Treatment of liver failure using personalized progenitor cells and derivatives thereof

Abstract

Methods and compositions of matter useful for preventing or reversing liver failure. Autologous hepatic progenitors are generated from pluripotent stem cells and administered in a manner to augment and / or replace endogenous regenerative processes. Generation of "adjuvant cells" to enhance engraftment and function of hepatic progenitors. Compositions and methods of reversing liver fibrosis whether caused by viral or chemical toxicity.

Description

TREATMENT OF LIVER FAILURE USING PERSONALIZEDPROGENITOR CELLS AND DERIVATIVES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 604,864, filed on November 30, 2023, entitled "TREATMENT OF LIVER FAILURE USING PERSONALIZED PROGENITOR CELLS AND DERIVATIVES THEREOF", the contents of which are incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention pertains to the field of liver regeneration, more specifically to treatment of liver failure through immune modulation and regeneration. More particularly, the invention pertains to utilization of autologous pluripotent stem cell derived cells that have been endowed with immune modulatory properties to stimulate liver regeneration while at the same time reducing liver fibrosis.BACKGROU ND OF THE INVENTION

[0003] It is widely recognized that the condition known as liver failure is a major burden on our health care system and the 7th largest cause of death in industrialized countries. To date the only cure for liver failure is transplantation, which is severely limited by lack of donors and adverse effects of chronic immune suppression. Liver failure is caused by a number of acute and chronic clinical inciting factors, including drug / alcohol-induced hepatotoxicity, viral infections, vascular injury, autoimmune disease, or genetic predisposition.

[0004] Causes of liver failure include fulminant acute hepatitis, chronic hepatitis, or cirrhosis. Subsequent to various acute insults to the liver, the organ regenerates due to its unique self-renewal activity. If the insult is continuously occurring, the liver's capacity to regenerate new cells is overwhelmed and fibrotic non-functional tissue is deposited which takes over the function of the hepatic parenchyma. The subsequent reduction of hepatocyte function can give rise to metabolic instability combined with disruption of essential bodily functions (i.e., energy supply, acid-base balance and coagulation). If not rapidly addressed, complications of hepatic dysfunction such as uncontrolled bleeding and sepsis occur, and dependent organs such as the brain and kidneys cease to function because of accumulation of toxic metabolites.

[0005] In critical cases, such as when patients progress to Acute-to-Chronic Live Failure(ACLF), liver transplant is considered the standard treatment. However, there are often serious difficulties to obtain a suitable donor and many complications arise after transplantation, including rejection and long-term adherence to immunosuppressant regimes. Although stem cell therapies are currently in development for treatment of liver failure, these possess numerous shortcomings. Embryonic and iPS derived stem cells are all difficult to grow in large quantities and possess the possibility of carcinogenesis or teratoma formation. Additionally, ectopic tissue differentiation in the hepatic microenvironment could have devastating consequences. Adult stem cells offer the possibility of inducing some clinical benefit, however responses to date have not been profound. This is in part because of the inability of adult stem cells to fully take over hepatic tissue.

[0006] The current invention is based around the notion of using immune modulation, whether by adult stem cells, or by immunocytes, as a means of inhibiting liver failure and inducing regression of disease.SUMMARY OF THE INVENTION

[0007] Disclosed are methods and compositions of matter useful for preventing or reversing liver failure. In one embodiment autologous hepatic progenitors are generated from pluripotent stem cells and administered in a manner to augment and / or replace endogenous regenerative processes. In another embodiment, methods are provided for generating"adjuvant cells" to enhance engraftment and function of autologous hepatic progenitors. The invention further provides methods of reversing liver fibrosis in a subject whether caused by viral or chemical toxicity.

[0008] A summary is provided below based on numbered aspects of the invention.

[0009] 1. A method of treating liver failure comprising administration of autologous pluripotent stem cell derived hepatic progenitors.

[0010] 2. The method of aspect 1, wherein said hepatic progenitors express OV6.

[0011] 3. The method of aspect 1, wherein said hepatic progenitors express CD133.

[0012] 4. The method of aspect 1, wherein said hepatic progenitors express SERPINB3.

[0013] 5. The method of aspect 1, wherein said hepatic progenitors are silenced for expression of prominin-1.

[0014] 6. The method of aspect 1, wherein said hepatic progenitors express KRT8.

[0015] 7. The method of aspect 6, wherein said hepatic progenitors express KRT8 and IL-3 receptor.

[0016] 8. The method of aspect 1, wherein said hepatic progenitors express KRT18.

[0017] 9. The method of aspect 8, wherein said hepatic progenitors express KRT18 and IL-3 receptor.

[0018] 10. The method of aspect 1, wherein said hepatic progenitors express EpCAM.

[0019] 11. The method of aspect 10, wherein said hepatic progenitors express EpCAM andIL-3 receptor.

[0020] 12. The method of aspect 10, wherein said hepatic progenitors express EpCAM and c-met.

[0021] 13. The method of aspect 10, wherein said hepatic progenitors express EpCAM, IL-3 receptor and c-met.

[0022] 14. The method of aspect 1, wherein said hepatic progenitors express KRT7 when exposed to a toll like receptor agonist.

[0023] 15. The method of aspect 14, wherein said toll like receptor antagonist is Poly IC.

[0024] 16. The method of aspect 14, wherein said toll like receptor antagonist is beta glucan.

[0025] 17. The method of aspect 14, wherein said toll like receptor antagonist is lipopolysaccharide.

[0026] 18. The method of aspect 14, wherein said toll like receptor antagonist is flagellin.

[0027] 19. The method of aspect 14, wherein said toll like receptor antagonist is imiquimod.

[0028] 20. The method of aspect 14, wherein said toll like receptor antagonist is CpGDNA.

[0029] 21. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated amphiregulin.

[0030] 22. The method of aspect 21, wherein said treatment with amphiregulin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0031] 23. The method of aspect 21, wherein said treatment with amphiregulin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0032] 24. The method of aspect 21, wherein said treatment with amphiregulin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0033] 25. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with VEGF.

[0034] 26. The method of aspect 25, wherein said treatment with VEGF is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0035] 27. The method of aspect 25, wherein said treatment with VEGF is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0036] 28. The method of aspect 25, wherein said treatment with VEGF is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0037] 29. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with FGF-1.

[0038] 30. The method of aspect 29, wherein said treatment with FGF-1 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0039] 31. The method of aspect 29, wherein said treatment with FGF-1 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0040] 32. The method of aspect 29, wherein said treatment with FGF-1 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0041] 33. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with FGF-2.

[0042] 34. The method of aspect 33, wherein said treatment with FGF-2 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0043] 35. The method of aspect 33, wherein said treatment with FGF-2 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0044] 36. The method of aspect 33, wherein said treatment with FGF-2 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0045] 37. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with FGF-5

[0046] 38. The method of aspect 37, wherein said treatment with FGF-5 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0047] 39. The method of aspect 37, wherein said treatment with FGF-5 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0048] 40. The method of aspect 37, wherein said treatment with FGF-5 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0049] 41. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with hepatocyte growth factor.

[0050] 42. The method of aspect 41, wherein said treatment with hepatocyte growth factor.is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0051] 43. The method of aspect 41, wherein said treatment with hepatocyte growth factor.is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0052] 44. The method of aspect 41, wherein said treatment with hepatocyte growth factor.is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0053] 45. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with scatter factor.

[0054] 46. The method of aspect 45, wherein said treatment with scatter factor.is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0055] 47. The method of aspect 45, wherein said treatment with scatter factor is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0056] 48. The method of aspect 45, wherein said treatment with scatter factor is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0057] 49. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with epidermal growth factor

[0058] 50. The method of aspect 49, wherein said treatment with epidermal growth factor.is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0059] 51. The method of aspect 49, wherein said treatment with epidermal growth factor is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0060] 52. The method of aspect 49, wherein said treatment with epidermal growth factor is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0061] 53. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with angiopoietin.

[0062] 54. The method of aspect 53, wherein said treatment with angiopoietin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0063] 55. The method of aspect 53, wherein said treatment with angiopoietin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0064] 56. The method of aspect 53, wherein said treatment with angiopoietin is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0065] 57. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-3.

[0066] 58. The method of aspect 57, wherein said treatment with interleukin-3 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0067] 59. The method of aspect 57, wherein said treatment with interleukin-3 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0068] 60. The method of aspect 57, wherein said treatment with interleukin-3 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0069] 61. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-6.

