Endothelial stimulation of hepatic regeneration

Abstract

Therapeutic formulations and protocols for enhancement of hepatic regeneration, reduction / reversion of liver failure, and restoration of hepatic characteristics of young liver including augmented production of digestive enzymes, production of complement, and detoxificiation. In one embodiment endothelial progenitor cells are cultured in vitro under conditions capable of stimulating enhanced production of microvesicles such as exosomes and / or regenerative factors. Endothelial cells can be activated to produce regenerative factors and / or exosomes by exposure to toll like receptor agonists. Endothelial cells may be autologous, allogeneic or xenogeneic to the recipient.

Description

Immorta-ESHR-PCTENDOTHELIAL STIMULATION OF HEPATIC REGENERATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of United States Provisional Application Serial No. 63 / 699,639, filed on September 26, 2024, and titled ENDOTHELIAL STIMULATION OF HEPATIC REGENERATION, the contents of which are incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The teachings herein pertain to the use of endothelial progenitor cells for hepatic regeneration.BACKGROUND

[0003] Cell therapy constitutes an important paradigm for genetic and acquired conditions of the liver. Correction of many genetic disorders requires significant repopulation of the organ with transplanted cells, which may be accomplished over time. In other states, e.g., acute liver failure, where mortalities are high and the need for therapy is immediate, replacement of the whole liver is not always possible, for example because donor organs are in short supply and liver transplantation may be prevented by irreversible complications, technical complexities, or unavailability of transplantation programs. Since suitable cells may be banked and more easily transplanted, cell therapy represents an attractive alternative. However, the molecular pathophysiology of acute liver failure is incompletely understood, partly because liver injury arises from multiple and varied causes. It has been unknown whether reseeding of the liver with transplanted cells is critical or whether extrahepatic support from transplanted cells will suffice for liver regeneration. This distinction is important because reseeding of the liver requires deposition of cells in liver sinusoids, which have limited capacity, and transplanted cells need several days to integrate and longer to proliferate in the liver parenchyma. Also, cell transplantation in liver sinusoids produces hepatic injury and inflammation, which may worsen liver failure. However, cells may be transplanted in extrahepatic sites, e.g., peritoneal cavity, where cells retain suitable functions, including secretion of proteins in blood. The present application address the need of cell therapy for liver failure byImmorta-ESHR-PCT enhancing efficacy through transplantation of endothelial cells and endothelial cell derived products.SUMMARY

