2 inhibitor, a bcl-xL inhibitor, dasatinib, imatinib, rapamycin, fisetin, quercetin, dasatinib and quercetin, fk-506, natural antibody, and IgM antibody and combinations thereof.

14. The method of claim 1 , wherein said hematopoietic stem cell is administered intravenously or intra-osseously.

15. The method of claim 12, wherein said mesenchymal stem cell is administered intravenously or intra-osseously.

16. The method of claim 12, wherein said mesenchymal stem cell is conditioned to possess enhanced hematopoiesis promoting activities by gene engineering to express one or more hematopoietic growth factors selected from the group consisting of BMP-2, BMP-4, IL-4, IL-6, IL-6-IL-6 receptor complex, IL-7, IL-9, IL- 10, IL-11 , thrombopoietin, IL-13, IL-15, IL-20, IL-22, IL-35, IL-37, IL-38, EGF, IGF-1 , VEGF, KLOTHO, PDGF, GDF-11 , GDF-15, oncostatin, HGF, stem cell factor, M-CSF, G-CSF, GM-CSF, GC-MAF, placental growth factor, interleukin-1 receptor antagonist, ciliary neurotrophic growth factor, brain derived growth factor, and nerve growth factor and combinations thereof.

17. The method of claim 12, wherein said mesenchymal stem cell is activated before administration by treatment with one or more agents that increase nuclear translocation of NF-kappa B or HIF-1 alpha.

18. The method of claim 17, wherein said activation comprises treatment with an agent selected from the group consisting of interleukin 1 beta, unmethylated CpG DNA, flagellin, HMGB1 , interleukin-35, Klotho, pluripotent stem cell cytoplasm, and oocyte cytoplasm, for a period of time and concentration sufficient to increase expression of one or more factors selected from the group consisting ofIMMORTA-SOHASCE-PCTMMP-3, MMP-9, angiopoietin, VEGF, M-CSF, G-CSF, GM-CSF, IL-11 , PDGF- BB, KGF, JAGGED, HLA-G, soluble TNF-alpha receptor, LIF, MMP-13, EPO receptor, and GDF11 and combinations thereof by at least 25%, 50%, or 100%.

19. The method of claim 1 , wherein said reducing the number of senescent cells in said recipient comprises administering one or more senolytic agents.

20. The method of claim 19, wherein said senolytic agent is selected from the group consisting of a bcl-2 inhibitor, a bcl-xL inhibitor, dasatinib, imatinib, rapamycin, fisetin, quercetin, dasatinib and quercetin, fk-506, natural antibody, IgM antibody or a senolytic immunization means and combinations thereof.21 . The method of claim 20, wherein said senolytic immunization means is administration of one or more antigen presenting cells that have been pulsed with one or more senescence associated compounds.

22. The method of claim 21 , wherein said antigen presenting cells are dendritic cells and senescence associated compounds are lysates from senescent cells.

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