[0070] 62. The method of aspect 61, wherein said treatment with interleukin-6 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0071] 63. The method of aspect 61, wherein said treatment with interleukin-6 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0072] 64. The method of aspect 61, wherein said treatment with interleukin-6 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0073] 65. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-8.

[0074] 67. The method of aspect 65, wherein said treatment with interleukin-8 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0075] 68. The method of aspect 65, wherein said treatment with interleukin-8 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0076] 69. The method of aspect 65, wherein said treatment with interleukin-8 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0077] 70. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-10.

[0078] 71. The method of aspect 70, wherein said treatment with interleukin-10 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0079] 72. The method of aspect 70, wherein said treatment with interleukin-10 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0080] 73. The method of aspect 70, wherein said treatment with interleukin-10 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0081] 74. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-13.

[0082] 75. The method of aspect 74, wherein said treatment with interleukin-13 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0083] 76. The method of aspect 74, wherein said treatment with interleukin-13 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0084] 77. The method of aspect 74, wherein said treatment with interleukin-13 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0085] 78. The method of aspect 74, wherein said treatment with interleukin-13 is performed at a sufficient concentration and duration to increase expression of CD73 by more than 100%.

[0086] 79. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-15.

[0087] 80. The method of aspect 79, wherein said treatment with interleukin-15 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0088] 81. The method of aspect 79, wherein said treatment with interleukin-15 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0089] 82. The method of aspect 79, wherein said treatment with interleukin-15 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0090] 83. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-17.

[0091] 84. The method of aspect 83, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0092] 85. The method of aspect 83, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0093] 86. The method of aspect 83, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0094] 87. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-17.

[0095] 88. The method of aspect 87, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0096] 89. The method of aspect 87, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0097] 90. The method of aspect 87, wherein said treatment with interleukin-17 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0098] 91. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-18.

[0099] 92. The method of aspect 91, wherein said treatment with interleukin-18 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0100] 93. The method of aspect 91, wherein said treatment with interleukin-18 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0101] 94. The method of aspect 91, wherein said treatment with interleukin-18 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0102] 95. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-22.

[0103] 96. The method of aspect 95, wherein said treatment with interleukin-22 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0104] 97. The method of aspect 95, wherein said treatment with interleukin-22 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0105] 98. The method of aspect 95, wherein said treatment with interleukin-22 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0106] 99. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-35.

[0107] 100. The method of aspect 99, wherein said treatment with interleukin-35 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0108] 101. The method of aspect 99, wherein said treatment with interleukin-35 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0109] 102. The method of aspect 99, wherein said treatment with interleukin-35 is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0110] 103. The method of aspect 1, wherein said hepatic progenitor cells are selected for based on expression of CD133, and subsequently treated with interleukin-35.

[0111] 104. The method of aspect 101, wherein said treatment with TGF-beta is performed at a sufficient concentration and duration to increase expression of CD117 by more than 25%.

[0112] 105. The method of aspect 101, wherein said treatment with TGF-beta is performed at a sufficient concentration and duration to increase expression of CD117 by more than 50%.

[0113] 106. The method of aspect 101, wherein said treatment with TGF-beta is performed at a sufficient concentration and duration to increase expression of CD117 by more than 100%.

[0114] 107. The method of aspect 1, wherein said hepatic progenitor cells are generated by: a) culture of pluripotent stem cells in fibroblast-conditioned knockout serum replacement medium supplemented with 1-100 ng / ml of bFGF; b) culture for 1-5 days in RPMI-B27 supplemented with 10-200-ng / ml Activin A and 10-100-nM Torin2; c) culture in 10-100-ng / ml BMP4 and 1-20-ng / ml bFGF for 1 day; d) culture in 2-100-ng / ml oncostatin M and 1-200 ng / ml hepatocyte growth factor for 1-7 days.

[0115] 108. The method of aspect 107, wherein said hepatic progenitor cells are generated by: a) culture of pluripotent stem cells in fibroblast-conditioned knockout serum replacement medium supplemented with 20-50 ng / ml of bFGF; b) culture for 1-5 days in RPMI- B27 supplemented with 50-100-ng / ml Activin A and 50-100-nM Torin2; c) culture in 50-100- ng / ml BMP4 and 10-20-ng / ml bFGF for 2 hours to 72 hours; d) culture in 50-100-ng / ml oncostatin M and 50-200 ng / ml hepatocyte growth factor for 1-7 days.

[0116] 109. The method of aspect 108, wherein said procedure is performed under hypoxia.

[0117] 110. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of VEGF from said pluripotent stem cells by more than 25%.

[0118] 111. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of VEGF from said pluripotent stem cells by more than 50%.

[0119] 112. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of VEGF from said pluripotent stem cells by more than 100%.

[0120] 113. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of PD-L1 from said pluripotent stem cells by more than 25%.

[0121] 114. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of PD-L1 from said pluripotent stem cells by more than 50%.

[0122] 115. The method of aspect 109, wherein said hypoxia is of sufficient intensity and duration to increase expression of PD-L1 from said pluripotent stem cells by more than 100%.

[0123] 116. The method of aspect 108, wherein a GSK-3 inhibitor is added prior to induction of hepatic differentiation.

[0124] 117. The method of aspect 116, wherein said GSK-3 inhibitor is lithium.

[0125] 118. The method of aspect 116, wherein said GSK-3 inhibitor is lithium chloride.

[0126] 119. The method of aspect 116, wherein said GSK-3 inhibitor is lithium oxide.

[0127] 120. The method of aspect 116, wherein said GSK-3 inhibitor is a lithium salt.

[0128] 121. The method of aspect 1, wherein an adjuvant cell population is administered together with said hepatic progenitor cells.

[0129] 122. The method of aspect 121, wherein said cellular population is a pluripotent stem cell.

[0130] 123. The method of aspect 122, wherein said pluripotent stem cell is an induced pluripotent stem cell.

[0131] 124. The method of aspect 123, wherein said induced pluripotent stem cell is generated by transfection of one or more dedifferentiating factors into a differentiated cell.

[0132] 125. The method of aspect 124, wherein said differentiated cell is "semidifferentiated".

[0133] 126. The method of aspect 125, wherein said differentiated or semi-differentiated cell is an adherent cell.

[0134] 127. The method of aspect 126, wherein said adherent cell is a mesenchymal stem cell.

[0135] 128. The method of aspect 126, wherein said adherent cell is a monocyte.

[0136] 129. The method of aspect 126, wherein said adherent cell is a macrophage.

[0137] 130. The method of aspect 126, wherein said adherent cell is a type 2 macrophage.

[0138] 131. The method of aspect 130, wherein said type 2 macrophage expresses more arginase as compared to a naive macrophage.

[0139] 132. The method of aspect 130, wherein said type 2 macrophage expresses moreEGF as compared to a naive macrophage.

[0140] 133. The method of aspect 130, wherein said type 2 macrophage expresses more latency associated protein as compared to a naive macrophage.

[0141] 134. The method of aspect 130, wherein said type 2 macrophage expresses moreCD38 as compared to a naive macrophage.

[0142] 135. The method of aspect 130, wherein said type 2 macrophage expresses moreLRP as compared to a naive macrophage.

[0143] 136. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-10 as compared to a naive macrophage.

[0144] 137. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-4 as compared to a naive macrophage.

[0145] 138. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-9 as compared to a naive macrophage.

[0146] 139. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-13 as compared to a naive macrophage.

[0147] 140. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-1 receptor antagonist as compared to a naive macrophage.

[0148] 141. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-20 as compared to a naive macrophage.

[0149] 142. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-22 as compared to a naive macrophage.

[0150] 143. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-25 as compared to a naive macrophage.

[0151] 144. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-28 as compared to a naive macrophage.

[0152] 145. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-35 as compared to a naive macrophage.

[0153] 146. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-38 as compared to a naive macrophage.

[0154] 147. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-37 as compared to a naive macrophage.

[0155] 148. The method of aspect 130, wherein said type 2 macrophage expresses moreTGF-beta as compared to a naive macrophage.

[0156] 149. The method of aspect 130, wherein said type 2 macrophage expresses more endoglin as compared to a naive macrophage.

[0157] 150. The method of aspect 130, wherein said type 2 macrophage expresses moreIGF-2 as compared to a naive macrophage.

[0158] 151. The method of aspect 130, wherein said type 2 macrophage expresses more hepatocyte growth factor as compared to a naive macrophage.

[0159] 152. The method of aspect 130, wherein said type 2 macrophage expresses moreIGF-binding protein as compared to a naive macrophage.

[0160] 153. The method of aspect 130, wherein said type 2 macrophage expresses more angiopoietin as compared to a naive macrophage.