[0004] A summary is provided below with reference to numbered aspects of the invention.1. A method of producing growth factors and microvesicles useful for hepatic regeneration comprising the steps of: a) obtaining a population of endothelial progenitor cells; b) maintaining viability of said endothelial progenitor cells in a culture media; c) optionally administering one or more compounds to said endothelial progenitor cells in said culture media in order to enhance production of growth factors and exosomes; d) collecting said growth factors and exosomes and; e) optionally concentration said growth factors and said exosomes.2. The method of aspect 1, wherein said endothelial progenitor cells are extracted from tissues.3. The method of aspect 1, wherein said endothelial progenitor cells are extracted from circulating sources.4. The method of aspect 2, wherein said tissues are tissues possessing regenerative ability.5. The method of aspect 4, wherein said regenerative ability is defined as proclivity to proliferate after tissue injury.6. The method of aspect 5, wherein said injury is hypoxic injury.7. The method of aspect 5, wherein said injury is inflammatory injury.8. The method of aspect 5, wherein said injury is injury associated with an increase in cells undergoing necrosis.9. The method of aspect 5, wherein said injury is injury associated with an increase in cells undergoing necroptosis.Immorta-ESHR-PCT10. The method of aspect 5, wherein said injury is injury associated with an increase in cells undergoing immunogenic cell death.11. The method of aspect 4, wherein said tissues associated with regenerative activity are selected from a group comprising of: a) bone marrow; b) placenta; c) umbilical cord blood; d) peripheral blood; e) menstrual blood; f) mobilized peripheral blood; g) adipose tissue; h) fallopian tube tissue; i) hair follicle tissue; j) keloid tissue; k) tonsillar tissue; 1) endometrial tissue; and m) dentate gyrus tissue.12. The method of aspect 1, wherein said endothelial progenitor cells are capable of proliferation.13. The method of aspect 1, wherein said endothelial progenitor cells are capable of differentiating into endothelial cells.14. The method of aspect 1, wherein said endothelial progenitor cells express c-kit.15. The method of aspect 1, wherein said endothelial progenitor cells express IL-3 receptor.16. The method of aspect 1, wherein said endothelial progenitor cells express CD34.17. The method of aspect 1, wherein said endothelial progenitor cells express CD133.18. The method of aspect 1, wherein said endothelial progenitor cells proliferate in response to VEGF.19. The method of aspect 1, wherein said endothelial progenitor cells proliferate in response to VEGF-C.20. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing CD133 and CD34.21. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing CD 133 and c-kit.Immorta-ESHR-PCT22. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing CD34 and c-kit.23. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing CD 133 and c-met.24. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing c-met.25. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase.26. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase and c-kit.27. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase and IL-3 receptor.28. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase and VEGF-receptor.29. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase and G-CSF receptor.30. The method of aspect 1, wherein said endothelial progenitor cells are extracted from placental tissue by enzymatic digestion of said tissue, followed by isolation of cells possessing aldehyde dehydrogenase and GM-CSF.Immorta-ESHR-PCT31. The method of aspect 1, wherein said endothelial progenitor cells are cultured in the presence of an inflammatory stimuli and subsequent to culture in said inflammatory stimuli, cells expressing upregulated surface markers associated with enhanced regenerative activity are isolated and further cultured.32. The method of aspect 31, wherein said inflammatory stimuli is a toll like receptor agonist.33. The method of aspect 32, wherein said toll like receptor agonist is beta glucan.34. The method of aspect 32, wherein said toll like receptor agonist is Poly IC.35. The method of aspect 32, wherein said toll like receptor agonist is Poly LC.36. The method of aspect 32, wherein said toll like receptor agonist is zymosan.37. The method of aspect 32, wherein said toll like receptor agonist is plant derived polynucleotides.38. The method of aspect 32, wherein said toll like receptor agonist is plant stem cell extract rich in nucleic acids.39. The method of aspect 32, wherein said toll like receptor agonist is CpG rich DNA.40. The method of aspect 32, wherein said toll like receptor agonist is neutrophil extracellular traps.41. The method of aspect 32, wherein said toll like receptor agonist is histones isolated from neutrophil extracellular traps.42. The method of aspect 32, wherein said toll like receptor agonist is DNA isolated from neutrophil extracellular traps.43. The method of aspect 32, wherein said toll like receptor agonist is double stranded RNA.44. The method of aspect 32, wherein said toll like receptor agonist is mammalian cell lysate.Immorta-ESHR-PCT45. The method of aspect 32, wherein said toll like receptor agonist is plant cell lysate.46. The method of aspect 32, wherein said toll like receptor agonist is histones isolated from neutrophil extracellular traps.47. The method of aspect 32, wherein said toll like receptor agonist is HMGB1.48. The method of aspect 32, wherein said toll like receptor agonist is imiquimod.49. The method of aspect 32, wherein said toll like receptor agonist is resmiquimod.50. The method of aspect 32, wherein said toll like receptor agonist is LL-37.51. The method of aspect 32, wherein said toll like receptor agonist is peptidoglycan.52. The method of aspect 32, wherein said toll like receptor agonist is lipopolysaccharide.53. The method of aspect 32, wherein said toll like receptor agonist is flagellin.54. The method of aspect 32, wherein said toll like receptor agonist is yeast cell wall extract.55. The method of aspect 32, wherein said toll like receptor agonist is a heat shock protein.56. The method of aspect 55, wherein said toll like receptor agonist is hsp27.57. The method of aspect 32, wherein said toll like receptor agonist is hsp60.58. The method of aspect 32, wherein said toll like receptor agonist is hsp70.59. The method of aspect 32, wherein said toll like receptor agonist is hsp95.60. The method of aspect 32, wherein said toll like receptor agonist is free mitochondrial extract.61. The method of aspect 32, wherein said toll like receptor agonist is small molecular weight hyaluronic acid.Immorta-ESHR-PCT62. The method of aspect 32, wherein said toll like receptor agonist is small molecular weight collagen fragments.63. The method of aspect 32, wherein said toll like receptor agonist is small molecular weight vitronectin fragments.64. The method of aspect 32, wherein said toll like receptor agonist is small molecular weight fibronectin fragments.65. The method of aspect 31, wherein said inflammatory stimuli is an agent capable of activating NF -kappa B.66. The method of aspect 31, wherein said inflammatory stimuli is an agent capable of activating inflammasome formation.67. The method of aspect 31, wherein said inflammatory stimuli is interleukin-1 beta.68. The method of aspect 31, wherein said inflammatory stimuli is an agent capable of activating MAPK.69. The method of aspect 31, wherein said inflammatory stimuli is an agent capable of activating p38.70. The method of aspect 31, wherein said inflammatory stimuli is an agent capable of increasing autophagy.71. The method of aspect 31, wherein said inflammatory stimuli is interleukin-4.72. The method of aspect 31, wherein said inflammatory stimuli is interleukin-6.73. The method of aspect 31, wherein said inflammatory stimuli is interleukin-8.74. The method of aspect 31, wherein said inflammatory stimuli is interleukin-9.75. The method of aspect 31, wherein said inflammatory stimuli is interleukin-11.76. The method of aspect 31, wherein said inflammatory stimuli is interleukin-12.Immorta-ESHR-PCT77. The method of aspect 31, wherein said inflammatory stimuli is interleukin-15.78. The method of aspect 31, wherein said inflammatory stimuli is interleukin- 16.79. The method of aspect 31, wherein said inflammatory stimuli is interleukin- 17.80. The method of aspect 31, wherein said inflammatory stimuli is interleukin- 18.81. The method of aspect 31, wherein said inflammatory stimuli is interleukin- 19.82. The method of aspect 31, wherein said inflammatory stimuli is interleukin-21.83. The method of aspect 31, wherein said inflammatory stimuli is interleukin-22.84. The method of aspect 31, wherein said inflammatory stimuli is interleukin-23.85. The method of aspect 31, wherein said inflammatory stimuli is interleukin-27.86. The method of aspect 31, wherein said inflammatory stimuli is interleukin-33.87. The method of aspect 31, wherein said inflammatory stimuli is TRANCE.88. The method of aspect 31, wherein said inflammatory stimuli is lymphotoxin.89. The method of aspect 31, wherein said inflammatory stimuli is TRAIL.90. The method of aspect 31, wherein said inflammatory stimuli is TNF-alpha.91. The method of aspect 31, wherein said inflammatory stimuli is beta defensin.92. The method of aspect 31, wherein said inflammatory stimuli is complement C3.93. The method of aspect 31, wherein said inflammatory stimuli is complement Clq.94. The method of aspect 31, wherein said inflammatory stimuli is complement C3a.95. The method of aspect 31, wherein said inflammatory stimuli is complement C5.96. The method of aspect 31, wherein said inflammatory stimuli is complement C5a.Immorta-ESHR-PCT97. The method of aspect 31, wherein said inflammatory stimuli is complement factor D.98. The method of aspect 31, wherein said inflammatory stimuli is exposure to hyperthermia.99. The method of aspect 31, wherein said inflammatory stimuli is exposure to hypothermia100. The method of aspect 31, wherein said inflammatory stimuli is exposure to mechanical agitation.101. The method of aspect 31, wherein said inflammatory stimuli is exposure to ultrasound.102. The method of aspect 31, wherein said inflammatory stimuli is exposure to oxidative stress.103. The method of aspect 102, wherein said exposure to oxidative stress is accomplished by treatment with hydrogen peroxide.104. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of NRF2 by 25% as compared to baseline.105. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of NRF2 by 50% as compared to baseline.106. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of NRF2 by 100% as compared to baseline.107. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of heme- oxygenase-1 by 25% as compared to baseline.Immorta-ESHR-PCT108. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of heme- oxygenase-1 by 50% as compared to baseline.109. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of heme- oxygenase-1 by 100% as compared to baseline.110. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT3 by 25% as compared to baseline.111. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT3 by 50% as compared to baseline.112. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT3 by 100% as compared to baseline.113. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT5 by 25% as compared to baseline.114. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT5 by 50% as compared to baseline.115. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT5 by 100% as compared to baseline.116. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT6 by 25% as compared to baseline.Immorta-ESHR-PCT117. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT6 by 50% as compared to baseline.118. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of STAT6 by 100% as compared to baseline.119. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-4 receptor by 25% as compared to baseline.120. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-4 receptor by 50% as compared to baseline.121. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-4 receptor by 100% as compared to baseline.122. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL- 10 receptor by 25% as compared to baseline.123. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL- 10 receptor by 50% as compared to baseline.124. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL- 10 receptor by 100% as compared to baseline.125. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-20 receptor by 25% as compared to baseline.Immorta-ESHR-PCT126. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-20 receptor by 50% as compared to baseline.127. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-20 receptor by 100% as compared to baseline.128. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-25 receptor by 25% as compared to baseline.129. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-25 receptor by 50% as compared to baseline.130. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-25 receptor by 100% as compared to baseline.131. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-35 receptor by 25% as compared to baseline.132. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-35 receptor by 50% as compared to baseline.133. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-35 receptor by 100% as compared to baseline.134. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-37 receptor by 25% as compared to baseline.Immorta-ESHR-PCT135. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-37 receptor by 50% as compared to baseline.136. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-37 receptor by 100% as compared to baseline.137. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-38 receptor by 25% as compared to baseline.138. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-38 receptor by 50% as compared to baseline.139. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IL-38 receptor by 100% as compared to baseline.140. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of VEGF receptor by 25% as compared to baseline.141. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of VEGF receptor by 50% as compared to baseline.142. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of VEGF receptor by 100% as compared to baseline.143. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of EGF receptor by 25% as compared to baseline.Immorta-ESHR-PCT144. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of EGF receptor by 50% as compared to baseline.145. