Treatment of liver failure by hepatogenic monocytes

Abstract

Compositions of matter and therapeutic methods for preventing, reducing, or reversing liver pathologies. Autologous pluripotent stem cells for generating monocytes or macrophages that are capable of suppressing liver failure. Methods of administering monocytes that are engineered or induced to facilitate hepatic regeneration. Hepatogenic cells such as monocytes are generated from induced pluripotent stem cells. Hepatogenic cells can be generated by overexpression of one or a plurality of factors associated with an M2 phenotype in pluripotent stem cell-derived monocytes such as signal transducer and activator of transcription 6 (STAT6) and transforming growth factor-beta. Hepatogenic monocytes can be generated in an environment that simulates liver injury.

Description

Immorta-LivMon-PCT TREATMENT OF LIVER FAILURE BY HEPATOGENIC MONOCYTES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from U.S. Provisional Application No. 63 / 644,449, entitled “TREATMENT OF LIVER FAILURE BY HEPATOGENIC MONOCYTES” and filed on May 8, 2024, the entire contents of which are hereby incorporated by reference. FIELD OF THE INVENTION

[0002] The invention relates to the field of regenerative medicine. More specifically, the invention relates to cell therapies for treating liver failure. BACKGROUND

[0003] Cell therapy for liver failure represents an important paradigm shift for genetic and acquired conditions. Conditions such as 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. The present application addresses the need of cell therapy for liver failure.Immorta-LivMon-PCT SUMMARY

[0004] Methods for treating a subject with liver failure are provided, wherein the method comprises generating or obtaining a monocyte and utilizing or modifying said monocyte to create a therapeutic cell.

[0005] Disclosed herein are compositions of matter and therapeutic methods for preventing, reducing, or reversing liver pathologies. In one embodiment, the invention teaches the utilization of autologous pluripotent stem cells for generating monocytes or macrophages that are capable of suppressing liver failure. In one embodiment, the invention provides hepatogenic monocytes that are engineered or induced to facilitate hepatic regeneration. In certain embodiments, hepatogenic monocytes are generated from induced pluripotent stem cells. In certain embodiments, monocytes are generated by overexpression of one or a plurality of factors associated with an M2 phenotype and are rendered hepatogenic in the presence of growth factors that simulate liver injury.

[0006] In one specific embodiment of the invention, a method for treating a subject with liver failure is provided, the method comprising: a) identifying a subject with liver failure; b) isolating a primary somatic cell from a biological fluid or tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce dedifferentiation into an induced pluripotent stem cell; d) providing a second culture system, wherein the induced pluripotent stem cell is provided with culture conditions to induce differentiation into a monocyte; e) providing a third cell culture system, wherein the differentiated monocyte is provided with culture conditions to induce the expression of one or a plurality of hepatogenic factors; f) isolating the hepatogenic monocyte from the third cell culture system; and g) administering the hepatogenic monocyte to the subject, wherein the hepatogenic monocyte exerts regenerative functions, immune modulatory functions, or both. In certain embodiments, the hepatogenic monocyte is capable of reversing liver failure in the subject. In one embodiment, the primary somatic cell is selected from the group consisting of: a fibroblast, a lymphocyte, a myeloid cell, a neutrophil, an eosinophil, a basophil, a myeloblast, a monocyte, a macrophage, a mesenchymal stem cell, a keratinocyte, an epithelial cell, a renal epithelial cell, an epithelial progenitor cell, a hematopoietic stem cell, a common myeloid progenitor, a natural killer (NK) cell, an NK T cell, a common lymphoid progenitor, an endothelial cell, an endothelial progenitor cell, a synovial cell, a mobilized CD34+ peripheral blood mononuclear cell, a blood mononuclear cell, or aImmorta-LivMon-PCT tissue-specific stem or progenitor cell. In one embodiment, a hepatogenic monocyte shares one or a plurality of morphological, phenotypic, and / or functional characteristics in common with a primary hepatocyte isolated from liver. In one embodiment, the hepatogenic monocyte expresses one or a plurality of transcription factors selected from the group comprising a) signal transducer and activator of transcription 6 (STAT6); b)Peroxisome proliferator-activated receptor gamma (PPAR- -like factor 4(KLF4); d) c-Maf, e) MafB; and f) SP1. In another embodiment, the hepatogenic monocyte produces one or a plurality of molecules selected from the group comprising a) albumin; b) fibrinogen; c) a clotting factor (e.g., factor V, VII, IX, X, XI, XII, protein C, protein S, antithrombin, or a combination thereof); d) transferrin; e) plasminogen; f) ceruloplasmin; g) a complement protein; h) a bactericidal protein; i) an opsonin; j) an iron-sequestering protein; k) bile; l) bicarbonate; m) fetuin-B; and n) an aminotransferase (i.e., ALT and / or AST). In another embodiment, the hepatogenic monocyte produces one or a plurality of cytokines and chemokines selected from the group comprising a) hepatocyte growth factor (HGF); b) insulin-like growth factor-1 (IGF-1); c) fibroblast growth factor (FGF); d) platelet-derived growth factor (PDGF); e) transforming growth factor beta (TGF-beta); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; h) IL-10; i) colony-stimulating factor 1 (CSF1); j) Vascular endothelial growth factor (VEGF); k) Stromal cell-derived factor-1 (SDF-1); l) CXCR4; m) CCR2; n) CX3CR1; o) CXCL12; p) CC chemokine ligand 1 (CCL1); q) CXCR7; r) intercellular adhesionmolecule 1 (ICAM-1); s) tumor necrosis factor alpha (TNF- ); t) IL-22; u) Oncostatin M(OSM); and v) Stem cell factor (SCF). In one embodiment, the hepatogenic monocyte expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15. In one embodiment, the second culture system comprises monocyte conditioned medium, wherein the monocyte conditioned medium may be derived from culture of monocytes that have been stimulated with a toll-like receptor agonist. In one embodiment, the second culture system generates monocytes of the M2-type, wherein the M2-type monocytes are characterizedby production of IL-10, transforming growth factor-beta (TGF- ), or both. In anotherembodiment, the M2-type monocytes express of signal transducer and activator of transcription 6 (STAT6). In one embodiment, the second culture system comprises one or a plurality of factors selected from the group comprising hepatocyte growth factor (HGF),Immorta-LivMon-PCT fibroblast growth factor-1 (FGF-1), FGF-2, bone morphogenetic protein-2 (BMP-2), BMP-4, macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). In one embodiment, the third culture system comprises HGF. In one embodiment, the third culture system comprises one or a plurality of factors selected from the group comprising FGF-1, FGF-2, BMP-2, BMP-4, M-CSF GM-CSF, and LIF. In one embodiment, the induction of expression of one or a plurality of hepatogenic factors into the differentiated monocyte in the third cell culture system is performed by gene transfection. In one embodiment, the transfected genes in the third cell culture system are selected from the group comprising a) HNF4A; b) FOXA2; c) FOXA1; and d) C / EBP alpha. In one embodiment, the third cell culture system comprises serum harvested from a subject with liver failure. In one embodiment, the hepatogenic monocyte generated in the third cell culture system produces insulin-like growth factor 1 (IGF-1).

[0007] Various aspects of the invention are enumerated in the following paragraphs:

[0008] Aspect 1: A method of treating a liver disease or condition utilizing a hepatogenic monocyte capable of inhibiting, stopping or reversing liver damage, wherein the hepatogenic monocyte is generated from an immature progenitor cell or stem cell.

[0009] Aspect 2: The method of Aspect 1, wherein said monocyte is generated from a pluripotent stem cell.

[0010] Aspect 3: The method of Aspect 2, wherein said pluripotent stem cell expresses the marker NANOG.

[0011] Aspect 4: The method of Aspect 2, wherein said pluripotent stem cell expresses the marker OCT4.

[0012] Aspect 5: The method of Aspect 2, wherein said pluripotent stem cell expresses the marker KLF4.

[0013] Aspect 6: The method of Aspect 2, wherein said pluripotent stem cell expresses the marker Lin28.

[0014] Aspect 7: The method of Aspect 2, wherein said pluripotent stem cell expresses the marker SSEA4.

[0015] Aspect 8: The method of Aspect 2, wherein said pluripotent stem cell is an induced pluripotent stem cell.

[0016] Aspect 9: The method of Aspect 2, wherein said pluripotent stem cell is a parthenogenic derived pluripotent stem cell.Immorta-LivMon-PCT

[0017] Aspect 10: The method of Aspect 2, wherein the pluripotent stem cell is a somatic cell nuclear transfer-derived pluripotent stem cell.

[0018] Aspect 11: The method of Aspect 1, wherein the hepatogenic monocyte is generated by culture of a pluripotent stem cell in a media containing M-CSF, angiopoietin, and optionally, interleukin 6.

[0019] Aspect 12: The method of Aspect 1, wherein M-CSF is administered to the pluripotent stem cell at a concentration and duration sufficient to prime the pluripotent stem cell to differentiate into the monocytic lineage when cultured with angiopoietin.

[0020] Aspect 13: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of PU.1.

[0021] Aspect 14: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of PU.1 and TLR4.

[0022] Aspect 15: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of MyD88.

[0023] Aspect 16: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of CD14.

[0024] Aspect 17: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of CD16.

[0025] Aspect 18: The method of Aspect 12, wherein monocytic lineage differentiation is determined by expression of M-CSF receptor.

[0026] Aspect 19: The method of Aspect 12, wherein the pluripotent stem cells are seeded in a decellularized bone marrow matrix and allowed to differentiate into monocytic or monocytic-like cells by initial treatment with M-CSF, followed by GM- CSF, and optionally, followed by treatment with interleukin-6.

[0027] Aspect 20: The method of Aspect 19, wherein the bone marrow is decellularized with one or more alcohols.

[0028] Aspect 21: The method of Aspect 20, wherein the alcohol comprises ethanol.

[0029] Aspect 22: The method of Aspect 20, wherein the alcohol comprises isopropyl alcohol.

[0030] Aspect 23: The method of Aspect 19, wherein the bone marrow is decellularized with polyethylene glycol.

[0031] Aspect 24: The method of Aspect 19, wherein the pluripotent stem cell is introduced to a culture of decellularized bone marrow in a manner allowing for adherence of said pluripotent stem cells to the surface of said decellularized bone marrow.Immorta-LivMon-PCT

[0032] Aspect 25: The method of Aspect 19, wherein the pluripotent stem cell is introduced to a culture of decellularized bone marrow in a manner allowing for adherence of the pluripotent stem cells to the surface of the decellularized bone marrow.

[0033] Aspect 26: The method of Aspect 25, wherein the decellularized bone marrow is first seeded with endothelial cells and / or endothelial progenitor cells prior to introduction of the pluripotent stem cells.

[0034] Aspect 27: The method of Aspect 26, wherein the endothelial progenitor cells are isogenic to the pluripotent stem cells.

[0035] Aspect 28: The method of Aspect 26, wherein the endothelial progenitor cells are allogeneic to the pluripotent stem cells.

[0036] Aspect 29: The method of Aspect 26, wherein the endothelial progenitor cells are xenogeneic to the pluripotent stem cells.

[0037] Aspect 30: The method of Aspect 26, wherein the endothelial progenitor cells are derived from circulating blood progenitor cells.

[0038] Aspect 31: The method of Aspect 30, wherein the circulating blood progenitor cells are isolated by selection for cells expressing the marker CD133.

[0039] Aspect 32: The method of Aspect 30, wherein the circulating blood progenitor cells are isolated by selection for cells expressing the marker CD33.

[0040] Aspect 33: The method of Aspect 30, wherein the circulating blood progenitor cells are isolated by selection for cells expressing the marker CD34.

[0041] Aspect 34: The method of Aspect 30, wherein the circulating blood progenitor cells are isolated by selection for cells expressing the marker c-kit.

[0042] Aspect 35: The method of Aspect 30, wherein the circulating blood progenitor cells are isolated by selection for cells expressing the marker c-met.

[0043] Aspect 36: The method of Aspect 30, wherein said circulating blood progenitor cells are isolated by selection for cells expressing the marker M-CSF receptor.

[0044] Aspect 37: The method of Aspect 30, wherein the circulating endothelial progenitor cells are derived from a patient after treatment of the patient with one or a plurality of regimens to induce an increase in circulating endothelial progenitor cells.

[0045] Aspect 38: The method of Aspect 37, wherein the regimen to induce an increase in circulating endothelial progenitor cells comprises treatment with extracorporeal shock wave therapy.Immorta-LivMon-PCT

[0046] Aspect 39: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating SDF-1 by 25 percent or more.

[0047] Aspect 40: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating SDF-1 by 50 percent or more.

[0048] Aspect 41: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating SDF-1 by 100 percent or more.

[0049] Aspect 42: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating VEGF by 25 percent or more.

[0050] Aspect 43: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating VEGF by 50 percent or more.

[0051] Aspect 44: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating VEGF by 100 percent or more.

[0052] Aspect 45: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF1 by 25 percent or more.

[0053] Aspect 46: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF1 by 50 percent or more.

[0054] Aspect 47: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF1 by 100 percent or more.

[0055] Aspect 48: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF2 by 25 percent or more.

[0056] Aspect 49: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF2 by 50 percent or more.Immorta-LivMon-PCT

[0057] Aspect 50: The method of Aspect 38, wherein the extracorporeal shock wave therapy is applied for a time frame and intensity sufficient to increase the concentration of circulating FGF2 by 100 percent or more.