[0161] 154. The method of aspect 130, wherein said type 2 macrophage expresses more placental derived growth factor as compared to a naive macrophage.

[0162] 155. The method of aspect 130, wherein said type 2 macrophage expresses moreVEGF-C as compared to a naive macrophage.

[0163] 156. The method of aspect 130, wherein said type 2 macrophage expresses more interleukin-12 receptor as compared to a naive macrophage.

[0164] 157. The method of aspect 130, wherein said type 2 macrophage expresses more protein C receptor as compared to a naive macrophage.

[0165] 158. The method of aspect 130, wherein said type 2 macrophage expresses moreCD14 as compared to a naive macrophage.

[0166] 159. The method of aspect 130, wherein said type 2 macrophage expresses moreCD163 as compared to a naive macrophage.

[0167] 160. The method of aspect 130, wherein said type 2 macrophage expresses moreCD206 as compared to a naive macrophage.

[0168] 161. The method of aspect 130, wherein said type 2 macrophage expresses moreAnnexin-V receptor as compared to a naive macrophage.

[0169] 162. The method of aspect 130, wherein said type 2 macrophage expresses more urokinase plasminogen activator as compared to a naive macrophage.

[0170] 163. The method of aspect 130, wherein said type 2 macrophage expresses less interferon gamma as compared to a naive macrophage.

[0171] 164. The method of aspect 130, wherein said type 2 macrophage expresses less nitric oxide as compared to a naive macrophage.

[0172] 165. The method of aspect 130, wherein said type 2 macrophage expresses less interleukin-1 as compared to a naive macrophage.

[0173] 166. The method of aspect 130, wherein said type 2 macrophage expresses less interleukin-6 as compared to a naive macrophage.

[0174] 167. The method of aspect 130, wherein said type 2 macrophage expresses less interferon alpha as compared to a naive macrophage.

[0175] 168. The method of aspect 130, wherein said type 2 macrophage expresses lessTNF-alpha as compared to a naive macrophage.

[0176] 169. The method of aspect 130, wherein said type 2 macrophage expresses less myostatin as compared to a naive macrophage.

[0177] 170. The method of aspect 130, wherein said type 2 macrophage expresses less interleukin-11 as compared to a naive macrophage.

[0178] 171. The method of aspect 130, wherein said type 2 macrophage expresses lessGC-MAF as compared to a naive macrophage.

[0179] 172. The method of aspect 130, wherein said type 2 macrophage expresses lessHMGBl as compared to a naive macrophage.

[0180] 173. The method of aspect 130, wherein said type 2 macrophage expresses less IL-15 as compared to a naive macrophage.

[0181] 174. The method of aspect 130, wherein said type 2 macrophage expresses less IL-17 as compared to a naive macrophage.

[0182] 175. The method of aspect 130, wherein said type 2 macrophage expresses less IL-21 as compared to a naive macrophage.

[0183] 176. The method of aspect 130, wherein said type 2 macrophage expresses less IL-23 as compared to a naive macrophage.

[0184] 177. The method of aspect 130, wherein said type 2 macrophage expresses less IL-27 as compared to a naive macrophage.

[0185] 178. The method of aspect 130, wherein said type 2 macrophage expresses less IL-33 as compared to a naive macrophage.

[0186] 179. The method of aspect 130, wherein said type 2 macrophage expresses lessMMP-3 as compared to a naive macrophage.

[0187] 180. The method of aspect 130, wherein said type 2 macrophage expresses lessMMP-5 as compared to a naive macrophage.

[0188] 181. The method of aspect 130, wherein said type 2 macrophage expresses lessMMP-7 as compared to a naive macrophage.

[0189] 182. The method of aspect 130, wherein said type 2 macrophage expresses lessMMP-9 as compared to a naive macrophage.

[0190] 183. The method of aspect 130, wherein said type 2 macrophage expresses lessMMP-13 as compared to a naive macrophage.

[0191] 184. The method of aspect 130, wherein said type 2 macrophage expresses lessADAMs as compared to a naive macrophage.

[0192] 185. The method of aspect 130, wherein said type 2 macrophage expresses lessTNF-alpha receptor p55 as compared to a naive macrophage.

[0193] 186. The method of aspect 130, wherein said type 2 macrophage expresses lessTNF-alpha receptor p75 as compared to a naive macrophage.

[0194] 187. The method of aspect 130, wherein said type 2 macrophage expresses less platelet activating factor as compared to a naive macrophage.

[0195] 188. The method of aspect 130, wherein said type 2 macrophage expresses lessCD40 as compared to a naive macrophage.

[0196] 189. The method of aspect 130, wherein said type 2 macrophage expresses lessCD80 as compared to a naive macrophage.

[0197] 190. The method of aspect 130, wherein said type 2 macrophage expresses lessCD86 as compared to a naive macrophage.

[0198] 191. The method of aspect 130, wherein said type 2 macrophage expresses less complement C3 receptor as compared to a naive macrophage.

[0199] 192. The method of aspect 130, wherein said type 2 macrophage expresses less complement C5 receptor as compared to a naive macrophage.

[0200] 193. The method of aspect 130, wherein said type 2 macrophage expresses more complement factor H as compared to a naive macrophage.

[0201] 194. The method of aspect 130, wherein said type 2 macrophage expresses more decay accelerating factor as compared to a naive macrophage.

[0202] 195. The method of aspect 130, wherein said type 2 macrophage expresses moreCD55 as compared to a naive macrophage.

[0203] 196. The method of aspect 130, wherein said type 2 macrophage expresses moreSMAD2 as compared to a naive macrophage.

[0204] 197. The method of aspect 130, wherein said type 2 macrophage expresses moreSMAD4 as compared to a naive macrophage.

[0205] 198. The method of aspect 130, wherein said type 2 macrophage expresses moreSTAT3 as compared to a naive macrophage.

[0206] 199. The method of aspect 130, wherein said type 2 macrophage expresses lessSTAT5 as compared to a naive macrophage.

[0207] 200. The method of aspect 130, wherein said type 2 macrophage expresses morePGE2 as compared to a naive macrophage.

[0208] 201. The method of aspect 130, wherein said type 2 macrophage expresses moreGDF11 as compared to a naive macrophage.

[0209] 202. The method of aspect 130, wherein said type 2 macrophage expresses moreGDF15 as compared to a naive macrophage.

[0210] 203. The method of aspect 130, wherein said type 2 macrophage expresses moreGalectin-3 as compared to a naive macrophage.

[0211] 204. The method of aspect 130, wherein said type 2 macrophage expresses moreGalectin-7 as compared to a naive macrophage.

[0212] 205. The method of aspect 130, wherein said type 2 macrophage expresses moreGalectin-9 as compared to a naive macrophage.

[0213] 206. The method of aspect 1, wherein said hepatic progenitors are selected for expressilOon of KRT19.

[0214] 207. The method of aspect 1, wherein said hepatic progenitors are selected for expression of KRT19 and c-met.

[0215] 208. The method of aspect 1, wherein said hepatic progenitors are selected for expression of KRT19 and IL-3 receptor.

[0216] 209. The method of aspect 1, wherein said hepatic progenitors are selected for expression of KRT19 and TPO receptor.

[0217] 210. The method of aspect 1, wherein said hepatic progenitors are selected for expression of KRT19 and c-kit.

[0218] 211. The method of aspect 1, wherein said hepatic progenitors are selected for expression of Sox9.

[0219] 212. The method of aspect 1, wherein said hepatic progenitors are selected for expression of KRT8.

[0220] 213. The method of aspect 1, wherein said hepatic progenitors are selected for expression of alpha fetoprotein.

[0221] 214. The method of aspect 1, wherein said hepatic progenitors are selected for expression of CD34.

[0222] 215. The method of aspect 1, wherein said hepatic progenitors are selected for expression of CD90.

[0223] 216. The method of aspect 1, wherein said hepatic progenitors are selected for expression of c-kit.

[0224] 217. The method of aspect 1, wherein said hepatic progenitors are selected for expression of NCAM.

[0225] 218. The method of aspect 1, wherein said hepatic progenitors are selected for expression of CXCR4.DETAILED DESCRIPTION OF THE INVENTION

[0226] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in whch the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0227] The invention provides means of treating liver failure through utilization of autologous pluripotent stem cell derived progenitor cells alone or together with "regenerative adjuvants" which may include cells, genes, nucleic acids such as mRNA, or small molecules. In one embodiment, autologous personalized regenerative cells are generated through dedifferentiation of blood born amenable cells into cells possessing pluripotent activity. Said cells are subsequently differentiated into hepatic progenitor cells which are administered alone or together with regenerative adjuvants to a patient in need of therapy.