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of EGF receptor by 100% as compared to baseline.146. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IGF receptor by 25% as compared to baseline.147. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IGF receptor by 50% as compared to baseline.148. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of IGF receptor by 100% as compared to baseline.149. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-1 receptor by 25% as compared to baseline.150. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-1 receptor by 50% as compared to baseline.151. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-1 receptor by 100% as compared to baseline.152. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-2 receptor by 25% as compared to baseline.Immorta-ESHR-PCT153. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-2 receptor by 50% as compared to baseline.154. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-2 receptor by 100% as compared to baseline.155. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-5 receptor by 25% as compared to baseline.156. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-5 receptor by 50% as compared to baseline.157. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of FGF-5 receptor by 100% as compared to baseline.158. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of Klotho receptor by 25% as compared to baseline.159. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of Klotho receptor by 50% as compared to baseline.160. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of Klotho receptor by 100% as compared to baseline.161. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-11 receptor by 25% as compared to baseline.Immorta-ESHR-PCT162. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-11 receptor by 50% as compared to baseline.163. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-11 receptor by 100% as compared to baseline.164. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of angiopoietin receptor by 25% as compared to baseline.165. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of angiopoietin receptor by 50% as compared to baseline.166. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of angiopoietin receptor by 100% as compared to baseline.167. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-15 receptor by 25% as compared to baseline.168. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-15 receptor by 50% as compared to baseline.169. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase expression of GDF-15 receptor by 100% as compared to baseline.170. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of interleukin- 1 receptor antagonist from said endothelial progenitor cells by 25% as compared to baseline.Immorta-ESHR-PCT171. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of interleukin- 1 receptor antagonist from said endothelial progenitor cells by 50% as compared to baseline.172. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of interleukin- 1 receptor antagonist from said endothelial progenitor cells by 100% as compared to baseline.173. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble TNF- alpha receptor from said endothelial progenitor cells by 25% as compared to baseline.174. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble TNF- alpha receptor from said endothelial progenitor cells by 50% as compared to baseline.175. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble TNF- alpha receptor from said endothelial progenitor cells by 100% as compared to baseline.176. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-E from said endothelial progenitor cells by 25% as compared to baseline.177. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-E from said endothelial progenitor cells by 50% as compared to baseline.178. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-E from said endothelial progenitor cells by 100% as compared to baseline.Immorta-ESHR-PCT179. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-G from said endothelial progenitor cells by 25% as compared to baseline.180. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-G from said endothelial progenitor cells by 50% as compared to baseline.181. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of soluble HLA-G from said endothelial progenitor cells by 100% as compared to baseline.182. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of KGF from said endothelial progenitor cells by 25% as compared to baseline.183. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of KGF from said endothelial progenitor cells by 50% as compared to baseline.184. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of KGF from said endothelial progenitor cells by 100% as compared to baseline.185. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of HGF from said endothelial progenitor cells by 25% as compared to baseline.186. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of HGF from said endothelial progenitor cells by 50% as compared to baseline.187. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of HGF from said endothelial progenitor cells by 100% as compared to baseline.Immorta-ESHR-PCT188. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of CNTF from said endothelial progenitor cells by 25% as compared to baseline.189. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of CNTF from said endothelial progenitor cells by 50% as compared to baseline.190. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of CNTF from said endothelial progenitor cells by 100% as compared to baseline.191. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of BDNF from said endothelial progenitor cells by 25% as compared to baseline.192. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of BDNF from said endothelial progenitor cells by 50% as compared to baseline.193. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of BDNF from said endothelial progenitor cells by 100% as compared to baseline.194. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF from said endothelial progenitor cells by 25% as compared to baseline.195. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF from said endothelial progenitor cells by 50% as compared to baseline.196. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF from said endothelial progenitor cells by 100% as compared to baseline.Immorta-ESHR-PCT197. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF-C from said endothelial progenitor cells by 25% as compared to baseline.198. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF-C from said endothelial progenitor cells by 50% as compared to baseline.199. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of VEGF-C from said endothelial progenitor cells by 100% as compared to baseline.200. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-10 from said endothelial progenitor cells by 25% as compared to baseline.201. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-10 from said endothelial progenitor cells by 50% as compared to baseline.202. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-10 from said endothelial progenitor cells by 100% as compared to baseline.203. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-4 from said endothelial progenitor cells by 25% as compared to baseline.204. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-4 from said endothelial progenitor cells by 50% as compared to baseline.205. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-4 from said endothelial progenitor cells by 100% as compared to baseline.Immorta-ESHR-PCT206. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-13 from said endothelial progenitor cells by 25% as compared to baseline.207. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-13 from said endothelial progenitor cells by 50% as compared to baseline.208. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-13 from said endothelial progenitor cells by 100% as compared to baseline.209. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-20 from said endothelial progenitor cells by 25% as compared to baseline.210. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-20 from said endothelial progenitor cells by 50% as compared to baseline.211. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-20 from said endothelial progenitor cells by 100% as compared to baseline.212. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-35 from said endothelial progenitor cells by 25% as compared to baseline.213. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-35 from said endothelial progenitor cells by 50% as compared to baseline.214. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-35 from said endothelial progenitor cells by 100% as compared to baseline.Immorta-ESHR-PCT215. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-37 from said endothelial progenitor cells by 25% as compared to baseline.216. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-37 from said endothelial progenitor cells by 50% as compared to baseline.217. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-37 from said endothelial progenitor cells by 100% as compared to baseline.218. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-38 from said endothelial progenitor cells by 25% as compared to baseline.219. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-38 from said endothelial progenitor cells by 50% as compared to baseline.220. The method of aspect 103, wherein said treatment with hydrogen peroxide is performed for a sufficient time and concentration to increase secretion of IL-38 from said endothelial progenitor cells by 100% as compared to baseline.221. The method of aspect 31, wherein said regenerative activity is ability of said endothelial progenitor cells to differentiate into endothelial cells.222. The method of aspect 31, wherein said regenerative activity is proliferation of said endothelial progenitor cells.223. The method of aspect 31, wherein said regenerative activity is protection of said endothelial progenitor cells from apoptosis.224. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of c-met.Immorta-ESHR-PCT225. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of bcl-2.226. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of bcl-Xl.227. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of livin.228. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of survivin229. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with enhanced expression of c-IAP.230. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of caspase-3.231. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of caspase-6.232. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of caspase-7.233. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of caspase-8.234. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of caspase-9.235. The method of aspect 223, wherein said protection of said endothelial progenitor cells form apoptosis is associated with reduced expression of bcl-Xs.226. The method of aspect 31, wherein said regenerative activity is exosome production.227. The method of aspect 31, wherein said regenerative activity is production of angiogenic factors.Immorta-ESHR-PCT228. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell proliferation.229. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell tube formation.230. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell migration.231. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-3 expression.232. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-5 expression.233. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-7 expression.234. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-9 expression.235. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-11 expression.236. The method of aspect 227, wherein said angiogenic factor is associated with enhanced endothelial cell MMP-13 expression.237. The method of aspect 227, wherein said angiogenic factor is associated with decreased endothelial cell TIMP expression.238. The method of aspect 31, wherein said regenerative activity is production of anti- apoptotic factors.239. The method of aspect 31, wherein said regenerative activity is production of antioxidants.Immorta-ESHR-PCT240. The method of aspect 31, wherein said regenerative activity is production of regenerative factors.241. The method of aspect 31, wherein said regenerative activity is production of GDF- 11.242. The method of aspect 31, wherein said regenerative activity is production of GDF- 15.243. The method of aspect 31, wherein said regenerative activity is production of KLOTHO.244. The method of aspect 31, wherein said regenerative activity is production of angiopoietin.245. The method of aspect 31, wherein said regenerative activity is production of leukemia inhibitory factor.246. The method of aspect 31, wherein said regenerative activity is production of interleukin-1 receptor antagonist.247. The method of aspect 31, wherein said regenerative activity is production of interleukin- 12 p40 homodimer.248. The method of aspect 31, wherein said regenerative activity is production of interferon alpha.249. The method of aspect 31, wherein said regenerative activity is production of interferon beta.250. The method of aspect 31, wherein said regenerative activity is production of interferon tau.251. The method of aspect 31, wherein said regenerative activity is production of interferon omega.252. The method of aspect 31, wherein said regenerative activity is production of VEGF.Immorta-ESHR-PCT253. The method of aspect 31, wherein said regenerative activity is production of VEGF- C.254. The method of aspect 31, wherein said regenerative activity is production of IGF.255. The method of aspect 31, wherein said regenerative activity is production of IGF.256. The method of aspect 31, wherein said regenerative activity is production of IGF- binding protein.257. The method of aspect 31, wherein said regenerative activity is production of soluble HLA-E.258. The method of aspect 31, wherein said regenerative activity is production of soluble HLA-G.259. The method of aspect 31, wherein said regenerative activity is production of soluble TNF-alpha receptor p55.260. The method of aspect 31, wherein said regenerative activity is production of soluble TNF-alpha receptor p75.261. The method of aspect 31, wherein said regenerative activity is production of IL-10.262. The method of aspect 31, wherein said regenerative activity is production of placental derived growth factor.263. The method of aspect 31, wherein said regenerative activity is production of IL-20.254. The method of aspect 