[0058] Aspect 51: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells comprises treating the patient with G- CSF.

[0059] Aspect 52: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 25 percent or more.

[0060] Aspect 53: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 50 percent or more.

[0061] Aspect 54: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 100 percent or more.

[0062] Aspect 55: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 25 percent or more.

[0063] Aspect 56: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 50 percent or more.

[0064] Aspect 57: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 100 percent or more.

[0065] Aspect 58: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 25 percent or more.

[0066] Aspect 59: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 50 percent or more.

[0067] Aspect 60: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 100 percent or more.Immorta-LivMon-PCT

[0068] Aspect 61: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 25 percent or more.

[0069] Aspect 62: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 50 percent or more.

[0070] Aspect 63: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 100 percent or more.

[0071] Aspect 64: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of angiopoietin by 25 percent or more.

[0072] Aspect 65: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of angiopoietin by 50 percent or more.

[0073] Aspect 66: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of angiopoietin by 100 percent or more.

[0074] Aspect 67: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IGF-1 by 25 percent or more.

[0075] Aspect 68: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IGF-1 by 50 percent or more.

[0076] Aspect 69: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IGF-1 by 100 percent or more.

[0077] Aspect 70: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF-1 by 25 percent or more.

[0078] Aspect 71: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF-1 by 50 percent or more.Immorta-LivMon-PCT

[0079] Aspect 72: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF-1 by 100 percent or more.

[0080] Aspect 73: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of BDNF by 25 percent or more.

[0081] Aspect 74: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of BDNF by 50 percent or more.

[0082] Aspect 75: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of BDNF by 100 percent or more.

[0083] Aspect 76: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 25 percent or more.

[0084] Aspect 77: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 50 percent or more.

[0085] Aspect 78: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 100 percent or more.

[0086] Aspect 79: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 25 percent or more.

[0087] Aspect 80: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 50 percent or more.

[0088] Aspect 81: The method of Aspect 51, wherein the treatment with G-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 100 percent or more.

[0089] Aspect 82: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with GM- CSF.Immorta-LivMon-PCT

[0090] Aspect 83: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 25 percent or more.

[0091] Aspect 84: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 50 percent or more.

[0092] Aspect 85: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 100 percent or more.

[0093] Aspect 86: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-1 RA by 25 percent or more.

[0094] Aspect 87: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-1 RA by 50 percent or more.

[0095] Aspect 88: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-1 RA by 100 percent or more.

[0096] Aspect 89: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble HLA-G by 25 percent or more.

[0097] Aspect 90: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble HLA-G by 50 percent or more.

[0098] Aspect 91: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble HLA-G by 100 percent or more.

[0099] Aspect 92: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of LIF by 25 percent or more.

[0100] Aspect 93: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of LIF by 50 percent or more.Immorta-LivMon-PCT

[0101] Aspect 94: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of LIF by 100 percent or more.

[0102] Aspect 95: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of angiopoietin by 25 percent or more.

[0103] Aspect 96: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of angiopoietin by 50 percent or more.

[0104] Aspect 97: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of GMtin by 100 percent or more.

[0105] Aspect 98: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of endoglin by 25 percent or more.

[0106] Aspect 99: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of endoglin by 50 percent or more.

[0107] Aspect 100: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of endoglin by 100 percent or more.

[0108] Aspect 101: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 25 percent or more.

[0109] Aspect 102: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 50 percent or more.

[0110] Aspect 103: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 100 percent or more.

[0111] Aspect 104: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 25 percent or more.Immorta-LivMon-PCT

[0112] Aspect 105: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 50 percent or more.

[0113] Aspect 106: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF1 by 100 percent or more.

[0114] Aspect 107: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 25 percent or more.

[0115] Aspect 108: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 50 percent or more.

[0116] Aspect 109: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of FGF2 by 100 percent or more.

[0117] Aspect 110: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of stromelysin by 25 percent or more.

[0118] Aspect 111: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of stromelysin by 50 percent or more.

[0119] Aspect 112: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of stromelysin by 100 percent or more.

[0120] Aspect 113: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of CTNF by 25 percent or more.

[0121] Aspect 114: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of CTNF by 50 percent or more.

[0122] Aspect 115: The method of Aspect 82, wherein the treatment with GM-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of CTNF by 100 percent or more.Immorta-LivMon-PCT

[0123] Aspect 116: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with M-CSF.

[0124] Aspect 117: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 25 percent or more.

[0125] Aspect 118: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 50 percent or more.

[0126] Aspect 119: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of SDF-1 by 100 percent or more.

[0127] Aspect 120: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF by 25 percent or more.

[0128] Aspect 121: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF by 50 percent or more.

[0129] Aspect 122: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of EGF by 100 percent or more.

[0130] Aspect 123: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 25 percent or more.

[0131] Aspect 124: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 50 percent or more.

[0132] Aspect 125: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF by 100 percent or more.

[0133] Aspect 126: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of progesterone induced blocking factor by 25 percent or more.Immorta-LivMon-PCT

[0134] Aspect 127: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of progesterone induced blocking factor by 50 percent or more.

[0135] Aspect 128: The method of Aspect116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of progesterone induced blocking factor by 100 percent or more.

[0136] Aspect 129: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF-C by 25 percent or more.

[0137] Aspect 130: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF-C by 50 percent or more.

[0138] Aspect 131: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of VEGF-C by 100 percent or more.

[0139] Aspect 132: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of HGF by 25 percent or more.

[0140] Aspect 133: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of HGF by 50 percent or more.

[0141] Aspect 134: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of HGF by 100 percent or more.

[0142] Aspect 135: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p55 by 25 percent or more.

[0143] Aspect 136: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p55 by 50 percent or more.

[0144] Aspect 137: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p55 by 100 percent or more.Immorta-LivMon-PCT

[0145] Aspect 138: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p75 by 25 percent or more.

[0146] Aspect 139: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p75 by 50 percent or more.

[0147] Aspect 140: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TNF-alpha receptor p75 by 100 percent or more.

[0148] Aspect 141: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble CD25 by 25 percent or more.

[0149] Aspect 142: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble CD25 by 50 percent or more.

[0150] Aspect 143: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble CD25 by 100 percent or more.

[0151] Aspect 144: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble PD-L1 by 25 percent or more.

[0152] Aspect 145: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-L1 by 50 percent or more.

[0153] Aspect 146: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-L1 by 100 percent or more.

[0154] Aspect 147: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-1 by 25 percent or more.

[0155] Aspect 148: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-1 by 50 percent or more.Immorta-LivMon-PCT

[0156] Aspect 149: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-1 by 100 percent or more.

[0157] Aspect 150: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-L2 by 25 percent or more.

[0158] Aspect 151: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-L2 by 50 percent or more.

[0159] Aspect 152: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of PD-L2 by 100 percent or more.

[0160] Aspect 153: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-10 by 25 percent or more.

[0161] Aspect 154: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-10 by 50 percent or more.

[0162] Aspect 155: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-10 by 100 percent or more.

[0163] Aspect 155: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-13 by 25 percent or more.

[0164] Aspect 156: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-13 by 50 percent or more.

[0165] Aspect 157: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-13 by 100 percent or more.

[0166] Aspect 158: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-20 by 25 percent or more.Immorta-LivMon-PCT

[0167] Aspect 159: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-20 by 50 percent or more.

[0168] Aspect 160: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-20 by 100 percent or more.

[0169] Aspect 161: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 25 percent or more.

[0170] Aspect 162: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 50 percent or more.

[0171] Aspect 163: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-22 by 100 percent or more.

[0172] Aspect 164: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 25 percent or more.

[0173] Aspect 165: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 50 percent or more.

[0174] Aspect 166: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-35 by 100 percent or more.

[0175] Aspect 167: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-37 by 25 percent or more.

[0176] Aspect 168: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-37 by 50 percent or more.

[0177] Aspect 169: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-37 by 100 percent or more.Immorta-LivMon-PCT

[0178] Aspect 170: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-38 by 25 percent or more.

[0179] Aspect 171: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-38 by 50 percent or more.

[0180] Aspect 172: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of IL-38 by 100 percent or more.

[0181] Aspect 173: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 alpha by 25 percent or more.

[0182] Aspect 174: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 alpha by 50 percent or more.

[0183] Aspect 175: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 alpha by 100 percent or more.

[0184] Aspect 176: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 beta by 25 percent or more.

[0185] Aspect 177: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 beta by 50 percent or more.

[0186] Aspect 178: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MIP-1 beta by 100 percent or more.

[0187] Aspect 179: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TREM2 by 25 percent or more.

[0188] Aspect 180: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TREM2 by 50 percent or more.Immorta-LivMon-PCT

[0189] Aspect 181: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of soluble TREM2 by 100 percent or more.

[0190] Aspect 182: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR155 by 25 percent or more.

[0191] Aspect 183: The method of Aspect 116,wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR155 by 50 percent or more.

[0192] Aspect 184: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR155 by 100 percent or more.

[0193] Aspect 185: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR146a by 25 percent or more.

[0194] Aspect 186: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR146a by 50 percent or more.

[0195] Aspect 187: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of miR146a by 100 percent or more.

[0196] Aspect 188: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP1 by 25 percent or more.

[0197] Aspect 189: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP1 by 50 percent or more.

[0198] Aspect 190: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP1 by 100 percent or more.

[0199] Aspect 191: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP3 by 25 percent or more.Immorta-LivMon-PCT

[0200] Aspect 192: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP3 by 50 percent or more.

[0201] Aspect 193: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP3 by 100 percent or more.

[0202] Aspect 194: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP7 by 25 percent or more.

[0203] Aspect 195: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP7 by 50 percent or more.

[0204] Aspect 196: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP7 by 100 percent or more.

[0205] Aspect 197: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP9 by 25 percent or more.

[0206] Aspect 198: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP9 by 50 percent or more.

[0207] Aspect 199: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP9 by 100 percent or more.

[0208] Aspect 200: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP11 by 25 percent or more.

[0209] Aspect 201: The method Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP11 by 50 percent or more.

[0210] Aspect 202: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP11 by 100 percent or more.Immorta-LivMon-PCT

[0211] Aspect 203: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP13 by 25 percent or more.

[0212] Aspect 204: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP13 by 50 percent or more.

[0213] Aspect 205: The method of Aspect 116, wherein the treatment with M-CSF is administered at a concentration and frequency sufficient in increase circulating concentration of MMP13 by 100 percent or more.

[0214] Aspect 206: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with FLT3.

[0215] Aspect 207: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-1.

[0216] Aspect 208: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-2.

[0217] Aspect 209: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with a TLR4 agonist.

[0218] Aspect 210: The method of Aspect 209, wherein said TLR4 agonist comprises lipopolysaccharide.

[0219] Aspect 211: The method of Aspect 210, wherein said lipopolysaccharide is administered together with one or a plurality of antioxidants.

[0220] Aspect 212: The method of Aspect 211, wherein the antioxidant comprises n- acetylcysteine.

[0221] Aspect 213: The method of Aspect 211, wherein the antioxidant comprises bucillamine.

[0222] Aspect 214: The method of Aspect 211, wherein the antioxidant comprises resveratrol.

[0223] Aspect 215: The method of Aspect 211, wherein the antioxidant comprises sulforaphane.

[0224] Aspect 216: The method of Aspect 211, wherein the antioxidant comprises ascorbic acid.Immorta-LivMon-PCT

[0225] Aspect 217: The method of Aspect 211, wherein the antioxidant comprises alpha-tocopherol.

[0226] Aspect 218: The method of Aspect 211, wherein the antioxidant comprises an omega 3 fatty acid.

[0227] Aspect 219: The method of Aspect 211, wherein the antioxidant comprises retinoic acid.

[0228] Aspect 220: The method of Aspect 211, wherein the antioxidant comprises all trans retinoic acid.

[0229] Aspect 221: The method of Aspect 211, wherein the antioxidant comprises lenalidomide.

[0230] Aspect 222: The method of Aspect 211, wherein the antioxidant comprises thalidomide.

[0231] Aspect 223: The method of Aspect 211, wherein the antioxidant comprises EGCG.

[0232] Aspect 224: The method of Aspect 211, wherein the antioxidant comprises pterostilbene.

[0233] Aspect 225: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-5.

[0234] Aspect 226: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-6.

[0235] Aspect 227: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-8.

[0236] Aspect 228: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-11.

[0237] Aspect 229: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-12.

[0238] Aspect 230: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-15.Immorta-LivMon-PCT

[0239] Aspect 231: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-17.

[0240] Aspect 232: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interleukin-18.

[0241] Aspect 233: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interferon alpha.

[0242] Aspect 234: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with interferon gamma.

[0243] Aspect 235: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with TNF- alpha.

[0244] Aspect 236: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with lymphotoxin.

[0245] Aspect 237: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with TRANCE.

[0246] Aspect 238: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with RANK ligand.

[0247] Aspect 239: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with flagellin.

[0248] Aspect 240: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with Poly (I:C).

[0249] Aspect 241: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with Poly (A:U).Immorta-LivMon-PCT

[0250] Aspect 242: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with DNA CpG motifs.

[0251] Aspect 243: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with beta glucan.

[0252] Aspect 244: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with monocyte conditioned media.

[0253] Aspect 245: The method of Aspect 37, wherein the regimen stimulating an increase in circulating endothelial progenitor cells is treatment of the patient with mixed lymphocyte reaction .