[0228] Unless defined otherwise, the technical terms used herein have the same meaning as is commonly understood by one of skill in the art.

[0229] As used herein, the term "subject" refers to a human or an animal.

[0230] As used herien, "angiogenesis" refers to any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion. This includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction, or flow properties to improve blood perfusion of tissues. As used herein the terms, "angiogenesis," "revascularization," "increased collateral circulation," and "regeneration of blood vessels" are considered as synonymous.

[0231] As used herein, "chronic wound" refers to a wound that has not completely closed in a period of time, e.g., twelve weeks since the occurrence of the wound in a patient having a condition, disease or therapy associated with defective healing. Conditions, diseases, or therapies associated with defective healing include, for example, diabetes, arterial insufficiency, venous insufficiency, chronic steroid use, cancer chemotherapy, radiotherapy, radiation exposure, and malnutrition. A chronic wound includes defects resulting in inflammatory excess (e.g., excessive production of lnterleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and MMPs), a deficiency of important growth factors needed for proper healing, bacterial overgrowth and senescence of fibroblasts. A chronic wound has an epithelial layer that fails to cover the entire surface of the wound and is subject to bacterial colonization.

[0232] As used hererin, "cell culture" refers an artificial in vitro system containing viable cells, whether quiescent, senescent or (actively) dividing. In a cell culture, cells are grown and maintained at an appropriate temperature, typically a temperature of 370C. and under an atmosphere typically containing oxygen and carbion dioxide. Culture methods and conditions may vary widely for each cell type though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly-varied factor in cell culture systems is the growth medium. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0233] As used herein "therapeutically effective amount" refers to the amounts / quantities or concentrations of cells, conditioned medium or exosomes that, when administered to a mammal for treating a chronic wound, or angiogenic insufficiency is sufficient to affect such treatment. The "therapeutically effective amount" may vary depending on the size of the wound, and the age, weight, physical condition, and responsiveness of the mammal to be treated.

[0234] As used herien, "therapeutic agent" may refer to an agent that has "therapeutic efficacy" in modulating angiogenesis and / or wound healing and an amount of the therapeutic is said to be a "angiogenic modulatory amount", if administration of that amount of the therapeutic is sufficient to cause a significant modulation (i.e., an increase or decrease) in angiogenic activity when administered to a subject needing modulation of angiogenesis.

[0235] As used herein, "growth factor" may refer to a naturally occurring, endogenous or exogenous protein, or recombinant protein, capable of stimulating cellular proliferation and / or cellular differentiation and cellular migration.

[0236] The terms "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value.

[0237] As used herein, "pharmaceutically acceptable" refers to a natural or synthetic substance means that the substance has an acceptable toxic effect in view of its much greaterbeneficial effect, while the related the term, "physiologically acceptable," means the substance has relatively low toxicity.

[0238] As used herein, "endothelial cell mitogen" refers to a protein, protein, polypeptide, variant or portion thereof that is capable of, directly or indirectly, inducing endothelial cell growth. Such proteins include, for example, acidic and basic fibroblast growth factors (aFGF) (GenBank Accession No. NP. sub. -149127) and bFGF (GenBank Accession No. AAA52448), vascular endothelial growth factor (VEGF) (GenBank Accession No. AAA35789 or NP.sub.- 001020539), epidermal growth factor (EGF) (GenBank Accession No. NP. sub. -001954), transforming growth factor .alpha. (TGF-.alpha.) (GenBank Accession No. NP. sub. -003227) and transforming growth factor .beta. (TFG-.beta.) (GenBank Accession No. 1109243A), platelet- derived endothelial cell growth factor (PD-ECGF) (GenBank Accession No. NP. sub. -001944), platelet-derived growth factor (PDGF) (GenBank Accession No. 1109245A), tumor necrosis factor .alpha. (TNF-.alpha.) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF) (GenBank Accession No. BAA14348), insulin like growth factor (IGF) (GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF) (GenBank Accession No. AAB59527), granulocyte / macrophage CSF (GM-CSF) (GenBank Accession No. NP. sub. -000749), monocyte chemotactic protein-1 (GenBank Accession No. P13500) and nitric oxide synthase (NOS) (GenBank Accession No. AAA36365). See, Klagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991); Folkman, et al., J. Biol. Chem., 267:10931-10934 (1992) and Symes, et al., Current Opinion in Lipidology, 5:305-312 (1994). Variants or fragments of a mitogen may be used as long as they induce or promote endothelial cell or endothelial progenitor cell growth. Preferably, the endothelial cell mitogen contains a secretory signal sequence that facilitates secretion of the protein. Proteins having native signal sequences, e.g., VEGF, are preferred. Proteins that do not have native signal sequences, e.g., bFGF, can be modified to contain such sequences using routine genetic manipulation techniques. See, Nabel et al., Nature, 362:844 (1993).

[0239] As used herein, the terms "hepatocyte lineage" cell, "hepatoblastoid" cell and "hepatoembryoid" cell may be used in reference to the differentiated cells of this invention, obtained by differentiating pluripotent cells in the manner described. In certain embodiments, the differentiated cells have at least one of a variety of distinguishing phenotypic characteristics of known hepatocyte precursor cells, hepatoblasts, and hepatocytes, as provided in this disclosure. By the use of these terms, no particular limitation is implied with respect to cell phenotype, cellular markers, cell function, or proliferative capacity, except where explicitly required or stated.

[0240] As used herein, "hepatocyte precursor cell" or a "hepatocyte stem cell" refers to a cell that can proliferate and further differentiate into a hepatocyte, under suitable environmental conditions. Such cells may on occasion have the capacity to produce other types of progeny, such as oval cells, bile duct epithelial cells, or additional hepatocyte precursor cells.

[0241] As used herein, "hepatocyte differentiation agent" and "hepatocyte maturation factor" are two terms with different meanings used in this disclosure to represent a collection of compounds that can be used in preparing and maintaining the differentiated cells of this invention. These agents are further described and exemplified in the sections that follow. The terms are not meant to imply a particular mode or timing of action, and no such limitation should be inferred.

[0242] As used herein, "hepatocyte proliferative factor" is a biological or synthetic compound (a peptide, oligosaccharide, or the like) that promotes the proliferation of hepatocytes and / or hepatocyte precursor cells.

[0243] As used herein, "induced pluripotent stem cell" (iPSC) refers to a type of pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., from skin or blood cells, or from another tissue source).

[0244] As used herein, "mesenchymal stem cell" (MSC) refers to cells that are (1) adherent to plastic, (2) express CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) possess ability to differentiate to osteogenic, chondrogenic and adipogenic lineage. As used herein, "mesenchymal stromal cell" or "MSC" can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and tooth. As used herein, "mesenchymal stromal cell" or "MSC" includes cells that are CD34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally. As used herein, "MSC" includes cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof, and satisfy the ISCT criteria either before or after expansion. As used herein, "mesenchymal stromal cell" or "MSC" includes cells described in the literature as bone marrow stromal stem cells (BMSSC), marrow-isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MultiStem®, Prochymal®, remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX-PAD, AlloStem®, Astrostem®, Ixmyelocel-T, MSC-NTF, NurOwn™, Stemedyne™-MSC, Stempeucel®,StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, Revascor®, Cardiorel®, Cartistem®, Pneumostem®, Promostem®, Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs). In some embodiments of the invention, mesenchymal stem cells are administered prior to, and / or concurrent with, and / or subsequent to administration of hepatic progenitor cells. In some embodiments mesenchymal stem cells are allogeneic, in some they are autologous. Specifically, in some embodiments mesenchymal stem cells are generated from the same iPSC that are utilized to generate the hepatic progenitor cells.