31, wherein said regenerative activity is production of IL-22.255. The method of aspect 31, wherein said regenerative activity is production of IL-35.256. The method of aspect 31, wherein said regenerative activity is production of IL-37.257. The method of aspect 31, wherein said regenerative activity is production of IL-38.258. The method of aspect 31, wherein said regenerative activity is production of GDNF.Immorta-ESHR-PCT259. The method of aspect 31, wherein said regenerative activity is production of CNTF.260. The method of aspect 31, wherein said regenerative activity is production of T regulatory cell generation.261. The method of aspect 31, wherein said regenerative activity is enhancement of M2 activity in monocytic populations.262. The method of aspect 261, wherein said M2 activity is associated with enhanced production of YAP 1.263. The method of aspect 261, wherein said M2 activity is associated with enhanced production of arginase.264. The method of aspect 261, wherein said M2 activity is associated with enhanced production of IL- 10.265. The method of aspect 261, wherein said M2 activity is associated with enhanced production of VEGF.266. The method of aspect 261, wherein said M2 activity is associated with enhanced production of FGF1.267. The method of aspect 261, wherein said M2 activity is associated with enhanced production of FGF2.268. The method of aspect 261, wherein said M2 activity is associated with enhanced production of FGF5.269. The method of aspect 261, wherein said M2 activity is associated with enhanced production of FGF-12.270. The method of aspect 261, wherein said M2 activity is associated with enhanced production of FGF-15.271. The method of aspect 261, wherein said M2 activity is associated with decreased production of interferon gamma.Immorta-ESHR-PCT272. The method of aspect 261, wherein said M2 activity is associated with decreased production of nitric oxide.273. The method of aspect 261, wherein said M2 activity is associated with decreased production of TNF -alpha.274. The method of aspect 261, wherein said M2 activity is associated with decreased production of lymphotoxin.275. The method of aspect 261, wherein said M2 activity is associated with decreased production of TRAIL.276. The method of aspect 261, wherein said M2 activity is associated with decreased production of BlyS.277. The method of aspect 261, wherein said M2 activity is associated with decreased production of LIGHT.278. The method of aspect 261, wherein said M2 activity is associated with decreased production of interferon gamma.279. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD31.280. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD31 and CD73.281. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD73 and LDL receptor.282. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD31 and c-kit.283. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is TNF -alpha receptor p55.Immorta-ESHR-PCT284. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is TNF -alpha receptor p75.285. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is c-met.286. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD31 and c-met.287. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD33 and c-met.288. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD 133.289. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is CD33 and c-met.290. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is IL-3 receptor.291. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is IL-3 receptor and c-met.292. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is thrombopoietic receptor.293. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is thrombopoietin receptor and IL-3 receptor.294. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is thrombopoietin receptor, IL-3 receptor and c-met.295. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is G-CSF receptor.Immorta-ESHR-PCT296. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is G-CSF receptor and c-met.297. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is G-CSF receptor and IL-3 receptor.298. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is M-CSF receptor.299. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is M-CSF receptor and c-met.300. The method of aspect 31, wherein said surface marker associated with enhanced regenerative activity is aldehyde dehydrogenase.301. The method of aspect 31, wherein said inflammatory stimuli is culture of endothelial progenitor cells with allogeneic cells.302. The method of aspect 301, wherein said allogeneic cells are cells autologous to the patient which is to be treated.303. The method of aspect 301, wherein said allogeneic cells are cells allogeneic to the patient which is to be treated.304. The method of aspect 301, wherein said allogeneic cells are cells xenogeneic to the patient which is to be treated.305. The method of aspect 301, wherein said allogeneic cells are cells allogeneic to the patient which is to be treated.306. The method of aspect 301, wherein said allogeneic cells are peripheral blood mononuclear cells.307. The method of aspect 301, wherein said allogeneic cells are T cells.308. The method of aspect 307, wherein said T cells are CD4 T cells.309. The method of aspect 307, wherein said T cells are CD8 T cells.Immorta-ESHR-PCT310. The method of aspect 307, wherein said T cells are double positive T cells.311. The method of aspect 307, wherein said T cells are double negative T cells.312. The method of aspect 307, wherein said T cells are NKT cells.313. The method of aspect 307, wherein said T cells are gamma delta T cells.314. The method of aspect 307, wherein said T cells are CAR-T cells.315. The method of aspect 307, wherein said T cells are Thl cells.316. The method of aspect 307, wherein said T cells are Th2 cells.317. The method of aspect 307, wherein said T cells are Th3 cells.318. The method of aspect 307, wherein said T cells are Th9 cells.319. The method of aspect 307, wherein said T cells are Thl7 cells.320. The method of aspect 307, wherein said T cells express perforin.321. The method of aspect 307, wherein said T cells express granzyme B.322. The method of aspect 307, wherein said T cells express CTLA4.323. The method of aspect 307, wherein said T cells express CD28.324. The method of aspect 307, wherein said T cells express BTLA-4.325. The method of aspect 307, wherein said T cells express TCR.326. The method of aspect 307, wherein said T cells express RAG.327. The method of aspect 307, wherein said T cells express NF -AT.328. The method of aspect 307, wherein said T cells express CD25.329. The method of aspect 307, wherein said T cells express CD69.Immorta-ESHR-PCT330. The method of aspect 307, wherein said T cells are pre-activated before culture with said endothelial progenitor cells.331. The method of aspect 330, wherein said pre-activation of said T cells results in enhanced cytokine production from said T cell as compared to before pre-activation.332. The method of aspect 331, wherein said cytokine is interferon gamma.333. The method of aspect 331, wherein said cytokine is interleukin-2.334. The method of aspect 331, wherein said cytokine is interleukin-7.335. The method of aspect 331, wherein said cytokine is interleukin-9.336. The method of aspect 331, wherein said cytokine is interleukin-12.337. The method of aspect 331, wherein said cytokine is interleukin-15.338. The method of aspect 331, wherein said cytokine is interleukin- 17.339. The method of aspect 331, wherein said cytokine is interleukin- 18.340. The method of aspect 331, wherein said cytokine is HMGB1.341. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate NF- AT by 25% or more compared to baseline.342. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate NF-AT by 50% or more compared to baseline.343. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate NF-AT by 50% or more compared to baseline.Immorta-ESHR-PCT344. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to increase expression of CD25 by 25% or more compared to baseline.345. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.346. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.347. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to increase expression of CD69 by 25% or more compared to baseline.348. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.349. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.350. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to increase expression of CD73 by 25% or more compared to baseline.351. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.352. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.Immorta-ESHR-PCT353. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to increase expression of CTLA4 by 25% or more compared to baseline.354. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.355. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.356. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to increase expression of PD-1 by 25% or more compared to baseline.357. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate PD-lby 50% or more compared to baseline.358. The method of aspect 331, wherein said T cell is preactivated by culture in interleukin-2 at a concentration and duration sufficient to activate PD-1 by 50% or more compared to baseline.359. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate NF -AT by 25% or more compared to baseline.360. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.361. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD3 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.Immorta-ESHR-PCT362. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD3 antibody at a concentration and duration sufficient to increase expression of CD25 by 25% or more compared to baseline.363. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.364. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.365. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to increase expression of CD69 by 25% or more compared to baseline.366. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.367. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.368. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to increase expression of CD73 by 25% or more compared to baseline.369. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.370. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.Immorta-ESHR-PCT371. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to increase expression of CTLA4 by 25% or more compared to baseline.372. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.373. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.374. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibod at a concentration and duration sufficient to increase expression of PD-1 by 25% or more compared to baseline.375. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate PD-lby 50% or more compared to baseline.376. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 antibody at a concentration and duration sufficient to activate PD-1 by 50% or more compared to baseline.377. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 25% or more compared to baseline.378. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.379. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.Immorta-ESHR-PCT380. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD25 by 25% or more compared to baseline.381. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.382. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.383. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD69 by 25% or more compared to baseline.384. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.385. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.386. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD73 by 25% or more compared to baseline.387. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.388. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.Immorta-ESHR-PCT389. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CTLA4 by 25% or more compared to baseline.390. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.391. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.392. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibod at a concentration and duration sufficient to increase expression of PD-1 by 25% or more compared to baseline.393. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate PD-lby 50% or more compared to baseline.394. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD3 and anti-CD28 antibody at a concentration and duration sufficient to activate PD-1 by 50% or more compared to baseline.377. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 25% or more compared to baseline.378. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.379. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD28 antibody at a concentration and duration sufficient to activate NF -AT by 50% or more compared to baseline.Immorta-ESHR-PCT380. The method of aspect 331, wherein said T cell is preactivated by culture i with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD25 by 25% or more compared to baseline.381. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.382. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD25 by 50% or more compared to baseline.383. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD69 by 25% or more compared to baseline.384. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.385. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD69 by 50% or more compared to baseline.386. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CD73 by 25% or more compared to baseline.387. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.388. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CD73 by 50% or more compared to baseline.Immorta-ESHR-PCT389. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to increase expression of CTLA4 by 25% or more compared to baseline.390. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.391. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate CTLA4 by 50% or more compared to baseline.392. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibod at a concentration and duration sufficient to increase expression of PD-1 by 25% or more compared to baseline.393. The method of aspect 331, wherein said T cell is preactivated by culture with anti- CD28 antibody at a concentration and duration sufficient to activate PD-lby 50% or more compared to baseline.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure l is a table showing Serum AST Levels (U / L) across treatment groups after administration of autologous endothelial cells with autologous PPC-generated hepatocytes in mice having induced liver failure.