[0254] Aspect 246: The method of Aspect 1, wherein the hepatogenic monocyte is generated by culture of a pluripotent stem cell in the presence of a monocytic differentiation media, and wherein the monocytic differentiation media comprises one or a plurality of factors that induce the generation of monocytes with a Type 2 phenotype and function.

[0255] Aspect 247: The method of Aspect 246, wherein the monocytic differentiation media comprises one or a plurality of factors comprising FGF-1, FGF-2, BMP-2, BMP-4, M-CSF and GM-CSF.

[0256] Aspect 248: The method of Aspect 247, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 100% compared to baseline.

[0257] Aspect 249: The method of Aspect 247, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 200% compared to baseline.

[0258] Aspect 250: The method of Aspect 247, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 500% compared to baseline.

[0259] Aspect 251: The method of Aspect 247, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 100% compared to baseline.Immorta-LivMon-PCT

[0260] Aspect 252: The method of Aspect 247, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 200% compared to baseline.

[0261] Aspect 253: The method of Aspect 246, wherein the cells are treated initially with FGF-1 at a concentration and duration sufficient to increase expression of PU1 by 500% compared to baseline.

[0262] Aspect 254: The method of Aspect 246, wherein the monocytic differentiation media comprises monocyte conditioned media.

[0263] Aspect 255: The method of Aspect 254, wherein FGF-1 is added to the monocyte conditioned media.

[0264] Aspect 256: The method of Aspect 254, wherein FGF-2 is added to the monocyte conditioned media.

[0265] Aspect 257: The method of Aspect 254, wherein BMP-2 is added the monocyte conditioned media.

[0266] Aspect 258: The method of Aspect 254, wherein BMP-4 is added to the monocyte conditioned media.

[0267] Aspect 259: The method of Aspect 254, wherein M-CSF is added to the monocyte conditioned media.

[0268] Aspect 260: The method of Aspect 254, wherein GM-CSF is added to the monocyte conditioned media.

[0269] Aspect 261: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, and GM-CSF are added to the monocyte conditioned media.

[0270] Aspect 262: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, and GM-CSF are added to the monocyte conditioned media.

[0271] Aspect 263: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, HGF and GM-CSF are added to the monocyte conditioned media.

[0272] Aspect 264: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, LIF and GM-CSF are added to the monocyte conditioned media.

[0273] Aspect 265: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, VEGF and GM-CSF are added to the monocyte conditioned media.

[0274] Aspect 266: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, TGF-beta and GM-CSF are added to the monocyte conditioned media.

[0275] Aspect 267: The method of Aspect 254, wherein FGF-1, FGF-2, BMP-2, BMP-4, M-CSF, endoglin and GM-CSF are added to the monocyte conditioned media.Immorta-LivMon-PCT

[0276] Aspect 268: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating a culture of monocytes with one or more agents / treatments that increases nuclear translocation of NF-kappa B.

[0277] Aspect 269: The method of Aspect 268, wherein NF-kappa B comprises the rel A component.

[0278] Aspect 270: The method of Aspect 268, wherein NF-kappa B comprises the rel B component.

[0279] Aspect 271: The method of Aspect 268, wherein NF-kappa B comprises the rel c component.

[0280] Aspect 272: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating a culture of monocytes with one or more agents / treatments that increases expression of CD14.

[0281] Aspect 273: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating a culture of monocytes with one or more agents / treatments that increases expression of CD40.

[0282] Aspect 274: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of CD80.

[0283] Aspect 275: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of CD86.

[0284] Aspect 276: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of CD56.

[0285] Aspect 277: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of CD57.

[0286] Aspect 278: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of TREM1.

[0287] Aspect 279: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of calreticulin.Immorta-LivMon-PCT

[0288] Aspect 280: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of HMGB1.

[0289] Aspect 281: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of hsp90.

[0290] Aspect 282: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of human IL-1 receptor antagonist.

[0291] Aspect 283: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-6.

[0292] Aspect 284: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-8.

[0293] Aspect 285: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-11.

[0294] Aspect 286: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-12.

[0295] Aspect 287: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-15.

[0296] Aspect 288: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-18.

[0297] Aspect 289: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-21.

[0298] Aspect 290: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-17.Immorta-LivMon-PCT

[0299] Aspect 291: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-23.

[0300] Aspect 292: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-27.

[0301] Aspect 293: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of IL-33.

[0302] Aspect 294: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of TRANCE.

[0303] Aspect 295: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of TRAIL.

[0304] Aspect 296: The method of Aspect 254, wherein the monocyte conditioned media is generated by stimulating monocytes with one or more agents / treatments that increases expression of LIGHT.

[0305] Aspect 297: The method of Aspect 254, wherein the monocyte conditioned media is generated by a first step of treating a population of monocytes to polyinosinic- polycytidylic acid [Poly (I:C)] or its derivative stabilized with carboxymethylcellulose and poly-L-lysine (poly-ICLC) to induce the expression of one or a plurality of factors by the monocytes, and a second step of culturing the monocytes to generate the conditioned media comprising the one or plurality of factors.

[0306] Aspect 298: The method of Aspect 297, wherein the first step is conducted by adding poly(I:C) or poly-ICLC to a culture of monocytes having one or a plurality of the following phenotypic characteristics: a) plastic adherence; b) >90% CD14-positive cells; c) >90% CD16-positive cells adherent; and d) >90% toll-like receptor 4 (TLR4)-positive cells.

[0307] Aspect 299: The method of Aspect 297, wherein poly(I:C) or poly-ICLC are provided to a culture of monocytes at a concentration and duration that is sufficient to induce an increase in production of hepatocyte growth factor (HGF) that is at least 25%, at least 50%, or at least 100% increased as compared to production by control monocytes,Immorta-LivMon-PCT wherein control monocytes comprise a population of monocytes that has not been treated with poly(I:C) or poly-ICLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0308] Figure 1 is a bar graph showing the induction of albumin-producing hepatogenic monocytes from induced pluripotent stem cells.

[0309] Figure 2 is a bar graph showing hepatogenic activity of hepatogenic monocytes in vivo.

[0310] Figure 3 is a bar graph showing hepatogenic activity of iPSC derived monocytes augmented in c-kit expressing monocytes. DETAILED DESCRIPTION

[0311] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0312] Liver failure poses a considerable health and economic burden worldwide and has limited treatment options. Liver transplantation is a therapeutic option that is severely limited due to the scarcity of transplantable donor organs. Transplantation of primary hepatocytes represents an effective alternative approach however large numbers of functional cells are required to reconstitute the inflamed and injured liver milieu. Expansion of primary hepatocytes has been challenging due to difficulties in growing these cells while retaining their functions in vitro. Accordingly. alternative sources of hepatocytes or hepatocyte-like cells are being explored as therapeutic options. Cell therapy utilizing non-hepatocyte cell types that have been generated or engineered to have hepatocyte functions remains a promising means for treating or limiting the progression of liver diseases by restoring the defective or deficient functions of endogenous hepatocytes. There is a need to address various liver diseases or conditions, including acute liver diseases, chronic liver diseases, and inherited metabolic disorders, that have different therapeutic requirements for facilitating hepatocyte regeneration or repair.Immorta-LivMon-PCT

[0313] As used herein, the terms “liver disease” or “liver condition” may be used interchangeably to refer to a range of conditions that affect the liver’s function, which may range from mild to severe and may be acute or chronic / progressive in nature. In certain embodiments, liver disease may result from one or a plurality of factors including but not limited to Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, alcoholic fatty liver disease, alcoholic cirrhosis, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), hemochromatosis, Wilson’s Disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, fibrolamellar carcinoma, angiosarcoma, hemangiosarcoma, metastatic liver cancer, other infections, inherited metabolic disorders, autoimmunity, exposure to toxins, substance abuse such as drug use, or liver damage that arises as a complication of another condition or injury. The terms “liver disease” or “liver condition” may also be used to refer to any specific degree of liver damage or dysfunction including but not limited to liver fibrosis, liver cirrhosis, and liver failure. In certain embodiments, a liver disease or condition may be defined or identified by the presence of specific cellular changes or molecular biomarkers in the organ including but not limited to tissue necrosis, oxidative stress, and inflammation.

[0314] As used herein, “liver failure” refers to acute or chronic damage to the liver that reduces or interferes with one or a plurality of the essential functions of this organ such as filtering blood, producing bile, and others. In the context of the invention, liver failure may be diagnosed in a subject based on one or a plurality of tests including blood tests to measure liver enzymes (e.g., ALT and AST), bilirubin levels, ultrasound, CT scan, MRI, liver biopsy, or other assessments.

[0315] As used herein, “liver fibrosis” refers to the accumulation of scar tissue in the liver due to inflammation, injury, or damage. Liver fibrosis may be identified using techniques such as a liver biopsy and histology to detect extracellular matrix deposition within liver parenchyma, and semi-quantitative scoring systems to categorize the severity, e.g., the METAVIR scale for hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, or Brunt criteria for nonalcoholic steatohepatitis (NASH), assign ordinal scores ranging from 0 to 4: 0 for no fibrosis; 1 for mild fibrosis; 2 for significant fibrosis; 3 for advanced fibrosis; 4 for cirrhosis. Liver fibrosis may also be identified by FibroScan or ultrasound (US) elastography or magnetic resonance (MR) elastography to quantify liver stiffness.Immorta-LivMon-PCT

[0316] As used herein, “liver cirrhosis” refers to a more advanced stage of liver fibrosis, wherein there is widespread scarring in the liver that may involve irreversible changing to liver structure or function. In certain embodiments, the degree of liver fibrosis in a subject can be monitored by measuring liver markers in blood, ultrasound, CT scan, MRI, or elastography.

[0317] As used herein, “liver regeneration” refers to the process by which the liver replaces damaged or lost cells or tissue. This term may be used to describe the cellular and biological mechanisms by which a damaged liver undergoes repair. These processes may involve one or a plurality of processes including but not limited to upregulation of hepatocyte genes involved in cell cycle progression, DNA repair, and mitochondrial function, proliferation of hepatocytes, proliferation of progenitor cells known as oval cells, differentiation of oval cells into hepatocytes, differentiation of oval cells into cholangiocytes, recruitment or activity of immune cells, release of cytokines, chemokines, or angiogenic factors, and proliferation or cytokine production by other non-parenchymal cell populations such as biliary epithelial cells.

[0318] As used herein, “monocyte” refers to a white blood cell that is naturally produced in the bone marrow, is present in circulation, and can localize to injured and inflamed tissues and differentiate locally into diverse myeloid cell populations, manifesting such functions as phagocytosis, antiviral immunity, antigen presentation, immune suppression and tissue repair. In certain embodiments, a monocyte can be produced from an induced pluripotent stem cell in vitro.

[0319] As used herein, “monocyte derivative” refers to a cell that has differentiated from a monocyte or has been induced or modified from a monocyte.

[0320] As used herein, “monocyte precursor” refers to a cell that can give rise to a monocyte either naturally in the body or from differentiation or induction in vitro (e.g., a monoblast, a committed precursor, or a hematopoietic stem cell)

[0321] As used herein, “macrophage” refers to an immune cell in the body that may have roles in the regulation of homeostasis, inflammation, immunity, or immune suppression in a tissue-specific and context-dependent manner. Macrophages may be classified into classically activated (also called “M1”) macrophages, which are induced -- -like receptor (TLR) ligand, and alternatively activated (also called “M2”) macrophages that result from the stimulation of Th2 signature cytokines such as interleukin-4 (IL-4) and / or IL-13. Macrophage M2 polarization involves tyrosine phosphorylation andImmorta-LivMon-PCT activation of a signal transducer and activator of transcription 6 (STAT6), which induces transcriptional activation of specific genes including but not limited to arginase 1 (Arg1),mannose receptor 1 (Mrc1), resistin- Retnla, Fizz1), chitinase-like protein 3 (Chil3,Ym1), and the chemokine genes Ccl17 and Ccl24.

[0322] As used herein, “M2-type” of “M2” monocyte or macrophage refers to a polarized cell type that may have roles in tissue repair, suppression of inflammation, T cell suppression, induction of regulatory T cells, promoting angiogenesis, and stimulating proliferation or growth of specific cell types. An M2 type cell exerts opposing or contrasting functions to an M1-type monocyte or macrophage that is involved in inducing inflammation.

[0323] As used herein “hepatogenic cell” refers to a cell type that has differentiated or has been induced through genetic engineering or other means outside of normal in vivo biological processes to resembles a cell in the liver. In certain contexts, a hepatogenic cell may not be identical to a hepatocyte from the liver but may share one or a plurality of morphological or phenotypic characteristics and / or functions in common with a hepatocyte. In certain embodiments, a hepatogenic cell may be derived from a pluripotent stem cell (such as an induced pluripotent stem cell) or from an embryonic stem cell. A hepatogenic cell may also be referred to as a “hepatocyte-like” cell.