[0245] Embodiments of the invention provide cell culture methods for generating hepatic progenitor cells that are useful for downstream therapeutic applications. In one embodiment of the invention, pluripotent stem cells are treated with hepatic differentiation agents to generate hepatic progenitor cells. In certain embodiments, n-Butyrate provided to a cell culture system as a hepatocyte differentiation agent, illustrated in the examples that follow. Those skilled in the art will readily recognize that a number of homologs of n-butyrate can readily be identified that have a similar effect and can be used as substitutes in the practice of this invention. One class of homologs consists of other hydrocarbons that have similar structural and physicochemical properties to those of n-butyrate. Some of such homologs are acidic hydrocarbons comprising 3- 10 carbon atoms in branched, straight-chain or cyclic form, and a conjugate base selected from the group consisting of a carboxylate, a sulfonate, a phosphonate, and other proton donors. In one embodiment, pluripotent stem cells are treated with hepatic differentiation agents comprising one or a plurality fo the following compounds: n-butyric acid, isobutyric acid, 2- butenoic acid, 3-butenoic acid, propanoic acid, propenoic acid, pentanoic acid, pentenoic acid, other short -chain fatty acids that are either saturated or unsaturated, amino butyric acid, phenyl butyric acid, phenyl propanoic acid, phenyl acetic acid, phenoxyacetic acid, cinnamic acid, and dimethylbutyrate. Also of interest is a hydrocarbon sulfonate or phosphonate that is isosteric with such compounds, particularly propanesulfonic acid and propanephosphonic acid, which are isosteric to n-butyrate. In the naming of such compounds, it is understood that all stereoisomers are included unless explicitly stated otherwise. Compounds with acidic groups may be provided in the acidic form or as the conjugate base, with any acceptable opposing counter-ion. Since the use of sodium n-butyrate would increase the ionic strength of the environment it is used in, the action of other agents may be augmented by providing a change in ionic strength, by adding a salt, if necessary. Another class of homologs are derivatives of butyrate and butyrate homologs, including conjugates with other molecules, such as amino acids, monosaccharides, and other acceptable conjugate pairs. Many such derivatives have beendeveloped as butyrate prodrugs that are transformed to the active form in vivo or in situ by the presence of a suitable converting enzyme— for example, a protease or a glycosidase. By way of illustration, members of this class include arginine butyrate, lysine butyrate, other butyrate amides, glucose pentabutyrate, tributyrin, diacetone glucose butyrate, other butyrate saccharides, aminobutyric acid, isobutyramide, pivaloyloxymethyl butyrate, l-(2-hydroxyethyl)4- )l-oxobutyl)-piperazine butyrate, other piperazine derivatives of butyrate, and piracetam (2-oxo- 1-pyrrolidine acetamide, Notropyl™), a cyclic derivative of gamma-amino butyrate. A further class of homologs are inhibitors of histone deacetylase. Non-limiting examples include trichostatin A, 5-azacytidine, trapoxin A, oxamflatin, FR901228, cisplatin, and MS-27-275. The reader is also referred to antiprotosoal cyclic tetrapeptides in U.S. Pat. No. 5,922,837; antibacterial agents in U.S. Pat. No. 5,925,659; corepressor inhibitors in WO 99 / 23885; and cyclic peptide derivatives in WO 99 / 11659. Methods to identify compounds with histone deacetylase inhibitors can be identified by de-repression of hormone receptor compounds (WO 98 / 48825).

[0246] In the context of the invention, the hepatocyte differentiation activity of n- butyrate may rely at least in part on an ability to inhibit histone deacetylase. Assays for histone deacetylase activity can be used as a preliminary screen to select candidates for other differentiation agents. Many such assays are available. For example, U.S. Pat. No. 5,922,837 (col. 3 ff.) describes an assay using tritiated N-desmethoxyapicidin and a parasite or chick liver S100 solution as a source of deacetylase activity. The candidate compound is added to the reaction mixture, and tritium release is measured using a filter method. A scintillation proximity assay may be performed using a peptide from histone H4, with lysine e-amino groups acetylated with tritium, and bound to an SPA bead that scintillates proportionately to the amount of proximal tritium. Histone deacetylase activity (obtained from extracts of HeLa cell nuclei) releases the labeled acetyl groups and decreases scintillation, and the presence of a deacetylase inhibitor maintains scintillation. A non-isotopic assay for histone deacetylase activity may also be performed. A fluorescent substrate has been developed that is an aminocoumarine derivative of O-acetylated lysine. This permits quantitation of substrate in the nanomolar concentration range, which allows for high throughput screening of histone deacetylase inhibitors. A definitive test for a suitable differentiation agent is its ability to transform iPSC cell cultures into cultures rd enriched for cells of the hepatocyte lineage, as described in this disclosure. Candidate compounds, optionally prescreened according to one or more of the above-listed criteria, are added to cultures of iPSC cells or embryoid bodies in a manner similar to what is known to be effective for n-butyrate. Any compound that can at least promote differentiation of iPSC cells down the hepatocyte lineage, or preferentially permit the growth of hepatoblast-type cells, orpreferentially remove cells of other lineages, will be beneficial in deriving certain differentiated cell populations embodied in this invention.

[0247] Following these guidelines, the ability of particular compound or combination of compounds to act as hepatocyte differentiation agents comprises culturing a population of substantially undifferentiated iPSC cells, or a mixed population of differentiated iPSC cells (such as those obtained from embryoid bodies or by overgrowth of a iPSC culture) in the presence of the compound, and then determining the effect on cell morphology, marker expression, enzymatic activity, proliferative capacity, or other features of interest in relation to cells of the hepatocyte lineage. For optimum results, several concentrations of the test compound are evaluated. A suitable base concentration may be iso osmolar or isotonic with effective butyrate concentrations or have equivalent inhibitory capacity of another histone deacetylase. The compound can then be tested over a range of about l / 10th to 10 times the base concentration, or more, to determine if it has the desired hepatocyte differentiation capacity. A compound will be considered effective as a differentiation agent if it is capable of producing from a culture of iPSC cells or embryoid body cells a population of cells in which at least 40% of the cells have at least three characteristics of hepatoblasts or hepatocytes. Agents that produce more uniform populations having a greater number of hepatocyte characteristics are advantageous in some contexts. It is recognized that agents producing less uniform or less mature hepatocyte populations may also be advantageous if the cells retain another desirable feature (such as hardiness to manipulation, or proliferation capacity). As described below, such cell populations can be further enriched for the desired cell type by sorting or adsorption techniques.

[0248] Enrichment for differentiated cells using a hepatocyte differentiation agent can be supplemented, if desired, by the use of a separate compound or mixture of compounds that act as hepatocyte maturation factors. Such agents may augment the phenotype change promoted by the differentiation agent, or they may push the differentiation pathway further towards more mature cells, or they may help select for cells of the hepatocyte lineage (for example, by preferentially supporting their survival), or they may promote more rapid proliferation of cells with the desired phenotype. In certain embodiments, hepatocyte maturation factors are provided in a cell culture system comprising soluble growth factors (peptide hormones, cytokines, ligand-receptor complexes, and the like) that are capable of promoting the growth of cells of the hepatocyte lineage, wherein the hepatocyte maturation factors comprise one or a plurality of the following: epidermal growth factor (EGF), insulin, TGF-a, TGF-P, fibroblast growth factor (FGF), heparin, hepatocyte growth factor (HGF), Oncostatin M in the presence of dexamethazone, IL-1, IL-6, IGF-I, IGF-II, HBGF-1, and glucagon.

[0249] Another class of hepatocyte maturation factors are corticosteroids, particularly glucocorticoids. Such compounds are a steroid or steroid mimetic, and affects intermediary metabolism, especially promotion of hepatic glycogen deposition, and inhibiting inflammation. Included are naturally occurring hormones exemplified by cortisol, and synthetic glucocorticoids such as dexamethazone (U.S. Pat. No. 3,007,923) and its derivatives, prednisone, methylprednisone, hydrocortisone, and triamcinolone (U.S. Pat. No. 2,789,118) and its derivatives.

[0250] Another class of hepatocyte maturation factors are organic solvents like DMSO. Alternatives with similar properties include but are not limited to dimethylacetamide (DMA), hexmethylene bisacetamide, and other polymethylene bisacetamides. Solvents in this class are related, in part, by the property of increasing membrane permeability of cells. Also of interest are solutes such as nicotinamide. Testing for whether a candidate compound acts as a hepatocyte maturation factor for the purpose of this invention is performed empirically: iPSC cultures are differentiated into cells of the hepatocyte lineage using a hepatocyte differentiation agent described above, in combination with a model hepatocyte differentiation agent, such as a growth factor or DMSO (the positive control). In parallel, iPSC are subjected to a similar protocol using the same differentiation agent and the candidate maturation factor. Resultant cells are then compared phenotypically to determine whether the candidate agent has a similar effect to that of the positive control. In particular embodiments of this invention, the hepatocyte differentiation agent and the hepatocyte maturation factor are used simultaneously or sequentially. In one illustration, newly plated embryoid bodies or feeder-free iPSC cultures are placed in a medium containing both n-butyrate and DMSO, and cultured for 4, 6, or 8 days, or until characteristic features appear, replacing the medium periodically (say, every 24 h) with fresh medium containing n-butyrate and DMSO. In another illustration, EB or iPSC cultures are first cultured with n-butyrate and DMSO for 4, 6, or 8 days, then the medium is exchanged for a hepatocyte-friendly medium containing a cocktail of growth factors (perhaps in combination with n-butyrate) for long-term culture or assay. Following these guidelines, the ability of particular compound or combination of compounds to act as hepatocyte maturation factors comprises culturing a population of cells previously treated with a hepatocyte differentiation agent in the presence of the compound or including the compound in a culture of cells being treated with a hepatocyte differentiation factor. The effect of the compound on cell morphology, marker expression, enzymatic activity, proliferative capacity, or other features of interest is then determined in comparison with parallel cultures that did not include the candidate compound. For optimum results, several concentrations of the test compound areevaluated. A suitable base concentration for organic solvents may be iso osmolar or isotonic with effective DMSO concentrations. Suitable base concentrations for growth factors, cytokines, and other hormones may be concentrations known to have similar growth-inducing or hormone activity in other systems. The test compound can then be tested over a range of about l / 10th to 10 times the base concentration to determine if it has the desired effect on hepatocyte-directed maturation of iPSC cells.