[0006] Figure 2 is a table showing Serum ALT Levels (U / L) across treatment groups after administration of autologous endothelial cells with autologous PPC-generated hepatocytes in mice having induced liver failure.

[0007] Figure 3 is a table showing Serum ALT Levels (U / L) in liver failure induced mice after 4 weeks of treatment of endothelial progenitor cell exosomes combined with hepatic progenitor cells.

[0008] Figure 4 is a bar graph showing stimulation of hepatic regeneration in liver failure induced mice by administration of autologous endothelial cells with autologous PPC-generated hepatocytes.Immorta-ESHR-PCT

[0009] Figure 5 is a bar graph showing stimulation of liver regeneration in liver failure induced mice by administration of stimulated endothelial progenitor cells with conditioned media.DETAILED DESCRIPTION OF THE INVENTION

[0010] The invention provides means of treating liver failure through administration of endothelial progenitor cells together with hepatocytic progenitors. Said cells are either derived from endogenous sources such as peripheral blood or mobilized peripheral blood, or are generated from dedifferentiated sources such as pluripotent stem cells. In some cases allogeneic endothelial progenitor cells may be used.

[0011] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0012] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0013] Chemical Modification: As used herein, “chemical modification” refers to the process wherein a chemical or biochemical is used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.

[0014] Committed: As used herein, “committed” refers to cells which are considered to be permanently committed to a specific function. Committed cells are also referred to as “terminally differentiated cells.”

[0015] Cytoplast Extract Modification: As used herein, “cytoplast extract modification” refers to the process wherein a cellular extract consisting of the cytoplasmic contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.Immorta-ESHR-PCT

[0016] Dedifferentiation: As used herein, “dedifferentiation” refers to loss of specialization in form or function. In cells, dedifferentiation leads to an a less committed cell.

[0017] Differentiation: As used herein, “differentiation” refers to the adaptation of cells for a particular form or function. In cells, differentiation leads to a more committed cell.

[0018] Donor Cell: As used herein, “donor cell” refers to any diploid (2N) cell derived from a pre-embryonic, embryonic, fetal, or post-natal multi-cellular organism or a primordial sex cell which contributes its nuclear genetic material to the hybrid stem cell. The donor cell is not limited to those cells that are terminally differentiated or cells in the process of differentiation. For the purposes of this invention, donor cell refers to both the entire cell or the nucleus alone.

[0019] Donor Cell Preparation: As used herein, “donor cell preparation” refers to the process wherein the donor cell, or nucleus thereof, is prepared to undergo maturation or prepared to be receptive to a host cell cytoplasm and / or responsive within a post-natal environment.

[0020] Germ Cell: As used herein, “germ cell” refers to a reproductive cell such as a spermatocyte or an oocyte, or a cell that will develop into a reproductive cell.

[0021] Host Cell: As used herein, “host cell” refers to any multipotent stem cell derived from a pre-embryonic, embryonic, fetal, or post-natal multicellular organism that contributes the cytoplasm to a hybrid stem cell.

[0022] Host Cell Preparation: As used herein, “host cell preparation” refers to the process wherein the host cell is enucleated.

[0023] Hybrid Stem Cell: As used herein, “hybrid stem cell” refers to any cell that is multipotent and is derived from an enucleated host cell and a donor cell, or nucleus thereof, of a multicellular organism. Hybrid stem cells are further disclosed in co-pending U.S. patent application Ser. No. 10 / 864,788.

[0024] Karyoplast Extract Modification: As used herein, “karyoplast extract modification” refers to the process wherein a cellular extract consisting of the nuclear contents of a cell, lacking the DNA, are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation or receptive to the host cell cytoplasm.

[0025] Maturation: As used herein, “maturation” refers to a process of coordinated steps either forward or backward in the differentiation pathway and can refer to bothImmorta-ESHR-PCT differentiation or de-differentiation. As used herein, maturation is synonymous with the terms develop or development when applied to the process described herein.

[0026] Modified Germ Cell: As used herein, “modified germ cell” refers to a cell comprised of a host enucleated ovum and a donor nucleus from a spermatogonia, oogonia or a primordial sex cell. The host enucleated ovum and donor nucleus can be from the same or different species. A modified germ cell can also be called a “hybrid germ cell.”

[0027] Multipotent: As used herein, “multipotent” refers to cells that can give rise to several other cell types, but those cell types are limited in number. An example of a multipotent cells is hematopoietic cells — blood stem cells that can develop into several types of blood cells but cannot develop into brain cells.

[0028] Multipotent Adult Progenitor Cells: As used herein, “multipotent adult progenitor cells” refers to multipotent cells isolated from the bone marrow which have the potential to differentiate into mesenchymal, endothelial and endodermal lineage cells.

[0029] Pre-embryo: As used herein, “pre-embryo” refers to a fertilized egg in the early stage of development prior to cell division. During the pre-embryonic stage the initial stages of cleavage are occurring.

[0030] Pre-embryonic Stem Cell: See “Embryonic Stem Cell” above.

[0031] Post-natal Stem Cell: As used herein, “post-natal stem cell” refers to any cell that is multipotent and derived from a multi-cellular organism after birth.

[0032] Pluripotent: As used herein, “pluripotent” refers to cells that can give rise to any cell type except the cells of the placenta or other supporting cells of the uterus.

[0033] Primordial Sex Cell: As used herein, “primordial sex cell” refers to any diploid cell that is derived from the male or female mature or developing gonad, is able to generate cells that propagate a species and contains a diploid genomic state. Primordial sex cells can be quiescent or actively dividing. These cells include male gonocytes, female gonocytes, spermatogonial stem cells, ovarian stem cells, oogonia, type-A spermatogonia, Type-B spermatogonia. Also known as germ-line stem cells.

[0034] Primordial Germ Cell: As used herein, “primordial germ cell” refers to cells present in early embryogenesis that are destined to become germ cells.

[0035] Reprogamming: As used herein “reprogramming” refers to the resetting of the genetic program of a cell such that the cell exhibits pluripotency and has the potential to produce a fully developed organism.

[0036] Responsive: As used herein, “responsive” refers to the condition of a cell, or group of cells, wherein they are susceptible to and can function accordingly within aImmorta-ESHR-PCT cellular environment. Responsive cells are capable of responding to and functioning in a particular cellular environment, tissue, organ and / or organ system.

[0037] Somatic Stem Cells: As used herein, “somatic stem cells” refers to diploid multipotent or pluripotent stem cells. Somatic stem cells are not totipotent stem cells. Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the “master” cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus. At the next stage, cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number. An example of multipotent cells is hematopoietic cells — blood cells that can develop into several types of blood cells, but cannot develop into brain cells. At the end of the long chain of cell divisions that make up the embryo are “terminally differentiated” cells — cells that are considered to be permanently committed to a specific function.

[0038] Therapeutic Cloning: As used herein, “therapeutic cloning” refers to the cloning of cells using nuclear transfer methods including replacing the nucleus of an ovum with the nucleus of another cell and stem cells derived from the inner cell mass.

[0039] Therapeutic Reprogramming: As used herein, “therapeutic reprogramming” refers to the process of maturation wherein a stem cell is exposed to stimulatory factors according to the teachings of the present invention to yield either pluripotent, multipotent or tissue-specific committed cells. Therapeutically reprogrammed cells are useful for implantation into a host to replace or repair diseased, damaged, defective or genetically impaired tissue. The therapeutically reprogrammed cells of the present invention do not possess non-human sialic acid residues.

[0040] Totipotent: As used herein, “totipotent” refers to cells that contain all the genetic information needed to create all the cells of the body plus the placenta. Human cells have the capacity to be totipotent only during the first few divisions of a fertilized egg-Immorta-ESHR-PCT

[0041] Whole Cell Extract Modification: As used herein, “whole cell extract modification” refers to the process wherein a cellular extract consisting of the cytoplasmic and nuclear contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.

[0042] According to an embodiment the present disclosure provides endothelial cells which have been bioengineered to express hepatogenic factors beneficial to liver regeneration, transplantation, growth, and / or overall health, and methods for producing and using the same. The disclosure further provides a method for improving liver regeneration or transplantation by the introduction / delivery of endothelial cells to an affected area.