[0324] As used herein, “hepatogenic monocyte” or “hepatocyte-like monocyte” refers to a monocyte that has certain morphologic, phenotypic, functional, and / or molecular characteristics in common with a hepatocyte. By way of example, a hepatogenic monocyte may express or synthesize one or a plurality of liver-specific proteins such as albumin, alpha-1 antitrypsin, transferrin, transthyretin, cytokeratin-18,and / or hepatocyte nuclear factor 4 alpha (HNF4 ). As another non-limiting example, ahepatogenic monocyte may possess one or a plurality of functions of hepatocytes including but not limited to glycogen storage, urea production, and drug metabolism. In certain contexts, a hepatogenic monocyte may promote regeneration, repair, or maintenance of liver tissue. In certain contexts, a hepatogenic monocyte may circulate to the liver to facilitate repair in that tissue. A hepatogenic monocyte may be inducible to undergo differentiation into another cell type, for example, a specialized tissue-specific macrophage such as a Kupffer cell in the liver. A hepatogenic monocyte may produce cytokines or growth factors. A hepatogenic monocyte may be generated by inducing a stem cell or a derivative thereof to differentiate along the hepatic lineage, by manipulatingImmorta-LivMon-PCT or engineering a stem cell using specific growth factors, cytokines, small molecules, or by introducing genes and / or by transfecting a monocyte to express certain genes associated with liver cell development or hepatocyte phenotype or function. In certain embodiments, a hepatogenic monocyte expresses phenotypic or functional characteristics of a monocyte. In certain embodiments, a hepatogenic monocyte does not fully express all the genes and functions of either a monocyte or a hepatocyte. In certain embodiments, a hepatogenic monocyte is generated or induced from a stem cell (e.g., an induced pluripotent stem cell), wherein the hepatogenic monocyte may not fully express all the genes and functions of an adult hepatocyte. In certain embodiments, a hepatogenic monocyte may refer to a cell that is engineered or created in vitro to mimic the characteristics of a hepatocyte, or of another cell type of the liver.

[0325] As used herein, “myeloid lineage cell” refers to a common myeloid progenitor or its differentiated or terminally differentiated cell types, which may include monocytes, granulocytes, and dendritic cells.

[0326] As used herein, “myeloid-derived suppressor cell” (“MDSC”) refers an immature myeloid cell that exerts one or a plurality of immune suppressive or anti- inflammatory functions. By way of example, a myeloid-derived suppressor cell may suppress cytotoxic T cell and natural killer (NK) cell responses, induce regulatory T cells, and suppress effector T cell responses. In certain contexts, myeloid-derived suppressor cells may suppress inflammation in the liver.

[0327] 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). This technology was pioneered by the introduction of four specific genes encoding transcription factors Oct3 / 4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under embryonic stem (ES) cell culture conditions.

[0328] As used herein, “mesenchymal stem cell” (“MSC”) refers to a cell that possesses the following characteristics: (1) adherence to plastic, (2) expression of CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) possessing the ability to differentiate into the osteogenic, chondrogenic and adipogenic lineages. 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, “MSC” includes cells that are isolated from tissues using cell surface markers selectedImmorta-LivMon-PCT 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).

[0329] As used herein the terms "subject” or “patient” are used interchangeably, and refer to any animal, such as a mammal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient or subject is an animal or a human.

[0330] As used herein, the term "therapeutic" refers to a treatment or a prophylaxis. A therapeutic effect is obtained by suppression, remission, reduction, or eradication of a disease state.

[0331] As used herein, the term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0332] As used herein, “dedifferentiation” refers to loss of specialization in form or function. In cells, dedifferentiation leads to an a less committed cell.

[0333] 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.Immorta-LivMon-PCT

[0334] 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.

[0335] 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.

[0336] 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 a cellular environment. Responsive cells respond to and function in a particular cellular environment, tissue, organ and / or organ system.

[0337] Throughout this disclosure, various aspects of the invention can be presented in a range format. Any description presented herein in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0338] 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.

[0339] Throughout this application, the term “about” is used according to its plain and ordinary meaning in cell and molecular biology to indicate that a value includes the standard deviation of error for the method or procedure being employed to determine the value.

[0340] The present invention discloses methods of treating and preventing liver conditions including liver failure in a subject. In one embodiment, the invention provides cell therapies comprising cell types that have been endowed with biological capabilities to promote liver regeneration or repair. In one embodiment, therapeutic cells are provided to replace damaged, apoptotic, or necrotic cells in the liver. In one embodiment, cell therapies are provided utilizing cells that modulate inflammation or otherwise support theImmorta-LivMon-PCT functions of essential liver cell types. In one embodiment, methods are provided for restoring or improving essential liver functions in a subject such as by regulating glucose homeostasis, lipid metabolism, detoxification and other physiological processes.

[0341] In one embodiment, the methods of the invention are applied to improve the numbers or functions of one or a plurality of cell types in the liver selected from the group comprising a) hepatocytes; b) cholangiocytes; c) hepatic stellate cells; d) Kupffer cells; e) liver sinusoid endothelial cells; f) liver progenitor cells, or oval cells; g) mesenchymal stem cells; f) myeloid-derived suppressor cells; g) myeloid lineage cells such as monocytes; and h) T cells. In one embodiment, methods are provided for improving the numbers or functions of monocytes or macrophages in the liver. In certain embodiments, it is desirable to increase or improve the numbers of M2-type (M2) macrophages in the liver, wherein the M2 macrophages are defined by expression of one or a plurality of surface molecules selected from the group comprising CD163, CD206, and arginase 1. In one embodiment, a therapeutic cell type comprising an M2-type monocyte or an M2-type macrophage is provided for treating a subject with liver failure. In certain embodiments, it is desirable to apply a method of the invention to increase the concentrations of monocytes in the liver, wherein the monocytes possess a proclivity to differentiate into M2 monocytes or M2 macrophages. In one embodiment, it is desirable to increase or improve the functions of M2-type monocytes or macrophages in the liver, wherein the functions of M2 monocytes or macrophages are defined by expression of one or a plurality of cytokines selected from the group comprising IL-1 receptor antagonist(IL-1RA), IL-4, IL-10, and TGF- . In one embodiment, methods are provided forimproving the numbers or functions of one or a plurality of T cells subsets in the liver that are selected from the group comprising a) CD4+ T cells; b) CD8+T cells; c) natural killerT (NKT) cells; d) gamma delta ( ) T cells; e) mucosal-associated invariant T (MAIT)cells; and f) FoxP3+ T cells.

[0342] In one embodiment, a therapeutic cell population is provided for treating a liver disease such as liver failure in a subject in need thereof, wherein the therapeutic cell population is selected from the cell type comprising a) a monocyte; b) a macrophage; c) a mesenchymal stem cell (MSC); d) a myeloid lineage cell; e) a myeloid-derived suppressor cell (MDSC); f) a dendritic cell; g) a neutrophil; h) an eosinophil; and i) a basophil. In one embodiment, a therapeutic cell is administered to a subject with liver failure or another liver condition.Immorta-LivMon-PCT

[0343] In one embodiment, a hepatogenic cell population is provided for treating a liver disease in a subject in need thereof, wherein the hepatogenic cell population is selected from the cell type comprising a) a monocyte; b) a macrophage; c) a mesenchymal stem cell; d) a myeloid lineage cell; e) a myeloid-derived suppressor cell; f) a dendritic cell; and g) a granulocyte such as a neutrophil, an eosinophil, or a basophil. In one embodiment, a hepatogenic cell is administered to a subject with liver failure.

[0344] In certain embodiments, a hepatogenic cell is provided that is not phenotypically or functionally identical to a primary hepatocyte isolated from liver tissue but shares one or a plurality of morphological or phenotypic characteristics and / or functions in common with a hepatocyte. For example, a hepatogenic cell produced by the methods of the invention may comprise a monocyte that has phenotypic and molecular characteristics of both a monocyte and a hepatocyte.

[0345] Embodiments of the invention disclose methods for generating an iPSC from a somatic cell and subsequent methods and steps for differentiating the iPSC cell to ultimately generate a therapeutic cell type that is administered to a subject in need thereof. In some embodiments, a cell culture method is provided for differentiated an iPSC into a cell type that is hepatocyte-like. In some embodiments, a first cell culture method is provided for differentiating an iPSC into a lineage-specific cell such as a monocyte, and a second cell culture method is provided for differentiating or inducing the lineage-specific cell to be hepatocyte-like.

[0346] Embodiments of the invention provide iPSCs that are generated from somatic cells of a subject that are subsequently differentiated into autologous or allogeneic therapeutic cells. In one embodiment, the iPSC is derived from a somatic cell type that is selected from the group comprising a) a fibroblast; b) a peripheral blood cell; c) a peripheral blood lymphocyte; d) a monocyte; e) a macrophage; f) a dendritic cell; g) a granulocyte; h) a fibroblast; i) a keratinocyte; j) an epithelial cell; k) an endothelial cell; l) an umbilical cord stem cell; m) a hematopoietic stem cell; n) a mesenchymal stem cell; o) a placental cell; p) a T cell; q) a B cell; and r) a renal epithelial cell. In certain embodiments, the invention provides an iPSC-derived cell composition and methods of use thereof that is therapeutically advantageous for treating liver failure as compared to a primary cell of the same type or lineage from the subject that has not undergone reprogramming or modification. The invention thereby provides cells with specific properties that confer improved therapeutic potential over primary cells.Immorta-LivMon-PCT

[0347] In one embodiment, a hepatogenic cell comprises a cell that has differentiated or induced through genetic engineering or other means outside of normal in vivo biological processes to resembles a cell in the liver with respect to certain characteristics. In one embodiment, an induced pluripotent stem cell is transfected with genes encoding transcription factors that regulate liver development including but not limited to HNF4A, FOXA2, FOXA1, and C / EBP alpha. In another embodiment, a monocyte produced using the methods of the invention is transfected with one or a plurality of genes that are typically expressed by hepatocytes. In one embodiment, a monocyte is transfected with the gene for hepatocyte growth factor (HGF), which stimulates the growth of liver cells.

[0348] In one embodiment, a hepatogenic cell comprises a cell that has been induced to resemble a hepatocyte using a cell culture method. For example, specific growth factors, cytokines, and / or conditioned media may be provided to induce an iPSC or a derivative thereof such as a lineage-specific cell to become hepatocyte-like. In one embodiment, a hepatogenic cell is generated by exposure of an iPSC or a derivative thereof (e.g., an iPSC-derived monocyte) to monocyte differentiation medium, wherein the monocytic differentiation media comprises one or a plurality of factors selected from the group comprising fibroblast growth factor 1 (FGF-1), FGF-2, bone morphogenetic protein-2 (BMP-2), BMP-4, macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), angiopoietin, IL-6, and HGF. In one embodiment, the monocyte differentiation media that is provided for culturing an iPSC or a derivative thereof comprises conditioned medium. This conditioned medium may be generated from separate cultures of monocytes (e.g., primary human monocytes), wherein the monocytes have been stimulated under specific conditions to generate a medium that is rich in cytokines and growth factors. In one embodiment, the monocyte conditioned medium is generated by culturing monocytes in the presence of an inflammatory stimulus and / or a stimulus that mimics liver injury or infection, for example, by stimulating the monocytes with a toll- like receptor agonist [e.g., poly(I:C)], one or a plurality of agents / treatments that increasenuclear factor kappa B (NF- B) activation, and / or one or a plurality of agents / treatmentsthat increase cytokine production into the medium. In another embodiment, the monocyte conditioned medium is generated by culturing monocytes in the presence of serum from a subject with liver failure. The culture media from monocytes that is generated using theseImmorta-LivMon-PCT means is then harvested and applied to the methods of the invention. In one embodiment, the conditioned medium from monocytes that were stimulated with TLR agonists and / or other agents is applied for culturing an iPSC or a derivative thereof to generate a hepatogenic cell type. In another embodiment, the monocyte differentiation media that is provided for culturing an iPSC or a derivative thereof comprises serum that has been collected from a subject with liver disease.

[0349] In a specific embodiment, a hepatogenic monocyte is generated by culture of a pluripotent stem cell in the presence of a monocytic differentiation media, wherein the monocytic differentiation medium comprises one or a plurality of factors that induce the generation of monocytes from iPSC or another stem / progenitor cell. In one embodiment, the monocyte differentiation medium comprises conditioned medium from separate cultures of monocytes, wherein the monocytes are provided with conditions to stimulate the production and release of cytokines, growth factors, adhesion molecules, vesicles, proteins, or nucleic acids . In one embodiment, the monocyte differentiation medium comprises one or a plurality of molecules selected from the group comprising: a) fibroblast growth factor 1 (FGF-1); b) fibroblast growth factor 2 (FGF-2); c) bone morphogenetic protein-2 (BMP-2); d) BMP-4; e) macrophage colony-stimulating factor (M-CSF); f) granulocyte-macrophage colony-stimulating factor (GM-CSF); g) endoglin; h) vascular endothelial growth factor (VEGF); and i) transforming growth factor-beta(TGF- ). In one embodiment, the one or plurality of molecules are provided in arecombinant form. In one embodiment, one or a plurality of additional recombinant growth factors and cytokines are added to a monocyte differentiation medium or to a conditioned medium.

[0350] In one embodiment, a derivative of an iPSC (e.g., a lineage specific cell such as a myeloid lineage cell, a monocyte, or a mesenchymal stem cell) generated using the methods of the invention produces one or a plurality of cytokines or growth factors selected from the group comprising a) hepatocyte growth factor (HGF); b) insulin-like growth factor-1 (IGF-1); c) fibroblast growth factor (FGF); d) platelet-derived growth factor (PDGF); e) transforming growth factor beta (TGF-beta); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; h) IL-10; i) colony-stimulating factor 1 (CSF1); j) Vascular endothelial growth factor (VEGF); k) Stromal cell-derived factor-1 (SDF-1); l) CXCR4; m) CCR2; n) CX3CR1; o) CXCL12; p) CC chemokine ligand 1 (CCL1); q)Immorta-LivMon-PCT CXCR7; r) intercellular adhesion molecule 1 (ICAM-1); s) tumor necrosis factor alpha(TNF- ); t) IL-22; u) Oncostatin M (OSM); and v) Stem cell factor (SCF).