[0251] In certain embodiments, a method for treating liver failure using autologous pluripotent stem cell-derived hepatic progenitor cells is provided, the method comprising: a) identifying a subject with liver failure; b) generating a pluripotent stem cell population from the subject, wherein the pluripotent stem cell population comprises iPSCs; c) establishing a cell culture system comprising iPSCs, wherein the cell culture medium comprises one or a plurality of factors selected from the group consisting of: basic fibroblast growth factor (bFGF), activin, torin2, bone morphogenetic protein 4 (BMP4), oncostatin M, and hepatocyte growth factor (HGF), and wherein exposure of the cultured cells to the one or plurality of factors in the cell culture medium induces differentiation of hepatic progenitor cells; and d) administering a therapeutically effective amount of the hepatic progenitor cells to the subject

[0252] In one embodiment, a method for differentiation of iPSCs into hepatic progenitor cells comprises the following steps: a) exposure of cultured cells to a first culture medium, wherein the first culture medium comprises bFGF; b) exposure of cultured cells to a second culture medium, wherein the second culture medium comprises Activin A and Torin2; c) exposure of cultured cells to a third culture medium, wherein the third culture medium comprises BMP4 and bFGF; and d) exposure of cultured cells to a fourth culture medium, wherein the fourth culture medium comprises oncostatin M and HGF.

[0253] In a specific embodiment, a method for differentiation of iPSCs into hepatic progenitor cells comprises successive additions of one or a plurality of factors to a cell culture system as follows: a) culture of the starting population of iPSCs in 1-100 ng / mL bFGF; b) culture the cells in 10-200 ng / mL activin A and 10-100 nM torin2 for 1-5 days; c) culture the cells in 10- 100 ng / mL BMP4 and 1-20 ng / mL bFGF for 1 day; and then d) culture the cells in 2-100 ng / mL oncostatin M and 1-200 ng / mL HGF for between 1-7 days.

[0254] In another specific embodiment, a method for differentiation of iPSCs into hepatic progenitor cells comprises the following steps: a) culture the iPSCs in fibroblast-conditioned knockout serum replacement medium supplemented with 20-50 ng / mL bFGF; b) culture the cells in RPMI-B27 culture medium supplemented with 50-100 ng / mL Activin A and 50-100 nM Torin2 for 1-5 days; c) culture the cells with 50-100 ng / mL BMP4 and 10-20 ng / mL bFGF for between 2-72 hours; and d) culture the cells with 50-100 ng / mL oncostatin M and 50-200 ng / mL HGF for between 1-7 days.

[0255] In certain embodiments, the hepatic progenitor cells express one or a plurality of the following antigens or molecules: CD34, OV-6, SERPINB3 (squamous cell carcinoma antigen / SSCAl), keratin 18 (KRT18), IL-3 receptor, epithelial cell adhesion molecule (EPCAM), c- met (mesenchymal-epithelial transition factor), Keratin 7 (KRT7), CD117, CD90, c-kit, neural cell adhesion molecule (NCAM), C-X-C chemokine receptor 4 (CXCR4), alpha fetoprotein, keratin 8 (KRT8), Sox9, Keratin 19 (KRT19), and thrombopoietin (TPO) receptor.

[0256] In certain embodiments, the differentiated hepatic progenitor cells are treated with a toll-like receptor (TLR) agonist, and wherein the TLR agonist comprises one or a plurality of the following compounds and molecules: polyinosinic:polycytidylic acid [Poly( l:C)], beta glucan, lipopolysaccharide (LPS), flagellin, imiquimod, and CpG DNA.

[0257] In other embodiments, the differentiated hepatic progenitor cells are treated with one or a plurality of proteins or molecules selected from the following group: amphiregulin, vascular endothelial growth factor (VEGF), fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 5 (FGF-5), hepatocyte growth factor (HGF), epidermal growth factor, angiopoietin, interleukin-3 (IL-3), IL-6, IL-8, IL-10, IL-13, IL-15, IL-17, IL-18, IL-22, IL- 35, and transforming growth factor beta (TGF-P).

[0258] In some embodiments, treatment of the differentiated hepatic progenitor cell with one or a plurality of proteins or molecules in cell culture induces upregulated expression of CD117 by the hepatic progenitor cells.

[0259] In one embodiment, expression of CD117 by the hepatic progenitor cells is increased by at least 25%, at least 50%, or at least 100%.

[0260] In certain embodiments, a cell culture system comprising iPSCs is maintained under hypoxic conditions for differentiation into hepatic progenitor cells.

[0261] In some embodiments, iPSCs under hypoxic conditions exhibit high expression levels of VEGF in culture.

[0262] In some embodiments, the iPSCs under hypoxic conditions exhibit high expression levels of programmed death ligand 1 (PD-L1) in culture.

[0263] In one embodiment, differentiated hepatic progenitor cells in the culture medium are CD133-positive.

[0264] In a specific embodiment, CD133-positive hepatic progenitor cells are selected from the culture medium for administration at a therapeutically effective amount to the subject.

[0265] In certain embodiments, iPSCs generated from the subject are treated with an inhibitor of the enzyme glycogen synthase kinase-3 (GSK-3) prior to differentiation into hepatic progenitor cells.

[0266] In one embodiment, the GSK-3 inhibitor comprises one or a plurality of the following agents: a) lithium; b) lithium chloride; c) lithium oxide; and d) a lithium salt.

[0267] Embodiments of the invention provide an adjuvant cell population that is administered to the subject in addition to hepatic progenitor cells.

[0268] In certain embodiments, the adjuvant cell population is provided to enhance the engraftment and function of the administered hepatic progenitor cells in the subject.

[0269] In certain embodiments, the adjuvant cell population comprises iPSCs, wherein the iPSCs are generated by transfection of one or more dedifferentiation factors into a differentiated cell.

[0270] In certain embodiments, iPSCs are generated from a differentiated cell type that is selected from the group consisting of a mesenchymal stem cell, a monocyte, a macrophage, and a "type 2" macrophage. In certain embodiments, a type 2 macrophage is defined by expression of one or more of the following proteins or molecules: arginase, EGF, latency associated protein (LAP), CD38, low-density lipoprotein receptor-related protein (LRP), IL-10, IL-4, IL-9, signal transducer and activator of transcription 3 (STAT3), prostaglandin E2 (PGE2), IL-13, IL-1 receptor, IL-12 receptor, CD55, IL-20, IL-22, IL-25, IL-28, IL-35, IL-38, IL-37, TFG-P, IGF-2, HGF, endoglin, IGF-binding protein, angiopoietin, placental-derived growth factor, protein C receptor, CD14, VEGF, CD163, CD206, annexin V receptor, urokinase plasminogen activator, complement factor H, decay accelerating factor (DAF), SMAD2, SMAD4, growth differentiation factor 11 (GDF11), GDF15, galectin-3, galectin-7, and galectin-9.

[0271] Aspects of the invention provide methods for selecting a desired cell population from a cell culture system. Once cells of the desired phenotype are obtained, the cells can be harvested for any desired use. In certain differentiated cell populations of this invention, the cells are sufficiently uniform in phenotype that they can be harvested simply by releasing the cells from the substrate (e.g., using collagenase or by physical manipulation), and optionally washing the cells free of debris. If desired, the harvested cells can be further processed by positive selection for desired features, or negative selection for undesired features. For example, cells expressing surface markers or receptors can be positively or negatively selected by incubating the population with an antibody or conjugate ligand, and then separating out the bound cells— for example, by labeled sorting techniques, or adsorption to a solid surface.Negative selection can also be performed by incubating the population with a cytolytic antibodyT1specific for the undesired marker, in the presence of complement. If desired, harvested cells can be transferred into other culture environments, such as those described elsewhere for the propagation of other types of hepatocyte preparations. See, for example, U.S. Pat. Nos.5,030,105 and 5,576,207; EP Patent Application EP 953,633; Angelli et al., Histochem. J. 29:205, 1997; Gomez-Lechon et al., p.130 ff. in In vivo Methods in Pharmaceutical Research, Academic Press, 1997).