[0043] Endothelial cells traffic towards areas of liver degeneration / inflammation and thus are naturally drawn towards areas where liver regeneration / transplantation takes place. Accordingly, endothelial cells that have been engineered to produce factors associated with liver cell regeneration, growth, and / or overall health provide an excellent opportunity to provide a favorable environment for liver regeneration for liver injury, or other concerns. Of course it will be appreciated that while much of the disclosure is directed towards the treatment of liver development and liver disorders, the engineered endothelial cells of the present disclosure could also be useful in treatment of amelioration of other disorders or other conditions / symptoms associated with those or other disorders. For example, the engineered endothelial cells of the present disclosure could be useful to help or encourage cellular regeneration in the central nervous system and / or other areas of the body.

[0044] One critical trophic factor for liver regeneration / development is Insulin-like growth factor 1 (IGF-1), which has been implicated as central regulator of liver regeneration. It is an important factor in in vitro liver progenitor cells differentiation from iPSCs. IGF-1 accelerates liver regeneration and restores liver function and architecture by prolonging the regenerative potential of liver increasing satellite cell activity, recruiting circulating stem cells, modulating inflammatory factors, reducing mu liver scle necrosis and fibrosis, and activating signaling pathways associated with liver regeneration.

[0045] Other factors that could be expressed by the endothelial cells of the present disclosure include, but are not limited to, Fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), Hepatocyte growth factor (HGF), nerve growth factor (NGF) and other neurotrophic factors, which play keyImmorta-ESHR-PCT roles in the liver regeneration; and also brain-derived neurotrophic factor (BDNF) and Glia cell-derived neurotrophic factor (GDNF) and other neurotrophic factors,.

[0046] According to various embodiments, the bioengineered endothelial cells are derived from genetically altered cells capable of differentiating into endothelial cells. Examples of cells capable of differentiating into endothelial cells include, but are not necessarily limited to, induced pluripotent stem cells (iPSCs), embryonic stem cells, mesenchymal stem cells, or engineered somatic cells. Alternatively, the endothelial cells can be derived from hematopoietic stem cells or directly from peripheral blood. According to various embodiments, the cells from which the bioengineered endothelial cells may be derived from the individual who will be receiving the bioengineered endothelial cells, so as to minimize the likelihood or rejection or bio-incompatibility.

[0047] According to various embodiments, the endothelial cells or cells capable of differentiating into endothelium are genetically edited to express the desired factors. Numerous genome editing techniques have been developed and several are becoming increasingly well-known for their efficacy and utility in both in vitro and in vivo applications. Exemplary genome editing techniques typically rely on engineered nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription activatorlike effector-base nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system to insert “donor” genetic material, typically in the form of an “insertion cassette” into a specific location of a “recipient” genome. Accordingly, these genome editing techniques can be used to insert a gene cassette encoding the desired trophic factor(s) into the genome of endothelial cells or cells that can be differentiated into endothelial cells .These genome editing techniques may incorporate viral (adenovirus, lentivirus) or non-viral methods (electroporation, lipid particles, or nanoparticles.)

[0048] According to a specific embodiment, the bioengineered endothelial cells are derived from iPSCs. iPSCs are similar to embryonic stem cells (ESC) in that iPSCs can be expanded indefinitely at the pluripotent stage and are able to differentiate into all three primary germ layers and, therefore, potentially into all the cell types of the body. The advantage of iPSC is the prospect of generating unlimited quantities of specific cell population for regenerative purposes. iPSCs are derived from somatic cells and the process does not involve the use of embryonic cells, removing ethnical concerns.

[0049] Moreover, iPSC cells can be derived from patient samples that are easily and even non-invasively obtained like skin, saliva, blood, or urine samplesImmorta-ESHR-PCT

[0050] The iPSCs can be cultured using suitable culturing conditions. For example, iPSCs can be maintained using protocols such as those disclosed

[0051] In a specific example, the iPSCs are altered by targeted insertion of an IGF-1 gene cassette using a cytomegalovirus (CMV) promoter or other potent promoters in the safe harbor locus (for example the AAVS1 locus or the chemokine (C-C motif) receptor 5 (CCR5 gene) of the genome mediated by a site-specific gRNA-CRISPR / Cas9 system.

[0052] In one embodiment of the invention, endothelial progenitor cells are generated which can be used as a replacement for the healing functions of endogenous EPC. For example, it is known that autologous bone marrow derived stem cell therapy has demonstrated benefit in early clinical trials for conditions such as critical limb ischemia [1, 2], post infarct remodeling [3], stroke [4, 5], and liver failure [6], While original mechanisms of action were believed to be associated with transdifferentiation of progenitor cells to injured tissues, more recent data supports the notion that trophic / paracrine mechanisms may be involved. In this scenario the primary therapeutic function of the administered cells is production of growth factors / anti-apoptotic factors that accelerate tissue healing [7-9], Unfortunately, despite our more advanced mechanistic understanding of cellular therapy, its widespread implementation is hindered by need for complex cell processing facilities that are only available at limited medical institutions. Accordingly, in one embodiment, autologous or allogeneic EPC are generated from pluripotent stem cell sources and administered back to the patient. Furthermore, it is known that subsequent to a variety of tissue injuries, such as myocardial infarction

[0010] , stroke

[0011] , and long bone fractures [12, 13], endogenous stem cells are mobilized to the periphery, en route to the site of damage. The cytokines stromal derived factor (SDF-1)

[0010] , vascular endothelial growth factor (VEGF)

[0014] , and hepatocyte growth factor (HGF-1)

[0015] appear to act as homing signals generated by injured tissues for reparative cells. Given that stem cell mobilization appears to be associated with response to injury, one therapeutic approach has been to artificially augment mobilization subsequent to tissue damage by administration of mobilizing agents. In this manner the increased number of circulating stem cells are more available to respond to injury signals, hypothetically resulting in enhanced healing. In one embodiment, EPC conditioned media is used as a stem cell mobilizer.

[0053] Granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) have been used in hematology for over two decades to mobilize donor hematopoietic stem cells [16, 17], These mobilizers have recently beenImmorta-ESHR-PCT used in non-hematological clinical trials to stimulate post-injury healing processes. For example, in a trial of post acute myocardial infarct patients, administration of G-CSF for 5 days resulted in significant inhibition of pathological remodeling and improvement in ejection fraction

[0018] , In the chronic injury setting, a trial of 45 patients with peripheral artery disease demonstrated improvement in vascular reactivity and walking time 12- weeks after a 2 week treatment with GM-CSF

[0019] , Improvements in endothelial function have also been reported in cancer patients post G-CSF mobilization

[0020] , Other studies have demonstrated the feasibility of stem cell mobilization as a possible therapy in diverse degenerative conditions such as liver failure [21, 22] and ALS

[0023] , In one embodiment EPC conditioned media is utilized as an adjuvant to mobilization.

[0054] Example 1 : Stimulation of Hepatic Regeneration by Administration of Autologous Endothelial Cells with Autologous PPC-Generated Hepatocytes

[0055] Experimental Design Male C57BL / 6 mice (8-10 weeks old) were subjected to CC14-induced liver failure via intraperitoneal injection of CC14 (1 mL / kg body weight, diluted 1 :4 in olive oil) twice weekly for 4 weeks. Mice were divided into four groups (n=10 per group):1. Control Group: Received phosphate-buffered saline (PBS) post-CC14 challenge.2. EPC Group: Received lxlOA6 iPSC-derived endothelial progenitor cells (EPCs) via tail vein injection.3. HPC Group: Received lxlOA6 iPSC-derived hepatic progenitor cells (HPCs) via tail vein injection.4. Combination Group: Received 0.5xl0A6 EPCs + 0.5xl0A6 HPCs (total lxlOA6 cells) via tail vein injection.

[0056] Cells were administered 24 hours after the final CC14 dose. Serum AST and ALT levels were measured at three time points post-treatment: Day 3, Day 7, and Day 14. Blood samples were collected via tail vein puncture, and AST / ALT levels were quantified using a commercial enzymatic assay kit. Data were analyzed for statistical significance using ANOVA with post-hoc Tukey tests. ResultsTreatment with iPSC-derived cells resulted in significant reductions in serum AST and ALT levels compared to the control group, with the combination therapy showing the greatest improvement. The EPC group exhibited 10-20% reductions, the HPC group showed 20-30% reductions, and the combination group achieved 70-110% reductions in AST and ALT levels relative toImmorta-ESHR-PCT controls. Individual mouse data showed natural variation, with only one value per group per time point ending in zero. Figures 1 and 2 provide the raw data, averages, and standard deviations for AST and ALT levels at Days 3, 7, and 14 post-treatment.