[0351] In one embodiment, a hepatogenic cell generated using the methods of the invention produces one or a plurality of cytokines or growth factors selected from the group comprising a) hepatocyte growth factor (HGF); b) insulin-like growth factor-1 (IGF-1); c) fibroblast growth factor (FGF); d) platelet-derived growth factor (PDGF); e) transforming growth factor beta (TGF-beta); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; h) IL-10; i) colony-stimulating factor 1 (CSF1); j) Vascular endothelial growth factor (VEGF); k) Stromal cell-derived factor-1 (SDF-1); l) CXCR4; m) CCR2; n) CX3CR1; o) CXCL12; p) CC chemokine ligand 1 (CCL1); q) CXCR7; r)intercellular adhesion molecule 1 (ICAM-1); s) tumor necrosis factor alpha (TNF- ); t)IL-22; u) Oncostatin M (OSM); and v) Stem cell factor (SCF).

[0352] In one embodiment, an iPSC-derived cell generated using the methods of the invention expresses one or a plurality of transcription factors selected from the group comprising a) signal transducer and activator of transcription 6 (STAT6); b) Peroxisomeproliferator-activated receptor gamma (PPAR- -like factor 4 (KLF4); d) c-Maf, e) MafB; and f) SP1.

[0353] In one embodiment, a hepatogenic cell generated using the methods of the invention expresses one or a plurality of transcription factors selected from the group comprising a) signal transducer and activator of transcription 6 (STAT6); b) Peroxisomeproliferator-activated receptor gamma (PPAR- -like factor 4 (KLF4); d) c-Maf, e) MafB; and f) SP1.

[0354] In one embodiment, a monocyte derived from an iPSC expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15. In one embodiment, a hepatogenic cell comprises an M2 monocyte.

[0355] In one embodiment, a hepatogenic cell comprises a monocyte, wherein a hepatogenic monocyte expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15. In one embodiment, a hepatogenic cell comprises an M2 monocyte.Immorta-LivMon-PCT

[0356] In one embodiment, a hepatogenic cell comprises a macrophage, wherein the hepatogenic macrophage expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15. In one embodiment, a hepatogenic cell comprises an M2 macrophage.

[0357] In one embodiment, a hepatogenic cell possesses one or a plurality of the following functions: a) carbohydrate metabolism; b) lipid metabolism; c) protein synthesis; d) detoxification; and e) immune regulation.

[0358] In one embodiment, a hepatogenic cell expresses or produces one or a plurality of the following molecules: a) soluble CD14; b) soluble MD-2; c) transferrin; d) lipocalin-2; e) hepcidin; f) hemopexin; g) fibrinogen; h) PGLYP2; i) LBP; j) a complement component such as C1r / s, C2, C4, C3, factor B, MBL, MASP1-3, Map19; and k) an opsonin such as CRP, SAA and SAP.

[0359] In one embodiment, a hepatogenic cell possesses one or a plurality of phenotypic and functional characteristics of one or a plurality of the following liver cell types: a) a hepatocyte; b) a non-parenchymal cell; c) a sinusoidal endothelial cell; d) a Kupffer cell; e) a stellate cell; and f) a cholangiocyte. In one embodiment, the hepatogenic cell is administered to a subject as a therapeutic cell. In another embodiment, the hepatogenic cell is induced in vivo by administration of a treatment regimen to the subject.

[0360] In one embodiment, a hepatogenic cell possesses one or a plurality of phenotypic and functional characteristics of a hepatocyte, wherein the hepatogenic cell expresses or produces one or a plurality of the following molecules: a) albumin; b) fibrinogen; c) a clotting factor (e.g., factor V, VII, IX, X, XI, XII, protein C, protein S, antithrombin); d) transferrin; e) plasminogen; f) ceruloplasmin; g) a complement protein; h) a hepatokine such as hepatocyte growth factor; i) IL-6; j) IL-22; k) a chemokine; l) a bactericidal protein; m) an opsonin; n) an iron-sequestering protein; o) bile; p) bicarbonate; q) fetuin-B; and r) an aminotransferase (i.e., ALT and / or AST).

[0361] In certain embodiments, a hepatogenic monocyte is derived from an iPSC. In certain embodiments, a hepatogenic monocyte is engineered in vitro to mimic one or a plurality of functional characteristics of a hepatocyte, or of another cell type of the liver. By way of example, a hepatogenic cell such as a hepatogenic monocyte may express or synthesize one or a plurality of liver-specific proteins including but not limited toImmorta-LivMon-PCT albumin, alpha-1 antitrypsin, transferrin, transthyretin, cytokeratin-18, and / or hepatocytenuclear factor 4 alpha (HNF4 ).

[0362] One embodiment of the invention discloses a three-step culture method for generating a hepatogenic cell type such as a monocyte that is a suitable therapeutic cell for treating a liver disease. In one embodiment, a method for generating a hepatogenic monocyte for treatment of liver failure is provided, the method comprising: a) isolating a somatic cell from a subject; b) providing a first culture system comprising one or a plurality of agents to induce dedifferentiation of the somatic cell into an iPSC; c) providing a second culture system comprising one or a plurality of agents for inducing differentiation of the iPSC into a monocyte; and d) providing a third culture system comprising one or a plurality of agents for inducing the expression of hepatogenic factors in the monocyte.

[0363] Another embodiment discloses a two-step culture method for generating a hepatogenic monocyte that is useful for administration to a subject with liver failure. In this embodiment, a method for generating a hepatogenic monocyte for treatment of liver failure is provided, the method comprising: a) isolating a somatic cell from a subject; b) providing a first culture system comprising one or a plurality of agents to induce dedifferentiation of the somatic cell into an iPSC; and c) providing a second culture system comprising one or a plurality of agents for inducing iPSC differentiation into a monocyte and for inducing the expression of hepatogenic factors in the monocyte.

[0364] In one embodiment, a therapeutic cell comprising a monocyte is derived from endogenous sources such as peripheral blood or mobilized peripheral blood. In another embodiment, a therapeutic cell comprises a monocyte that has been generated or modified in vitro; for example, a monocyte differentiated from an induced pluripotent stem cell (iPSC). In one embodiment, a therapeutic cell comprises an autologous monocyte, wherein the autologous monocyte comprises a primary cell isolated from a tissue or blood, or a cultured cell derived from the subject in need of treatment. In another embodiment, a therapeutic cell comprises an allogeneic monocyte, wherein the allogeneic monocyte comprises a primary cell isolated from tissue or blood, or a cultured cell derived from a donor subject

[0365] One embodiment provides a therapeutic cell population for treating a liver disease or condition such as liver failure that is derived from induced human pluripotent stem cells, wherein the induced pluripotent stem cells are derived by dedifferentiation ofImmorta-LivMon-PCT somatic cells that are autologous or allogeneic. Embodiments of the invention disclose steps for providing an iPSC population that is suitable for differentiation into therapeutic cells of the invention, and the methods for subsequent generation of therapeutic cells. In one embodiment, somatic cell nuclear transfer is performed, wherein the somatic cell nuclear transfer is augmented by suppression of the p53 in iPSCs by RNA interference, and wherein RNA interference comprises treating the cells with short interfering RNA, short hairpin RNA, double stranded RNA, antisense oligonucleotides, a ribozyme, morpholino oligonucleotides or an aptamer to silence the gene. In one embodiment, generation of iPSCs is performed by reprogramming somatic cells, wherein reprogramming of somatic cells is performed by introducing genes encoding for one or a plurality of the following factors: PIM1, PIM3, Lin28 Sox-2, c-Myc, L-Myc, c-MET, k- ras, NF-kappa B, NANOG, KLF4, and OCT4. In one embodiment, the factors are delivered to somatic cells by protein transduction using protein transduction domain containing proteins such as cell penetrating peptides. In one embodiment, cell penetrating peptides are delivered by co-injection of DNA or protein, and wherein a cell penetrating peptide may comprise one or a plurality of the following: LL37, TAT, penetratin, polyarginine, PEP-1, TAT-H2, Hph-1, HP4, LAH4, LAH4-L1, vectofusin, low molecular weight protamine, and VP22. In one embodiment, RNA nanoparticles comprising RNA encoding one or a plurality of the factors capable of inducing cellular dedifferentiation and / or reprogramming are introduced to the somatic cells to induce cellular dedifferentiation and / or reprogramming.

[0366] In one embodiment, monocytes are provided that 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 monocyte cells to an affected area.

[0367] The invention leverages the capability of monocytes to traffic towards areas of liver degeneration / inflammation and to be drawn to areas where liver regeneration / transplantation takes place. Accordingly, monocyte 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. While much of the disclosure is directed towards the treatment of liver development and liver disorders, it will be appreciated by one of ordinary skill in the art that the engineered monocytes of theImmorta-LivMon-PCT 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 monocytes of the present disclosure could be useful to help or encourage cellular regeneration in the central nervous system or other areas of the body.

[0368] Embodiments of the invention provide a composition of monocytes for treating a subject and methods of use thereof, wherein the monocytes are identifiable based on expression of cell surface molecules, and wherein the cell surface molecules are selected from the group comprising CD14, CD11b, CD45, CD33, CD163, CD80, CD206, 25F9, CD1d, CD9, CD36, CD99, CD163, CLEC5A, CD15, and CD16.

[0369] In certain embodiments, the invention provides a therapeutic cell population comprising monocytes, wherein the monocytes express one or a plurality of trophic factors such as growth factors, cytokines, chemokines, vesicles, or other signaling molecules. In certain embodiments, a therapeutic cell population expresses or produces one or a plurality of trophic factors selected from the group comprising: a) insulin-like growth factor-1 (IGF-1); b) fibroblast growth factor (FGF); c) platelet-derived growth factor (PDGF); d) transforming growth factor beta (TGF-beta); e) hepatocyte growth factor (HGF); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; i) IL-10; j) brain-derived neurotrophic factor (BNDF); k) glial cell line-derived neurotrophic factor (GDNF); l) colony-stimulating factor 1 (CSF1); m) Vascular endothelial growth factor (VEGF); n) Stromal cell-derived factor-1 (SDF-1); o) CXCR4; p) CCR2; q) CX3CR1; r) CXCL12; s) CC chemokine ligand 1 (CCL1); t) CXCR7; u) intercellular adhesionmolecule 1 (ICAM-1); v) tumor necrosis factor alpha (TNF- ); w) IL-22; x) OncostatinM (OSM); and y) Stem cell factor (SCF).

[0370] In one embodiment, a therapeutic cell population is provided comprising a hepatogenic monocyte, wherein the monocyte expresses or produces insulin-like growth factor 1 (IGF-1), which has been implicated as central regulator of liver regeneration. In one embodiment, a monocyte expressing IGF-1 is provided to accelerate 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 necrosis and fibrosis, and activating signaling pathways associated with liver regeneration.