[0272] Cells can be characterized according to a number of phenotypic criteria. The criteria include but are not limited to the detection or quantitation of expressed cell markers, and enzymatic activity, and the characterization of morphological features and intercellular signaling. Certain differentiated iPSC cells embodied in this invention have morphological features characteristic of hepatocytes. The features are readily appreciated by those skilled in evaluating such things, and include any or all of the following: a polygonal cell shape, a binucleate phenotype, the presence of rough endoplasmic reticulum for synthesis of secreted protein, the presence of Golgi-endoplasmic reticulum lysosome complex for intracellular protein sorting, the presence of peroxisomes and glycogen granules, relatively abundant mitochondria, and the ability to form tight intercellular junctions resulting in creation of bile canalicular spaces. A number of these features present in a single cell is consistent with the cell being a member of the hepatocyte lineage. Unbiased determination of whether cells have morphologic features characteristic of hepatocytes can be made by coding micrographs of differentiated iPSC cells, adult or fetal hepatocytes, and one or more negative control cells, such as a fibroblast, or RPE (Retinal pigment epithelial) cells— then evaluating the micrographs in a blinded fashion and breaking the code to determine if the differentiated iPSC cells are accurately identified. One embodiment the invention teaches the generation of a cell population obtained by differentiating pluripotent stem cells in such a manner that a significant proportion of cells in the population have characteristics of cells of the hepatocyte lineage. Desirable characteristics are listed later in the description. The cells may demonstrate any or all of the following: antibody- detectable expression of al-antitrypsin or albumin; absence of antibody-detectable expression of a-fetoprotein; expression of asialoglycoprotein receptor at a level detectable by reverse PCR amplification; evidence of glycogen storage; evidence of cytochrome p450 or glucose-6- phosphatase activity; and morphological features of hepatocytes. Preferred cell populations have more of these hepatocyte characteristics in a greater proportion of the cells in the population. It is understood that the cells may replicate to form progeny, both during differentiation, and in subsequent manipulation. Such progeny also fall within the scope of the invention in all instances where not explicitly excluded. Exemplary cells are obtained bydifferentiating pluripotent stem cells obtained from cultures that originated from human cells that are adult in origin but have been dedifferentiatied. The differentiated cells are generated by culturing the pluripotent stem cells, such as iPSC in a growth environment that comprises a hepatocyte differentiation agent, such as n-butyric acid or other differentiation agent outlined in the disclosure. The differentiation agent can be added directly to undifferentiated pluripotent stem cells cultured with or without feeder cells. Alternatively, the pluripotent stem cells are allowed to differentiate into a mixed cell population (e.g., by forming embryoid bodies or by culture overgrowth), and the differentiation agent is added to the mixed population. What emerges is a less heterogeneous population, in which a substantial proportion of the cells have the desired phenotype. In some instances, the culture method also includes hepatocyte maturation factors such as those exemplified in the disclosure, which include solvents like DMSO, growth factors like FGF, EGF, and hepatocyte growth factor, and glucocorticoids like dexamethasone. Another embodiment of the invention is a differentiated cell having characteristics of a cell of the hepatocyte lineage, which is either harvested from a differentiated cell population of this invention, or is the progeny of a cell harvested from such a population. Exemplary is a differentiated cell produced by providing human pluripotent cells in a growth environment essentially free of feeder cells; culturing the pluripotent stem cells in a medium containing a hepatocyte differentiation agent under conditions that produce a cell population enriched for cells with characteristic features of hepatocytes; and subsequently harvesting the differentiated cell from the enriched cell population. Another embodiment of the invention is a method of treating said pluripotent stem cells to obtain differentiated cells that can be maintained in an in vitro culture, by providing a culture of the pluripotent stem cells, and culturing the cells on a substrate in a culture medium containing a hepatocyte differentiation agent under conditions that permit enrichment of the differentiated cells. Beneficial techniques and reagents for use in the context of such methods are detailed later in the disclosure. Also embodied in the invention is a differentiated cell produced according to a method of this invention, particularly those having characteristics of cells of the hepatocyte lineage.

[0273] In one embodiment of the invention, iPSC generated mesenchymal stem cells are utilized as a source of liver progenitor cells. In other embodiments, mesenchymal stem cells generated from iPSC are utilized as a "regenerative adjuvant" by providing growth factor support for newly implanted hepatic progenitors. Various populations of mesenchymal stem cells may be used for the practice of the invention, in addition to bone marrow, adipose, or umbilical cord derived mesenchymal stem cells, amniotic membrane mesenchymal stem cells may be utilized as immune modulatory cells. In one specific embodiment, 8_8 cm2 sections of amnioticmembrane are obtained. They were washed with 1.0M phosphate-buffered saline (PBS; pH 7.2) containing 300 lU / ml penicillin and 300 mg / ml streptomycin (Gibco, Grand Island, NY, USA), and are immediately immersed in Dulbecco's modified Eagle's medium (DM EM) -high glucose (Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco), 300 HU / ml penicillin and 300 mg / ml streptomycin. All samples are processed within 12-15 h after collection. The amniotic membranes are treated with 0.1% collagenase I (Sigma-Aldrich, St Louis, MO, USA) in 1.0M PBS (pH 7.2) and are incubated at 37 _C for 20 min. Each amniotic membrane is washed three times with low-glucose DMEM (Gibco), and the detached cells are harvested after a gentle massage of the amniotic membrane. The cells are centrifuged at 300 g for 10 min at 37_C, and subsequently resuspended in RPMI 1640 medium with 10% FBS, then grown in 25 cm2 flasks at a density of l_106 cells / ml. After 24 h incubation, nonadherent cells are removed. The culture medium is replaced every 3 days. Adherent cells are cultured until they reached 80-90% confluence. Cells are subsequently selected based on quality control procedures including purity (eg >90% CD90 and CD105 positive), sterility (e.g., lack of endotoxin and mycoplasma / bacterial contamination) and potency (e.g., ability to immune modulate in vitro by suppressing production of inflammatory cytokines such as IFN-gamma). Cells may subsequently be utilized for perilymphatic or intralymphatic administration. The present application contemplates the collection and delivery of a naturally occurring population of MSC derived from intra alia, placental / umbilical cord, bone marrow, skin, or tooth pulp tissue. In accordance with the invention, the MSCs are generally an adherent cell population expressing markers CD90 and CD105 (>90%) and lacking expression of CD34 and CD45 and MHC class II (<5%) as detected by flow cytometry, although other markers described in the specification may be utilized.