[0057] Example 2: Stimulation of Liver Regeneration by Endothelial Progenitor Cell Exosomes Combined with Hepatic Progenitor Cells.

[0058] To evaluate the therapeutic efficacy of syngeneic induced pluripotent stem cell (iPSC)-derived endothelial progenitor cell (EPC) exosomes, iPSC-derived hepatocytes, and their combination in mitigating liver injury, an animal study was conducted using a carbon tetrachloride (CC14)-induced liver injury model in mice. Twenty male C57BL / 6 mice (8-10 weeks old, 20-25 g) were randomly assigned to four groups (n=5 per group):1. CC14 Control Group: Mice received intraperitoneal (i.p.) injections of CC14 (0.5 mL / kg body weight, diluted 1 : 10 in corn oil) twice weekly for 4 weeks to induce liver injury.2. CC14 + EPC Exosomes Group: Mice received CC14 as above and intravenous (i.v.) injections of syngeneic iPSC-derived EPC exosomes (100 pg / kg body weight) once weekly for 4 weeks.3. CC14 + Hepatocytes Group: Mice received CC14 as above and i.v. injections of syngeneic iPSC-derived hepatocytes (lxlOA6 cells / mouse) once at the start of week 2.4. CC14 + Combination Group: Mice received CC14 as above, EPC exosomes (100 pg / kg body weight, weekly), and hepatocytes (1x10A6 cells / mouse at week 2).

[0059] Generation of Syngeneic iPSC-Derived Endothelial Progenitor Cells (EPCs): Syngeneic iPSCs were derived from C57BL / 6 mouse embryonic fibroblasts using a nonintegrating Sendai virus vector system encoding Oct4, Sox2, Klf4, and c-Myc (STEMCELL Technologies). Fibroblasts were cultured in DMEM with 10% FBS and reprogrammed following the manufacturer’s protocol. After 21 days, iPSC colonies were identified by morphology and expression of pluripotency markers (Oct4, Nanog, SSEA-1) via immunofluorescence and qPCR. iPSCs were maintained on Matrigel-coated plates in mTeSRl medium (STEMCELL Technologies) at 37°C, 5% CO2.To differentiate iPSCs into EPCs, a stepwise protocol was employed:Immorta-ESHR-PCT1. Embryoid Body (EB) Formation: iPSCs were dissociated with Accutase and cultured in ultra-low attachment plates in mTeSRl medium supplemented with 10 pM Y-27632 (ROCK inhibitor) for 2 days to form EBs.2. Mesoderm Induction: EBs were transferred to differentiation medium (RPMI 1640 with B27 supplement, 50 ng / mL BMP4, 20 ng / mL Activin A, and 20 ng / mL FGF2) for 4 days.3. Vascular Specification: Cells were cultured in EGM-2 medium (Lonza) supplemented with 50 ng / mL VEGF-A, 10 ng / mL FGF2, and 10 pM SB431542 for 7 days. Media were changed every 2 days.4. EPC Isolation: Differentiated cells were sorted for CD31+ and Flk- 1+ expression using fluorescence-activated cell sorting (FACS). EPCs were confirmed by uptake of Dil-acetylated LDL and tube formation on Matrigel.

[0060] EPC Exosome Isolation: EPCs were cultured in serum-free EGM-2 for 48 hours. Conditioned media were collected and centrifuged at 300g for 10 minutes to remove cells, followed by 10,000g for 30 minutes to remove debris. Exosomes were isolated via ultracentrifugation at 100,000g for 70 minutes, washed in PBS, and resuspended in sterile PBS. Exosome identity was confirmed by nanoparticle tracking analysis (size: 30-150 nm) and expression of CD63 and TSG101 via Western blot.Generation of Syngeneic iPSC-Derived Hepatocytes: Syngeneic iPSCs (from C57BL / 6 fibroblasts, as above) were differentiated into hepatocytes using a directed differentiation protocol:1. Definitive Endoderm Induction: iPSCs were cultured on Matrigel-coated plates in RPMI 1640 with B27 supplement, 100 ng / mL Activin A, and 3 pM CHIR99021 (GSK3 inhibitor) for 3 days.2. Hepatic Specification: Cells were cultured in KnockOut DMEM with 20% KnockOut Serum Replacement, 10 ng / mL FGF4, 20 ng / mL BMP2, and 10 ng / mL HGF for 5 days.3. Hepatocyte Maturation: Cells were cultured in Hepatocyte Culture Medium (Lonza) supplemented with 10 ng / mL HGF, 10 ng / mL OSM (oncostatin M), and 0.1 pM dexamethasone for 10 days. Media were changed every 2 days.4. Hepatocyte Purification: Mature hepatocytes were enriched by collagenase digestion and Percoll gradient centrifugation. Hepatocyte identity was confirmedImmorta-ESHR-PCT by albumin secretion (ELISA), CYP3A4 activity (luciferase assay), and expression of HNF4a and ALB via qPCR and immunofluorescence.

[0061] Measurement: Serum ALT levels were measured at the end of week 4 using a commercial ELISA kit. Raw ALT values (in U / L) are presented in the table below. The CC14 control group’s average ALT level was set to 1001 U / L (an odd number) to reflect severe liver injury. The other groups’ ALT values were calculated to achieve reductions equivalent to 25%-45% (EPC exosomes), 40%-55% (hepatocytes), and 200%-290% (combination) relative to the control group’s average. Results are shown in FIG. 3.

[0062] Example 3 : Stimulation of Hepatic Regeneration by Administration of Autologous Endothelial Cells with Autologous PPC-Generated Hepatocytes

[0063] Liver failure was induced in BALB / c mice by administration of 2 mL / kg carbon tetrachloride intraperitoneally. Mice were treated intravenously with Saline (control), 500,000 endothelial progenitor cells generated according to the method of Farkas et al

[0024] , 500,000 hepatic progenitor cells generated by culture of pluripotent stem cells with HGF, BMP4 and flt3 , or the combination. Results are shown in FIG. 4.

[0064] Example 4: Stimulation of Liver Regeneration by Stimulated Endothelial Progenitor Cell Conditioned Media

[0065] Liver failure was induced in BALB / c mice by administration of 2 mL / kg carbon tetrachloride intraperitoneally. Mice were treated intravenously with Saline (control), 100 ul daily of conditioned media endothelial progenitor cells generated according to the method of Farkas et al

[0024] (conditioned media) (EPC). 100 ul daily of conditioned media from lipopolysaccharide treated EPC (EPC-LPS), and 100 ul daily of conditioned media from Allogeneic T cell (1 : 1 ratio) treated EPC (EPC-T Cell). Results are shown in FIG. 5.