[0371] In one specific embodiment of the invention, a method for treating a subject with liver failure is provided, the method comprising: a) identifying a subject with liverImmorta-LivMon-PCT failure; b) isolating a primary somatic cell from a biological fluid or tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce dedifferentiation into an induced pluripotent stem cell; d) providing a second culture system, wherein the induced pluripotent stem cell is provided with culture conditions to induce differentiation into a monocyte; e) providing a third cell culture system, wherein the differentiated monocyte is provided with culture conditions to induce the expression of one or a plurality of hepatogenic factors; f) isolating the hepatogenic monocyte from the third cell culture system; and g) administering the hepatogenic monocyte to the subject, wherein the hepatogenic monocyte exerts regenerative functions, immune modulatory functions, or both. In certain embodiments, the hepatogenic monocyte is capable of reversing liver failure in the subject. In one embodiment, the primary somatic cell is selected from the group consisting of: a fibroblast, a lymphocyte, a myeloid cell, a neutrophil, an eosinophil, a basophil, a myeloblast, a monocyte, a macrophage, a mesenchymal stem cell, a keratinocyte, an epithelial cell, a renal epithelial cell, an epithelial progenitor cell, a hematopoietic stem cell, a common myeloid progenitor, a natural killer (NK) cell, an NK T cell, a common lymphoid progenitor, an endothelial cell, an endothelial progenitor cell, a synovial cell, a mobilized CD34+ peripheral blood mononuclear cell, a blood mononuclear cell, or a tissue-specific stem or progenitor cell. In one embodiment, a hepatogenic monocyte shares one or a plurality of morphological, phenotypic, and / or functional characteristics in common with a primary hepatocyte isolated from liver. In one embodiment, the hepatogenic monocyte expresses one or a plurality of transcription factors selected from the group comprising a) signal transducer and activator of transcription 6 (STAT6); b)Peroxisome proliferator-activated receptor gamma (PPAR- -like factor 4(KLF4); d) c-Maf, e) MafB; and f) SP1. In another embodiment, the hepatogenic monocyte produces one or a plurality of molecules selected from the group comprising a) albumin; b) fibrinogen; c) a clotting factor (e.g., factor V, VII, IX, X, XI, XII, protein C, protein S, antithrombin, or a combination thereof); d) transferrin; e) plasminogen; f) ceruloplasmin; g) a complement protein; h) a bactericidal protein; i) an opsonin; j) an iron-sequestering protein; k) bile; l) bicarbonate; m) fetuin-B; and n) an aminotransferase (i.e., ALT and / or AST). In another embodiment, the hepatogenic monocyte produces one or a plurality of cytokines and chemokines selected from the group comprising a) hepatocyte growth factor (HGF); b) insulin-like growth factor-1 (IGF-1); c) fibroblast growth factor (FGF); d) platelet-derived growth factor (PDGF); e) transforming growthImmorta-LivMon-PCT factor beta (TGF-beta); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; h) IL-10; i) colony-stimulating factor 1 (CSF1); j) Vascular endothelial growth factor (VEGF); k) Stromal cell-derived factor-1 (SDF-1); l) CXCR4; m) CCR2; n) CX3CR1; o) CXCL12; p) CC chemokine ligand 1 (CCL1); q) CXCR7; r) intercellular adhesionmolecule 1 (ICAM-1); s) tumor necrosis factor alpha (TNF- ); t) IL-22; u) Oncostatin M(OSM); and v) Stem cell factor (SCF). In one embodiment, the hepatogenic monocyte expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15. In one embodiment, the second culture system comprises monocyte conditioned medium, wherein the monocyte conditioned medium may be derived from culture of monocytes that have been stimulated with a toll-like receptor agonist. In one embodiment, the second culture system generates monocytes of the M2-type, wherein the M2-type monocytes are characterizedby production of IL-10, transforming growth factor-beta (TGF- ), or both. In anotherembodiment, the M2-type monocytes express of signal transducer and activator of transcription 6 (STAT6). In one embodiment, the second culture system comprises one or a plurality of factors selected from the group comprising hepatocyte growth factor (HGF), fibroblast growth factor-1 (FGF-1), FGF-2, bone morphogenetic protein-2 (BMP-2), BMP-4, macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). In one embodiment, the third culture system comprises HGF. In one embodiment, the third culture system comprises one or a plurality of factors selected from the group comprising FGF-1, FGF-2, BMP-2, BMP-4, M-CSF GM-CSF, and LIF. In one embodiment, the induction of expression of one or a plurality of hepatogenic factors into the differentiated monocyte in the third cell culture system is performed by gene transfection. In one embodiment, the transfected genes in the third cell culture system are selected from the group comprising a) HNF4A; b) FOXA2; c) FOXA1; and d) C / EBP alpha. In one embodiment, the third cell culture system comprises serum harvested from a subject with liver failure. In one embodiment, the hepatogenic monocyte generated in the third cell culture system produces insulin-like growth factor 1 (IGF-1).

[0372] In certain embodiments, a therapeutic cell population comprises a monocyte that produces one or a plurality of other factors including but not limited to fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factorImmorta-LivMon-PCT beta (TGF-beta), hepatocyte growth factor (HGF), nerve growth factor (NGF) and other neurotrophic factors, which play key roles in the liver regeneration. In certain embodiments, monocytes express or produce one or a plurality of trophic factors that mediate regeneration in the liver and / or in extra-hepatic tissues including but not limited to brain-derived neurotrophic factor (BDNF) and Glia cell-derived neurotrophic factor (GDNF) and other neurotrophic factors. In certain embodiments, a monocyte expresses one or a plurality of trophic factors that mediate regenerative functions in the central nervous system such as neurodegenerative disorders (motor neuron disease, Parkinson’s disease, Alzheimer disease, spinocerebellar ataxia), neuroinflammatory diseases (such as Multiple sclerosis), and stroke.

[0373] According to one embodiment, the bioengineered monocytes are derived from genetically altered cells capable of differentiating into monocyte cells. Examples of cells capable of differentiating into monocytes include but are not limited to induced pluripotent stem cells (iPSCs), embryonic stem cells, mesenchymal stem cells, or engineered somatic cells. In certain embodiments, the monocytes are derived from hematopoietic stem cells or directly from peripheral blood. In certain embodiments, the cells from which the bioengineered monocytes may be derived from the individual who will be receiving the bioengineered monocytes to minimize the likelihood or rejection or bio-incompatibility.

[0374] In one embodiment, the monocytes or cells capable of differentiating into monocytes are genetically edited to express one or a plurality of factors such as cytokines, growth factors, adhesion molecules, signaling molecules, proteins, or nucleic acid molecules. 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 activator- like 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 monocytes or cells that can be differentiated into monocyte cells. These genome editing techniques may incorporate viral (adenovirus, lentivirus) or non-viral methods (electroporation, lipid particles, or nanoparticles.)Immorta-LivMon-PCT

[0375] In one embodiment, bioengineered monocytes 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. Moreover, iPSC cells can be derived from patient samples that are easily and even non-invasively obtained like skin, saliva, blood, or urine samples. The iPSCs can be cultured using suitable culturing conditions. For example, iPSCs can be maintained using protocols such as those disclosed. In a specific embodiment, 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.

[0376] In another embodiment, the invention teaches the manipulation of myeloid suppressor cells for use as therapeutic cells in liver failure by pretreatment with HGF. In other embodiments myeloid suppressor cells are derived from iPSC sources and utilized as an autologous or allogeneic therapeutic cell population.

[0377] In one embodiment, hepatogenic monocytes comprise myeloid derived suppressor cells. In certain embodiments, the invention teaches means of enhancing myeloid derived suppressor activity in order to induce immunological tolerance to liver cells from death. In some embodiments, the invention uses patient lymphocytes conditioned by stem cells to increase TGF-beta expression on myeloid suppressor cells, thereby increasing potency of myeloid suppressor cell inhibition of immunity. In some embodiments of the invention activation of IL-4 receptor myeloid derived suppressor cells is disclosed as a means of treating liver failure.

[0378] In one embodiment of the invention, liver failure is treated by compounds that stimulate activity of myeloid derived suppressor cells are given along with the regenerative cell reprogrammed PBMC. In one embodiment compounds such as IL-6, PGE-2, S100A9, exosomes, LPS and interferon gamma, GM-CSF, M-CSF, BCG, TLR-2 activators, other TLR activators, hepatic acute phase proteins such as serum amyloid A and Cxcl1 / K, Galectin-9, anti-CD137 antibodies are administered to a subject to augment activity of myeloid derived suppressor cells. In some embodiments, enhancement ofImmorta-LivMon-PCT myeloid derived suppressor cell function such as increasing arginine metabolism, is accomplished by activation of said cells with agent such as toll like receptor activators.

[0379] In one embodiment of the invention, IL-17 producing gamma delta T cells are utilized to generate myeloid suppressor cells which are subsequently administered to a subject in need thereof to prevent or treat liver failure. Additionally, simple IL-17 administration either directly, or through administration of cells secreting IL-17 may be used for stimulation of myeloid derived suppressor cells.

[0380] In some embodiments, a subject with liver failure is treated by administration of monocytes that are pre-conditioned in vitro using mesenchymal stem cells or myeloid derived suppressor cells.

[0381] In one embodiment, the invention provides a method for generating an autologous immunological composition for the treatment of liver failure in humans, wherein the method comprises: a) obtaining peripheral blood mononuclear cells from the subject in need of treatment; b) isolating and / or expanding a population of regulatory or suppressor T cells from the peripheral blood mononuclear cells, wherein the population of regulatory or suppressor T cells is enriched for T cells reactive to antigens expressed by liver cells; and c) inducing the regulatory or immune suppressive T cells to possess a tolerogenic and / or regenerative phenotype through incubation with a mesenchymal stem cell population in vitro. In certain embodiments, the regulatory T cells are used as a therapeutic cell population for administration to the subject with liver failure. In other embodiments, the regulatory T cells are used to condition a therapeutic cell population. By way of example, the regulatory T cells described herein may be used to condition an iPSC-derived monocyte population, wherein the iPSC-derived monocyte population is subsequently administered to the subject as a therapeutic cell population. In some embodiments, the antigens expressed by the liver cells are proteins or peptides. In some embodiments, these antigens may be identified by whole exome sequencing and RNAseq of pancreatic, beta cell, and alpha cell tissues the same individual, and HLA binding algorithms are applied to determine which liver cell-specific peptides bind HLA molecules.

[0382] In some embodiments, a subject undergoes an immunization procedure, wherein one or a plurality of hepatic antigens are administered to the subject as an adjunct to administration of a therapeutic cell population for the treatment of liver failure in the subject. In some embodiments, the immunization consists of intramuscular injection of hepatic antigen emulsified in an adjuvant. In one embodiment, the immunizationImmorta-LivMon-PCT procedure utilizes DNA vaccination plus electroporation. In one embodiment, immunization is performed using hepatic extracts comprising a mixed source of antigens derived from liver cells. Immunization of a subject with a composition of one or a plurality of hepatic antigens may be useful in augmenting the therapeutic activity of hepatogenic cells. Immunization of the subject may be performed prior to extraction of a cell population from the subject, wherein the cell population is subsequently subjected to in vitro culture methods and / or manipulation to generate a therapeutic cell type. In another embodiment, immunization of the subject may be performed following administration of a therapeutic cell population to the subject. In one embodiment, immunization of the subject is performed a single time or multiple times prior to or following administration of a therapeutic cell population to treat a subject with liver failure.

[0383] In one embodiment, administration of PGE-2, or agents or cells that induce expression of PGE2 is performed to increase activity and / or numbers of a therapeutic cell population that is administered to a subject for treating liver failure. Methods of administering PGE-2 are known to one of ordinary skill in the art. In certain embodiments, PGE2 is administered to induce increased numbers or activity of myeloid- derived suppressor cells in a subject, wherein the myeloid-derived suppressor cells may be naturally occurring in vivo or comprising a therapeutically administered cell population. In another embodiment, VEGF is administered to induce augmentation of activity and / or number of myeloid derived suppressor cells in a subject. In another embodiment low dose interleukin-2 is administered as a means of augmenting myeloid derived suppressor cell number and / or activity. In some embodiments, hepatogenic monocytes are monocytes derived from undifferentiated sources and administered together with other agents or molecules. In certain embodiments, a hepatogenic monocyte is administered to a subject in combination with one or a plurality of agents or molecules selected from a group comprising cyclosporine, rapamycin, campath-1H, ATG, Prograf, anti-IL-2R antibody, MMF, FTY, LEA, cyclosporin A, diftitox, denileukin, levamisole, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, and trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, and thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, and tegafur) fluocinolone,Immorta-LivMon-PCT triaminolone, anecortave acetate, fluorometholone, medrysone, prednislone, or combinations thereof. In another embodiment, the use of stem cell conditioned medium may be used to potentiate an existing anti-inflammatory agent. Anti-inflammatory agents may comprise one or more agents including NSAIDs, interleukin-1 antagonists,dihydroorotate synthase inhibitors, p38 MAP kinase inhibitors, TNF- -sequestration agents, and methotrexate. More specifically, anti-inflammatory agents maycomprise one or more of, e.g., anti-TNF- -antitrypsin (AAT),interleukin-10 (IL-10), pentoxyfilline, COX-2 inhibitors, 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino- acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (eg., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone,Immorta-LivMon-PCT oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), epsilon.-acetamidocaproic acid, s-adenosylmethionine, 3-amino-4-hydroxybutyric .acid, amixetrine, bendazac,benzydamine -bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol,guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, zileuton, candelilla wax, alpha bisabolol, aloe vera, Manjistha, Guggal, kola extract, chamomile, sea whip extract, glycyrrhetic acid, glycyrrhizic acid, oil soluble licorice extract, monoammonium glycyrrhizinate, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate, 1-beta-glycyrrhetic acid, stearyl glycyrrhetinate, and 3-stearyloxy-glycyrrhetinic acid.

[0384] In some embodiments of the invention, prior to expansion, a source of T cells is obtained from a subject. In some embodiments, the subject is a partially or fully HLA-matched healthy donor (i.e., non-cancerous donor). T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as ficoll separation. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, theImmorta-LivMon-PCT undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL.TM. gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3.sup.+, CD28.sup.+, CD4.sup.+, CD8.sup.+, CD45RA.sup.+, and CD45RO.sup.+ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3.times.28)-conjugated beads, such as DYNABEADS.TM., for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells. Further, use of longer incubation times can increase the efficiency of capture of T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

[0385] In one embodiment, enrichment of a hepatoprotective T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.Immorta-LivMon-PCT

[0386] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. In one embodiment, the concentration of cells used is 5.times.10.sup.6 / ml. In other embodiments, the concentration used can be from about 1.times.10.sup.5 / ml to 1.times.10.sup.6 / ml, and any integer value in between. In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10.degree. C. or at room temperature. If desired or necessary, T cell populations (i.e., CD3.sup.+ cells) may be depleted from blood preparations prior to ex vivo expansion by a variety of methodologies, including anti-CD3 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal, or by the use of counterflow centrifugal elutriation. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, for example, those produced by Dynal AS under the trade name Dynabeads.TM.. Exemplary Dynabeads.TM. in this regard are M-280, M-450, and M-500. In one aspect, other non- specific cells are removed by coating the paramagnetic particles with "irrelevant" proteins (e.g., serum proteins or antibodies). Irrelevant proteins and antibodies include thoseImmorta-LivMon-PCT proteins and antibodies or fragments thereof that do not specifically target the T cells to be expanded. In certain embodiments the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin. In brief, such depletion of monocytes is performed by preincubating PBMC isolated from whole blood or apheresed peripheral blood with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37.degree. C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL.TM. Magnetic Particle Concentrator (DYNAL MPC.TM.)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after said depletion. T cells for stimulation can also be frozen after the washing step, which does not require the monocyte-removal step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80.degree. C. at a rate of 1.degree. per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20.degree. C. or in liquid nitrogen. The culture of T cells may be performed in the presence of regenerative cells. In some embodiments said regenerative cells are pulsed or primed with an immune stimulatory agent. This is to enhance the ability of the regenerative cells to program T cells, or PBMC. In one ideal embodiment patient PMBC are extracted, incubated with regenerative cells and subsequently administered back to the patient. In other embodiments immune cells from the patient are cultured in the conditioned media ofImmorta-LivMon-PCT regenerative cells. In some embodiments cells are cultured under hypoxia. In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

[0387] In some embodiments, the lymphocytes are taken from a partially or fully HLA-matched, non-cancerous donor and used to activated hepatogenic monocytes. T cells are activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631 with the exception that they are cultured together with regenerative cells. Monocytes co-cultured with lymphocytes secreting cytokines IL-10, TGF-beta or IL-1 RA may be considered hepatogenic.