[0274] Cell expansion for cells originating from any of the abovementioned tissues above takes place in clean room facilities purpose built for cell therapy manufacture and meeting GMP clean room classification. In a sterile class II biologic safety cabinet located in a class 10,000 clean production suite, cells were thawed under controlled conditions and washed in a 15 mL conical tube with 10 ML of complete DMEM-low glucose media (cDMEM) (GibcoBRL, Grand Island, N.Y.) supplemented with 20% Fetal Bovine Serum (Atlas) from dairy cattle confirmed to have no BSE % Fetal Bovine Serum specified to have Endotoxin level less than or equal to 100 EU / mL (with levels routinely less than or equal to 10 EU / mL) and hemoglobin level less than or equal to 30 mg / dl (levels routinely less than or equal to 25 mg / dl). The serum lot used is sequestered and one lot was used for all experiments. Cells are subsequently placed in a T-225 flask containing 45 mL of cDMEM and cultured for 24 hours at 37 degrees Celcius at 5% CO2 in a fully humidified atmosphere. This allowed the MSC to adhere. Non-adherent cells were washed off using cDMEMby gentle rinsing of the flask. This resulted in approximately 6 million cells per initiating T-225 flask. The cells of the first flask were then split into 4 flasks. Cells were grown for 4 days after which approximately 6 million cells per flask were present (24 million cells total). This scheme was repeated but cells were not expanded beyond 10 passages, and were then banked in 6 million cell aliquots in sealed vials for delivery. All processes in the generation, expansion, and product production were performed under conditions and testing that was compliant with current Good Manufacturing Processes and appropriate controls, as well as Guidances issued by the FDA in 1998 Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy; the 2008 Guidance for FDA Reviewers and Sponsors Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs); and the 1993 FDA points-to-consider document for master cell banks were all followed for the generation of the cell products described. Donor cells are collected in sterile conditions, shipped to a contract manufacturing facility, assessed for lack of contamination and expanded. The expanded cells are stored in cryovials of approximately 6 million cells / vial, with approximately 100 vials per donor. At each step of the expansion quality control procedures were in place to ensure lack of contamination or abnormal cel growth.Without departing from the spirit of the invention, mesenchymal stem cells may be optimized to possess heightened immune modulatory properties. In one embodiment this may be performed by exposure of mesenchymal stem cells to hypoxic conditions, specifically hypoxic conditions can comprise an oxygen level of lower than 10%. In some embodiments, hypoxic conditions comprise up to about 7% oxygen. For example, hypoxic conditions can comprise up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1% oxygen. As another example, hypoxic conditions can comprise up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% oxygen. In some embodiments, hypoxic conditions comprise about 1% oxygen up to about 7% oxygen. For example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 2% oxygen up to about 7% oxygen; about 3% oxygen up to about 7% oxygen; about 4% oxygen up to about 7% oxygen; about 5% oxygen up to about 7% oxygen; or about 6% oxygen up to about 7% oxygen. As another example, hypoxic conditions can comprise 1% oxygen up to 7% oxygen; 2% oxygen up to 7% oxygen; 3% oxygen up to 7% oxygen; 4% oxygen up to 7% oxygen; 5% oxygen up to 7% oxygen; or 6% oxygen up to 7% oxygen. As another example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 1% oxygen up to about 6% oxygen; about 1% oxygen up to about 5% oxygen; about 1% oxygen up to about 4% oxygen; about 1% oxygen up to about 3% oxygen; or about 1% oxygen up to about 2% oxygen. As another example, hypoxic conditions cancomprise 1% oxygen up to 7% oxygen; 1% oxygen up to 6% oxygen; 1% oxygen up to 5% oxygen; 1% oxygen up to 4% oxygen; 1% oxygen up to 3% oxygen; or 1% oxygen up to 2% oxygen. As another example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 2% oxygen up to about 6% oxygen; or about 3% oxygen up to about 5% oxygen. As another example, hypoxic conditions can comprise 1% oxygen up to 7% oxygen; 2% oxygen up to 6% oxygen; or 3% oxygen up to 5% oxygen. In some embodiments, hypoxic conditions can comprise no more than about 2% oxygen. For example, hypoxic conditions can comprise no more than 2% oxygen.

Claims

Claims1. A method for treating liver failure using autologous pluripotent stem cell-derived hepatic progenitor cells, comprising: a) identifying a subject with liver failure; b) generating a pluripotent stem cell population from the subject, wherein the pluripotent stem cell population comprises induced pluripotent stem cells (iPSCs); c) establishing a cell culture system comprising iPSCs, wherein the cell culture medium comprises one or a plurality of factors selected from the group consisting of: basic fibroblast growth factor (bFGF), activin, torin2, bone morphogenetic protein 4 (BMP4), oncostatin M, and hepatocyte growth factor (HGF), and wherein exposure of the cultured cells to the one or plurality of factors in the cell culture medium induces differentiation of hepatic progenitor cells; and d) administering a therapeutically effective amount of the hepatic progenitor cells to the subject.

2. The method of Claim 1, wherein the method for differentiation of iPSCs into hepatic progenitor cells comprises the following steps: a) exposure of cultured cells to a first culture medium, wherein the first culture medium comprises bFGF; b) exposure of cultured cells to a second culture medium, wherein the second culture medium comprises Activin A and Torin2; c) exposure of cultured cells to a third culture medium, wherein the third culture medium comprises BMP4 and bFGF; and d) exposure of cultured cells to a fourth culture medium, wherein the fourth culture medium comprises oncostatin M and HGF.

3. The method of Claim 1, wherein the method for differentiation of iPSCs into hepatic progenitor cells involves successive additions of one or a plurality of factors to the cell culture system as follows: a) 1-100 ng / mL bFGF; b) 10-200 ng / mL activin A and 10-100 nM torin2 for 1-5 days; c) 10-100 ng / mL BMP4 and 1-20 ng / mL bFGF for 1 day; and d) 2-100 ng / mL oncostatin M and 1-200 ng / mL HGF for between 1-7 days.

4. The method of Claim 1, wherein the method for differentiation of iPSCs into hepatic progenitor cells comprises the following steps: a) culture of iPSCs in fibroblast-conditioned knockout serum replacement medium supplemented with 20-50 ng / mL bFGF; b) culture in RPMI-B27 culture medium that is supplemented with 50-100 ng / mL Activin A and 50-100 nM Torin2 for 1-5 days; c) culture in 50-100 ng / mL BMP4 and 10-20 ng / mL bFGF for between 2-72 hours; and d) culture in 50-100 ng / mL oncostatin M and 50-200 ng / mL HGF for 1-7 days.

5. The method of Claim 1, wherein the hepatic progenitor cell expresses one or a plurality of antigens or molecules selected from the group consisting of CD34, OV-6, SERPINB3 (squamous cell carcinoma antigen / SSCAl), keratin 18 (KRT18), IL-3 receptor, epithelial cell adhesion molecule (EPCAM), c-met (mesenchymal-epithelial transition factor), Keratin 7 (KRT7), CD117, CD90, c-kit, neural cell adhesion molecule (NCAM), C-X-C chemokine receptor 4 (CXCR4), alpha fetoprotein, keratin 8 (KRT8), Sox9, Keratin 19 (KRT19), and thrombopoietin (TPO) receptor.

6. The method of Claim 1, wherein the differentiated hepatic progenitor cells are treated with a toll-like receptor (TLR) agonist, and wherein the TLR agonist comprises one or a plurality of the following molecules or compounds: polyinosinic:polycytidylic acid [Poly( I :C)], beta glucan, lipopolysaccharide (LPS), flagellin, imiquimod, and CpG DNA.

7. The method of Claim 1, wherein the differentiated hepatic progenitor cells are treated with one or a plurality of proteins or molecules in cell culture selected from the group consisting of amphiregulin, vascular endothelial growth factor (VEGF), fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 5 (FGF-5), hepatocyte growth factor (HGF), epidermal growth factor, angiopoietin, interleukin-3 (IL-3), IL-6, IL-8, IL-10, IL-13, IL-15, IL- 17, IL-18, IL-22, IL-35, and transforming growth factor beta (TGF-P).

8. The method of Claim 7, wherein treatment of the differentiated hepatic progenitor cell with one or a plurality of the proteins or molecules in cell culture induces upregulated expression of CD117 by the hepatic progenitor cells.

9. The method of Claim 8, wherein expression of CD117 by the hepatic progenitor cells is increased by at least 25%, at least 50%, or at least 100%.

10. The method of Claim 1, wherein the cell culture system comprising iPSCs is maintained under hypoxic conditions.

11. The method of Claim 10, wherein the iPSCs under hypoxic conditions exhibit high expression levels of VEGF in culture.

12. The method of Claim 10, wherein the iPSCs under hypoxic conditions exhibit high expression levels of programmed death ligand 1 (PD-L1) in culture.

13. The method of Claim 1, wherein the differentiated hepatic progenitor cells in the culture medium are CD133-positive.

14. The method of Claim 13, wherein the CD133-positive cells are selected from the culture medium for administration at a therapeutically effective amount to the subject.

15. The method of Claim 1, wherein the iPSCs generated from the subject are treated with an inhibitor of the enzyme glycogen synthase kinase-3 (GSK-3) prior to differentiation into hepatic progenitor cells.

16. The method of Claim 15, wherein the GSK-3 inhibitor comprises one or a plurality of agents selected from the group consisting of lithium, lithium chloride, lithium oxide, and a lithium salt.

17. The method of Claim 1, wherein an adjuvant cell population is administered to the subject in addition to the hepatic progenitor cells.

18. The method of Claim 17, wherein the adjuvant cell population is provided to enhance the engraftment and function of the administered hepatic progenitor cells in the subject.

19. The method of Claim 17, wherein the adjuvant cell population comprises iPSCs, and wherein the iPSCs are generated by transfection of one or more dedifferentiation factors into a differentiated cell.

20. The method of Claim 19, wherein the differentiated cell is selected from the group consisting of a mesenchymal stem cell, a monocyte, a macrophage and a type 2 macrophage.