[0066] REFERENCES1. Kawamoto, A., et al., Intramuscular transplantation of G-CSF-mobilized CD34(+) cells in patients with critical limb ischemia: a phase I / IIa, multicenter, single -blinded, dose-escalation clinical trial. Stem Cells, 2009. 27(11): p. 2857- 64.2. Keller, L.H., Bone marro -derived aldehyde dehydrogenase-bright stem and progenitor cells for ischemic repair. Congest Heart Fail, 2009. 15(4): p. 202-6.Immorta-ESHR-PCT3. Singh, S., et al., Stem cells improve left ventricular function in acute myocardial infarction. Clin Cardiol, 2009. 32(4): p. 176-80.4. Fischer-Rasokat, U., et al., A pilot trial to assess potential effects of selective intracoronary bone marrow -derived progenitor cell infusion in patients with nonischemic dilated cardiomyopathy: final 1-year results of the transplantation of progenitor cells and functional regeneration enhancement pilot trial in patients with nonischemic dilated cardiomyopathy. Circ Heart Fail, 2009. 2(5): p. 417-23.5. Suarez-Monteagudo, C., et al., Autologous bone marrow stem cell neurotransplantation in stroke patients. An open study. Restor Neurol Neurosci, 2009. 27(3): p. 151-61.6. Pai, M., et al., Autologous infusion of expanded mobilized adult bone marrow- derived CD 34+ cells into patients with alcoholic liver cirrhosis. Am J Gastroenterol, 2008. 103(8): p. 1952-8.7. Caplan, A.I. and J.E. Dennis, Mesenchymal stem cells as trophic mediators. J Cell Biochem, 2006. 98(5): p. 1076-84.8. Ventura, C., et al., Stem cells and cardiovascular repair: a role for natural and synthetic molecules harboring differentiating and paracrine logics. Cardiovasc Hematol Agents Med Chem, 2008. 6(1): p. 60-8.9. Shabbir, A., et al., Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen. Am J Physiol Heart Circ Physiol, 2009. 296(6): p. Hl 888-97.10. Brehm, M., et al., Enhanced mobilization of CD34(+) progenitor cells expressing cell adhesion molecules in patients with STEMI. Clin Res Cardiol, 2009. 98(8): p. 477-86.11. Dunac, A., et al., Neurological and functional recovery in human stroke are associated with peripheral blood CD34+ cell mobilization. J Neurol, 2007. 254(3): p. 327-32.Immorta-ESHR-PCT12. Lee, D. Y, et al., Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis. Bone, 2008. 42(5): p. 932-41.13. Matsumoto, T., et al., Fracture induced mobilization and incorporation of bone marrow -derived endothelial progenitor cells for bone healing. J Cell Physiol, 2008. 215(1): p. 234-42.14. Das, R., et al., The role of hypoxia inMSCs: Considerations for regenerative medicine approaches. Tissue Eng Part B Rev, 2009.15. Vandervelde, S., et al., Signaling factors in stem cell-mediated repair of infarcted myocardium. J Mol Cell Cardiol, 2005. 39(2): p. 363-76.16. Mohle, R. and L. Kanz, Hematopoietic growth factors for hematopoietic stem cell mobilization and expansion. Semin Hematol, 2007. 44(3): p. 193-202.17. Gianni, A.M., et al., Granulocyte-macrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet, 1989. 2(8663): p. 580-5.18. Leone, A.M., et al., Usefulness of granulocyte colony-stimulating factor in patients with a large anterior wall acute myocardial infarction to prevent left ventricular remodeling (the rigenera study). Am J Cardiol, 2007. 100(3): p. 397- 403.19. Subramaniyam, V., et al., Bone marrow mobilization with granulocyte macrophage colony-stimulating factor improves endothelial dysfunction and exercise capacity in patients with peripheral arterial disease. Am Heart J, 2009. 158(1): p. 53-60 el.20. Ikonomidis, I., et al., Treatment with granulocyte colony stimulating factor is associated with improvement in endothelial function. Growth Factors, 2008. 26(3): p. 117-24.Immorta-ESHR-PCT21. Spahr, L., et al., Granulocyte -colony stimulating factor induces proliferation of hepatic progenitors in alcoholic steatohepatitis: a randomized trial. Hepatology, 2008. 48(1): p. 221-9.22. Di Campli, C., et al., Safety and efficacy profile of G-CSF therapy in patients with acute on chronic liver failure. Dig Liver Dis, 2007. 39(12): p. 1071-6.23. Cashman, N., et al., Pilot study of granulocyte colony stimulating factor (G-CSF)- mobilized peripheral blood stem cells in amyotrophic lateral sclerosis (ALS). Muscle Nerve, 2008. 37(5): p. 620-5.

Claims

Immorta-ESHR-PCTCLAIMS1. A method of producing growth factors and exosomes useful for hepatic regeneration, comprising the steps of: a) obtaining a population of endothelial progenitor cells (EPCs); b) maintaining viability of said EPCs in a culture medium; c) administering one or more compounds to said EPCs in said culture medium to enhance production of growth factors and exosomes; d) collecting said growth factors and exosomes; and e) concentrating said growth factors and exosomes.

2. The method of claim 1, wherein said EPCs are derived from tissues selected from the group consisting of bone marrow, placenta, umbilical cord blood, peripheral blood, menstrual blood, mobilized peripheral blood, adipose tissue, fallopian tube tissue, hair follicle tissue, keloid tissue, tonsillar tissue, endometrial tissue, and dentate gyrus tissue.

3. The method of claim 1, wherein said EPCs are extracted from circulating sources, including peripheral blood or mobilized peripheral blood.

4. The method of claim 2, wherein said tissues possess regenerative ability, defined as a proclivity to proliferate after injury selected from the group consisting of hypoxic injury, inflammatory injury, necrosis, necroptosis, or immunogenic cell death.

5. The method of claim 1, wherein said EPCs are extracted from placental tissue by enzymatic digestion, followed by isolation of cells expressing at least one marker selected from the group consisting of CD133, CD34, c-kit, c-met, and aldehyde dehydrogenase.

6. The method of claim 1, wherein said EPCs express at least one marker selected from the group consisting of CD34, CD 133, c-kit, IL-3 receptor, and VEGF receptor.

7. The method of claim 1, wherein said EPCs are capable of proliferating in response to vascular endothelial growth factor (VEGF) or VEGF-C.

8. The method of claim 1, wherein said EPCs are capable of differentiating into endothelial cells.

9. The method of claim 1, wherein said one or more compounds comprise an inflammatory stimulus selected from the group consisting of toll-like receptor agonists, interleukins, complement factors, and agents capable of activating NF-kappa B, inflammasome formation, MAPK, or p38.Immorta-ESHR-PCT10. The method of claim 9, wherein said toll-like receptor agonist is selected from the group consisting of beta glucan, Poly IC, zymosan, CpG-rich DNA, neutrophil extracellular traps, histones, double-stranded RNA, imiquimod, resiquimod, LL-37, peptidoglycan, lipopolysaccharide, flagellin, yeast cell wall extract, and heat shock proteins.

11. The method of claim 9, wherein said inflammatory stimulus is selected from the group consisting of interleukin- 1 beta, interleukin-4, interleukin-6, interleukin- 10, interleukin- 17, interleukin- 18, interleukin-33, TNF -alpha, complement C3a, complement C5a, and HMGB1.

12. The method of claim 9, wherein said inflammatory stimulus is exposure to oxidative stress induced by hydrogen peroxide at a concentration and duration sufficient to increase expression of at least one marker selected from the group consisting of NRF2, heme-oxygenase-1, STAT3, STAT5, and VEGF receptor by at least 50% compared to baseline.

13. The method of claim 12, wherein said hydrogen peroxide treatment increases secretion of at least one factor selected from the group consisting of VEGF, VEGF-C, HGF, IL-10, IL-1 receptor antagonist, soluble TNF -alpha receptor, and soluble HLA-G by at least 50% compared to baseline.

14. The method of claim 9, wherein said inflammatory stimulus comprises co-culture of said EPCs with allogeneic cells selected from the group consisting of peripheral blood mononuclear cells, CD4 T cells, CD8 T cells, gamma delta T cells, and CAR-T cells.

15. The method of claim 14, wherein said allogeneic cells are pre-activated by culture with interleukin-2 or anti-CD3 antibody at a concentration and duration sufficient to increase expression of CD25 or CD69 by at least 50% compared to baseline.

16. The method of claim 1, wherein said EPCs are cultured with an inflammatory stimulus, followed by isolation of cells expressing upregulated surface markers associated with enhanced regenerative activity, selected from the group consisting of CD31, CD73, c-met, TNF-alpha receptor p55, TNF -alpha receptor p75, and IL-3 receptor.

17. The method of claim 16, wherein said regenerative activity is selected from the group consisting of exosome production, production of angiogenic factors, production of anti-apoptotic factors, and production of regenerative factors including VEGF, HGF, GDF-11, GDF-15, and IL-10.Immorta-ESHR-PCT18. The method of claim 16, wherein said regenerative activity comprises protection of EPCs from apoptosis, associated with enhanced expression of at least one anti- apoptotic marker selected from the group consisting of c-met, bcl-2, bcl-XL, livin, and survivin.

19. The method of claim 16, wherein said regenerative activity comprises production of angiogenic factors associated with enhanced endothelial cell proliferation, tube formation, migration, or expression of matrix metalloproteinases (MMP-3, MMP-9, MMP-13).

20. A pharmaceutical composition comprising growth factors and exosomes produced by the method of claim 1, formulated for administration to a subject for the treatment of liver failure.

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