[0388] Infusion of the immune cell population of the present invention enhances, potentiates, or increases the hepatogenic capacity of the immune response, as well as evokes regenerative potential. Generally, the immune response can include the humoral immune response, the cell-mediated immune response, or both. For example, antigen presentation through an immunological pathway involving MHC II proteins or direct B- cell stimulation can produce a humoral response; and, antigens presented through a pathway involving MHC I proteins can elicit the cellular arm of the immune system. A humoral response can be determined by a standard immunoassay for antibody levels in a serum sample from the subject receiving the pharmaceutically acceptable composition. A cellular immune response is a response that involves T cells and can be determined in vitro or in vivo. For example, a general cellular immune response can be determined as the T cell proliferative activity in cells (e.g., peripheral blood leukocytes (PBLs)) sampled from the subject at a suitable time following the administering of a pharmaceutically acceptable composition. Following incubation of e.g., PBMCs with a stimulator for an appropriate period, [.sup.3H]thymidine incorporation can be determined. The subset of T cells that is proliferating can be determined using flow cytometry. T cell cytotoxicity (CTh) can also be determined.

[0389] The pharmaceutically acceptable composition can be administered in a therapeutically or a prophylactically effective amount, wherein the pharmaceutically acceptable composition comprises the lymphocyte population of T cells are enriched for T cells reactive to neo-antigens in the recipient and depleted of T cells reactive to antigens on non-cancerous tissues of the recipient, either alone or in combination withImmorta-LivMon-PCT one or more other antigens. Administering the pharmaceutically acceptable composition of the present invention to the subject can be carried out using known procedures, and at dosages and for periods of time sufficient to achieve a desired effect. For example, a therapeutically or prophylactically effective amount of the pharmaceutically acceptable composition, can vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted by one of ordinary skill in the art to elicit the desired immune response including immune responses that provide therapeutic or prophylactic effects.

[0390] Administering can be properly timed by the care giver (e.g., physician, veterinarian), and can depend on the clinical condition of the subject, the objectives of administering, and / or other therapies also being contemplated or administered. In some embodiments, an initial dose can be administered, and the subject monitored for either an immunological or clinical response, preferably both. Suitable means of immunological monitoring include using patient's peripheral blood lymphocyte (PBL) as responders and neoplastic cells as stimulators. An immunological reaction also can be determined by a delayed inflammatory response at the site of administering. One or more doses subsequent to the initial dose can be given as appropriate, typically on a monthly, semimonthly, or preferably a weekly basis, or a daily basis until the desired effect is achieved. Thereafter, additional booster or maintenance doses can be given as required, particularly when the immunological or clinical benefit appears to subside. The lymphocyte compositions of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical lymphocyte compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

[0391] Pharmaceutical compositions comprising of hepatogenic monocytes of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by suchImmorta-LivMon-PCT factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0392] In certain embodiments, the present invention provides a method of enhancing activity of hepatogenic myeloid derived suppressor cells by exposing said cells to patient lymphocytes that have been conditioned with regenerative cells. In one embodiment, myeloid derived suppressor cells are generated by a means comprising of: a) contacting pluripotent stem cell with an effective amount of kit ligand (KL) (stem cell factor), vascular endothelial growth factor (VEGF), FMS-like tyrosine kinase 3 (Flt3L), thrombopoietin (TPO), and macrophage colony-stimulating factor (M-CSF); and b) culturing said iPSC cells under conditions suitable for propagation of said cell, thereby obtaining a preparation of an isolated myeloid-derived suppressor cell. In certain embodiments, the method further comprises cryopreservation of said myeloid-derived suppressor cell. In yet additional embodiments, the iPSC cell is a mammalian cell. In certain embodiments, the iPSC cell is a human cell. In yet additional embodiments, the isolated myeloid-derived suppressor cell expresses at least one of the cell surface markers selected from the group consisting of CD33, CD115, F4 / 80, Ly-6C, CD11b, Gr-1, VEGF receptor, CD40 and IL-4R. Other means of generating myeloid-derived suppressor cell are disclosed such as a) contacting a hematopoietic stem cell (HSC) with an effective amount of kit ligand (KL) (stem cell factor), vascular endothelial growth factor (VEGF), FMS-like tyrosine kinase 3 (Flt3L), thrombopoietin (TPO), and macrophage colony- stimulating factor (M-CSF); and b) culturing said HSC under conditions suitable for propagation of said cell, thereby obtaining a preparation of an isolated myeloid-derived suppressor cell. In certain embodiments, the method further comprises cryopreservation of said myeloid-derived suppressor cell. In yet additional embodiments, the HSC is a mammalian HSC. In yet additional embodiments, the HSC is a human HSC. In yet additional embodiments, the isolated myeloid-derived suppressor cell expresses at least one of the cell surface markers selected from the group consisting of CD33, CD115, VEGF receptor, F4 / 80, Ly-6C, CD11b, Gr-1, CD40 and IL-4R. In other embodiments, the isolated myeloid-derived suppressor cell derived from a human ES cell or human HSC expresses at least one of the cell surface markers selected from the group consisting of CD11b, CD33, CD15, and CD16. In yet other embodiments, the isolated myeloid- derived suppressor cell expresses CD11b and CD33. In still other embodiments, the isolated myeloid-derived suppressor cell expresses CD11b and Gr-1. In yet additionalImmorta-LivMon-PCT embodiments, the invention provides an isolated myeloid-derived suppressor cell obtained by any of the methods described herein.

[0393] The invention provides novel stem cell types, methods of manufacture, and therapeutic uses. Provided are means of deriving stem cells possessing regenerative, immune modulatory, anti-inflammatory, and angiogenic / neurogenic activity from umbilical cord tissue such as Wharton’s Jelly. In some embodiments manipulation of stem cell “potency” is disclosed through hypoxic manipulation, growth on non- xenogeneic conditions, as well as addition of epigenetic modulators.

[0394] In one embodiment, the cells of the invention are cultured under hypoxia to induce and / or augment expression of chemokine receptors. One such receptor is CXCR- 4. The population of cells, including population of umbilical cord mesenchymal cells, may be enriched for CXCR-4, such as (or such as about) 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the population expressing CXCR-4, CD31, CD34, or any combination thereof. In addition, <1%, <2%, <3%, <4%, <5%, <6%, <7%, <8%, <9%, or <10% of the population of cells may express CD14 and / or CD45. The umbilical cord cells of the invention may further possess markers selected from the group consisting of STRO-1, CD105, CD54, CD56, CD106, HLA-I markers, vimentin, ASMA, collagen-1, fibronectin, LFA-3, ICAM-1, PECAM-1, P- selectin, L-selectin, CD49b / CD29, CD49c / CD29, CD49d / CD29, CD61, CD18, CD29, thrombomodulin, telomerase, CD10, CD13, STRO-2, VCAM-1, CD146, and THY-1, and a combination thereof. In some embodiments said placental cells of the invention are admixed with endothelial cells. Said endothelial cells may express one or more markers selected from the group consisting of: a) extracellular vimentin; b) CD133; c) c-kit; d) VEGF receptor; e) activated protein C receptor; and f) a combination thereof. In some embodiments, the population of endothelial cells comprises endothelial progenitor cells.

[0395] In certain embodiments, a therapeutic cell population that is administered to a subject with liver disease is allogeneic, autologous, or xenogenic to the subject. In some embodiments, the population of cells are matched by mixed lymphocyte reaction matching.

[0396] In some embodiments, a population of therapeutic cells used to treat a liver disease is derived from tissue selected from the group consisting of the placental body, placenta, umbilical cord tissue, peripheral blood, hair follicle, cord blood, Wharton's Jelly, menstrual blood, endometrium, skin, omentum, amniotic fluid, and a combination thereof. In some embodiments, the population of cells, the population of umbilicalImmorta-LivMon-PCT mesenchymal stem cells, or the population of endothelial cells comprises human umbilical cord derived adherent cells. In certain embodiments, human umbilical cord derived adherent cells express cytokines selected from the group consisting of ) FGF-1; b) FGF-2; c) HGF; d) interleukin-1 receptor antagonist; and e) a combination thereof. In some embodiments, the population of cells, the population of umbilical cord cells express arginase, indoleamine 2,3 deoxygenase, interleukin-10, and / or interleukin 35. In some embodiments, the population of cells, the population of umbilical cord cells, or the population of endothelial cells express hTERT and Oct-4 but does not express a STRO-1 marker. In certain embodiments, a population of cells is used to generate induced pluripotent stem cells, wherein the population of cells is selected from the group comprising the placental body, placenta, umbilical cord tissue, peripheral blood, hair follicle, cord blood, Wharton's Jelly, menstrual blood, endometrium, skin, omentum, and amniotic fluid. Subsequently, the induced pluripotent stem cells are used to generate a population of therapeutic cells that is administered to the subject.

[0397] In certain embodiments, a therapeutic cell population comprises mesenchymal stem cells, wherein the mesenchymal stem cells may be endogenously isolated cells and / or in vitro modified or engineered cells. In certain embodiments, mesenchymal stem cells are subjected to retroviral transfection to over express CXCR-4 to increase the migration of these cells to injured or hypoxic liver tissue. The functional activity of these cells may be evaluated using assays known in the art to define the growth profiles and differentiation ability of CXCR-4 overexpressing mesenchymal stem cells when compared to control transfected mesenchymal stem cells. In certain embodiments, a mesenchymal stem cell is pulsed with a brief period of hypoxia, and / or is exposed to one or a plurality of cytokines such as SCF, IL-6, Flt-3 ligand, HGF and IL-3. In certain embodiments, a therapeutic cell population comprises mesenchymal stem cells, genetically modified mesenchymal stem cells, or otherwise manipulated / treated mesenchymal stem cells that are administered intravenously to a subject in need thereof.

[0398] In certain embodiments, a cytokine or growth factor is administered to the subject in addition to a therapeutic cell of the invention for the purpose of enhancing the migration or trafficking of a therapeutic cell of the invention to a site of injury in the liver. One method involves providing an exogenous depot of angiogenic cytokines in proximity to the area where stem cell migration is desired. By way of example, a therapeutic cell population may be administered to a subject concurrent with fibroblasts or another cell type expressing stromal cell-derived factor 1 (SDF-1). Another method involvesImmorta-LivMon-PCT enhancing chemoattractant activity by concurrently administering exogenous stem cells, or by mobilizing endogenous bone marrow stem cells. By way of example, bone marrow stem cells may be mobilized by administering G-CSF to the subject. Other clinically used methods may be implemented to enhance stem cell trafficking. For example, erythropoietin (EPO) may be administered to a subject to stimulate responsiveness of bone marrow derived stem cells to SDF-1. These embodiments may be useful to provide trophic, angiogenic, anti-inflammatory and anti-apoptotic effects to the liver in a subject in need thereof.

[0399] In certain embodiments, a therapeutic cell is administered to a subject with liver failure, wherein the therapeutic cell expresses one or a plurality of molecules associated with pluripotency such as Nanog, Oct3, Oct4, Sox2, c-Myc, and Klf4. In certain embodiments, a therapeutic cell is transfected with a gene that is associated with pluripotency. The expression of pluripotency-associated factors may provide the therapeutic cell with superior expansion potential and ability to differentiate as compared to control non-transfected cells of the same lineage and type.

[0400] In some embodiments, the population of therapeutic cells of the invention undergoes cell division in less than 36 hours in a growth medium. In some embodiments, the population of therapeutic cells has the ability to proliferate at a rate of 0.9-1.2 doublings per 36 hours in growth media. In some embodiments, the population of therapeutic cells has an ability to proliferate at a rate of 0.9, 1.0, 1.1, or 1.2 doublings per 36 hours in growth media. The population of therapeutic cells may produce exosomes capable of inducing more than 50% proliferation when the exosomes are cultured with human umbilical cord endothelial cells. The induction of proliferation may occur when the exosomes are cultured with the human umbilical cord endothelial cells at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more exosomes per cell.

[0401] In some embodiments, a population of therapeutic cells of the invention may be administered via any suitable route, including as non-limiting examples, intramuscularly and / or intravenously.

[0402] In some embodiments, a population of umbilical cord cells is optionally obtained, the population is then optionally contacted via culturing with a population of progenitor for T regulatory cells, wherein the culturing conditions allow for the generation of T regulatory cells, then the generated T regulatory cells are administered to a subject in need thereof.Immorta-LivMon-PCT

[0403] Embodiments of the invention provide cell therapies for regeneration of the liver. Mechanistically, the process of liver regeneration occurs in three broad phases: a) Priming; b) Proliferation and c) Termination. It is important to note that hepatocytes are not terminally differentiated cells, but cells that reside in a state of proliferative quiescence. Specifically, they share features with other regenerative cells such hematopoietic stem cells, in that they are normally in the G0 phase of cell cycle. Embodiments of the invention provide cell therapies that are useful for the three phases of liver regeneration. As the name suggests, the “priming phase” is characterized by response to some type of damage or “danger” signal. During the priming phase, numerous injury signals are generated due to the underlying injury, these include activators of toll like receptors, complement degradation products, and Damage Associated Molecular Patterns (DAMPs). These signals stimulate various cells, primarily Kupffer cells, to produce cytokines and growth factors such as IL-6, TNF-alpha, and HGF which induce entry of hepatocytes into cell cycle. The Proliferation Phase of hepatic regeneration is associated with “primed” hepatocytes leaving G1 stage of cell cycle and entering S phase, which is accompanied by phosphorylation of the retinoblastoma protein (pRb) and by up-regulated expression of a number of proliferation-associated genes including cyclin E, cyclin A, and DNA polymerase. Key cytokines involved in stimulation of proliferation of the hepatocytes include hepatocyte growth factor (HGF) and epidermal growth factor (EGF). EGF signaling is also involved in entry into the proliferative phase post injury. The Termination Phase of liver regeneration occurs when the normal liver-mass / body-weight ratio of 2.5% has been restored and involves anti- inflammatory cytokines such as IL-10, which dampen proliferative stimuli. Additionally, cytokines with direct antiproliferative activity such as TGF-beta are generated, which result in cell cycle arrest of proliferating hepatocytes .

[0404] In one embodiment, a therapeutic cell population is generated using an MSC, for example, a bone marrow-derived MSC, wherein the MSC is used to generate a hepatogenic cell. In one embodiment, an MSC is isolated and subject to a two-step protocol for hepatocyte differentiation using culture in hepatocyte growth factor, followed by oncostatin M. After 3-6 weeks of induction, the cell culture can be assessed for the presence of spindle-like BM-MSC taking a cuboidal morphology, which is characteristic of hepatocytes and for initiation of expression of hepatic-specific genes in a time- dependent manner correlating with morphological changes. From a functional perspective, the cells can be assayed in vitro for features of liver cells, specificallyImmorta-LivMon-PCT albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In certain embodiments, methods to improve yield and potency of MSC-generated hepatocytes are provided, wherein conditioned media from cultured hepatocytes is included as part of the differentiation culture conditions. The differentiation process can be identified by an epithelioid, binucleated morphology. Gene assessment may also be performed to detect an increase in AFP, HNF-3beta, CK19, CK18, ALB, TAT, and G-6-Pase mRNA, which may be confirmed at the protein levels. Additionally, the cells may be assessed for hepatocyte functions including glycogen storage, and urea secretion activities. Upon administration of the cells to a subject in need thereof, the functionality of the artificially generated hepatic like cells can be evaluated on the basis of full or partial restoration of albumin activity and suppression of liver enzymes. Changes in fibrosis of the liver and other signs of liver damage can also be assessed using techniques such as elastography.

[0405] In one embodiment, a therapeutic cell population such as a hepatogenic monocyte is provided that is capable of synthesizing hepatocyte growth factor (HGF). In certain embodiments, a therapeutic cell population that produces HGF comprises an MSC. An HGF-producing cell may be desirable as a therapeutic cell population that stimulates hepatic regeneration, decreasing hepatocyte apoptosis, and reducing fibrosis / collagen deposition in the liver. In one embodiment, the regenerative effect of an HGF-producing cell population can be monitored in vitro or in preclinical models using methods that measure the levels of hepatic hydroxyproline, serum fibrosis markers, and histologic examination of the liver in the preclinical setting. In one embodiment, the engraftment of the therapeutic cell population can be monitored following administration of said therapeutic cell population by immunofluorescence, polymerase chain reaction, and / or fluorescence in situ hybridization analysis, to demonstrate the presence of donor- derived cells possessing epithelium-like morphology expressed albumin. In certain embodiments, functional recovery of the liver can be monitored by assessing the cytokine profile in the liver or systemically. In one embodiment, functional recovery of the liver is correlated with increased IL-10 and decreased IL-1, tumor necrosis factor alpha, and interferon gamma. In one embodiment, in vivo differentiation of a therapeutic cell population is examined in a preclinical model -fetoprotein, albumin, cytokeratin 18, cytochrome P4501A1 (CYP1A1), CYP1A2, glucose 6- phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF) 1 alpha, and HNF4 alpha in cells deriving from the administeredImmorta-LivMon-PCT hepatogenic cell. These embodiments provide means for characterizing and assessing the potency of a hepatogenic cell for treating a liver disease or condition.

[0406] In one embodiment, a preclinical model is provided for assessing the influence of a hepatogenic cell on the liver. In one embodiment, the carbon tetrachloride model, or other preclinical models of hepatic injury are employed for this purpose. In another embodiment, a hepatectomy recovery model is employed. In one embodiment, the effects of a hepatogenic cell are assessed in an acute model of liver failure is produced by administration to animals of D-galactosamine, a TNF-alpha stimulating hepatotoxin and lipopolysaccharide (LPS) a potent inflammatory stimulus that replicates translocation of gut bacteria often seen in liver failure.

[0407] In one embodiment, a hepatogenic cell is administered to a subject in need thereof by one of the following routes of administration: a) hepatic artery injection; b) portal vein injection; c) subcutaneous administration; d) intramuscular; e) intravenous; f) intradermal; and g) intraperitoneal. In one embodiment, the most effective delivery method for a hepatogenic cell is determined by assessing parameters of liver function in the subject.

[0408] In one embodiment, methods are provided for assessing a subject to whom a hepatogenic cell such as a hepatogenic monocyte has been administered. In certain embodiments, blood chemistry measurements are performed for a subject prior to and following the administration of one or a plurality of doses of a therapeutic cell population to the subject. By way of example, the levels of serum albumin, bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), prothrombin concentration, international normalized ratio (INR), or combinations thereof may be monitored in a subject. In one embodiment, the model for end-stage liver disease (MELD) score is utilized to evaluate a subject, wherein the score is calculated using methods known in the art using a formula based on lab values: bilirubin, creatinine, and INR (International Normalized Ratio). The MELD score may be applied to estimate the subject’s chance of survival. In one embodiment, the MELD score for a subject is assessed prior to and following the administration of a hepatogenic cell. In one embodiment, the MELD score is assessed prior to or following the administration of one or a plurality of doses of a hepatogenic cell, or both. In one embodiment, MELD-Na score, a modified version of the MELD score, is applied according to the methods of the invention, wherein measurement of serum sodium is included in the analysis. In one embodiment, the MELD or MELD-Na score for a subject is used to indicate the need for treatment with a method of theImmorta-LivMon-PCT invention. In one embodiment, the MELD or MELD-Na score for a subject is used to indicate the need for repeat or additional treatment with the methods of the invention, for example, to administer additional doses of a hepatogenic cell to the subject. In another embodiment, the Child-Pugh score is used to assess a subject, wherein the prognosis for chronic liver disease such as cirrhosis is predicted. In one embodiment, the Child-Pugh score for a subject is assessed prior to or following the administration of one or a plurality of doses of a hepatogenic cell, or both. In one embodiment, the Child-Pugh score for a subject is used to indicate the need for treatment with a method of the invention. In one embodiment, the Child-Pugh a score for a subject is used to indicate the need for repeat or additional treatment with the methods of the invention, for example, to administer additional doses of a hepatogenic cell to the subject. In certain embodiments, a quality of life score for the subject’s overall well-being is assessed, for example, using the SF-36 questionnaire or another equivalent tool having physical component and / or mental component scoring scales. In one embodiment, the quality of life score for a subject is assessed prior to or following the administration of one or a plurality of doses of a hepatogenic cell, or both.

[0409] In another embodiment, computerized tomography scan volumetry can be performed to measure the volume of the liver in a subject, wherein this imaging technique may be applied prior to and / or following the administration of one or a plurality of doses of a hepatogenic cell, or both.

[0410] In one embodiment, a heterogeneous population of therapeutic cell types is administered for treating a subject with a liver disease or condition. In one embodiment, bone marrow mononuclear cells (BMMC) are administered to a subject in need thereof, wherein the bone marrow mononuclear cells comprise one or a plurality of cell types. In one embodiment, a bone marrow mononuclear cell comprises a hepatogenic cell.

[0411] In one embodiment, CD133+ cells are administered to a subject in need thereof. In one embodiment, a CD133+ cell is derived from an iPSC. In another embodiment, a CD133+ cell comprises a myeloid lineage cell. In another embodiment, a CD133+ cell is derived from bone marrow. In one embodiment, a CD133+ cell comprises a hepatogenic cell.

[0412] In one embodiment, CD34+ cells are administered to a subject in need thereof. In one embodiment, a CD34+ cell is derived from an iPSC. In another embodiment, a CD34+ cell is derived from bone marrow. In one embodiment, a CD34+ cell comprises a hepatogenic cell.Immorta-LivMon-PCT

[0413] In one embodiment, MSC are administered to a subject in need thereof. In one embodiment, a MSC is derived from an iPSC. In another embodiment, an MSC is derived from bone marrow. In one embodiment, a MSC comprises a hepatogenic cell. In one embodiment, an MSC expresses one or a plurality of molecules selected from the group comprising CD29, CD44, CD71, CD90, CD105 / SH2, and SH3. In one embodiment, an MSC lacks expression of one or a plurality of molecules selected from the group comprising CD31, CD34, and CD45. In one embodiment, a MSC possesses hepatocyte differentiation ability. In one embodiment, a MSC possesses angiogenesis- stimulating ability. In another embodiment, a MSC has immune modulatory functions.

[0414] In one embodiment, an adipose-derived cell is administered as a therapeutic cell to a subject in need thereof. In one embodiment, an adipose-derived cell comprises an MSC. In one embodiment, an adipose-derived cell comprises a hepatogenic cell. In one embodiment, a hepatogenic adipose-derived cell has undergone modifications or engineering. While autologous MSC possess the benefit of lack of immunogenicity, a drawback may be a relative dysfunction of these cells given the poor health condition of the patients. Therefore, in certain embodiments, an allogeneic source of MSC is utilized as a source of therapeutic cells. In certain embodiments, adipose tissue is an attractive alternative to bone marrow as a source of therapeutic cells for treatment of liver failure for the following reasons: a) extraction of adipose derived cells is a simpler procedure that is much less invasive than bone marrow extraction; b) Adipose tissue contains a higher content of mesenchymal stem cells (MSC) as compared to bone marrow, therefore shorter in vitro expansion times are needed; and c) MSC from adipose tissue do not decrease in number with aging. In one embodiment, an adipose-derived cell is derived from collecting the stromal vascular fraction (SVF) from subject, which, in certain embodiments, is subjected to expansion protocols in vitro for generating a population of therapeutic cells.

[0415] In one embodiment, an umbilical cord-derived cell is administered as a therapeutic cell to a subject in need thereof. In one embodiment, an umbilical cord- derived cell comprises an MSC. In one embodiment, an umbilical cord-derived cell comprises a hepatogenic cell. In one embodiment, a hepatogenic umbilical cord-derived cell has undergone modifications or engineering. An umbilical cord-derived cell may be utilized according to the methods of the invention to treat liver failure, decompensated liver cirrhosis (e.g., in a subject with chronic hepatitis B), or primary biliary cirrhosis . In one embodiment, an umbilical cord-derived cell is administered to a subject to improveImmorta-LivMon-PCT liver function, as may be indicated by an increase of serum albumin levels, a decrease in - glutamyltransferase levels, and / or a decrease in the sodium model for end-stage liver disease scores. Adverse events and survival rates may also be monitored.

[0416] In one embodiment, biologically useful immune cells are generated after culture with regenerative cells, and / or stem cells are disclosed, of the mesenchymal or related lineages, which are therapeutically reprogrammed cells having minimal oxidative damage and telomere lengths that compare favorably with the telomere lengths of undamaged, pre-natal or embryonic stem cells (that is, the therapeutically reprogrammed cells of the present invention possess near prime physiological state genomes). Moreover, the therapeutically reprogrammed cells of the present invention are immunologically privileged and therefore suitable for therapeutic applications. Additional methods of the present invention provide for the generation of hybrid stem cells. Furthermore, the present invention includes related methods for maturing stem cells made in accordance with the teachings of the present invention into specific host tissues. For use in the current invention, the practitioner is thought that ontogeny of mammalian development provides a central role for stem cells. Early in embryogenesis, cells from the proximal epiblast destined to become germ cells (primordial germ cells) migrate along the genital ridge. These cells express high levels of alkaline phosphatase as well as expressing the transcription factor Oct4. Upon migration and colonization of the genital ridge, the primordial germ cells undergo differentiation into male or female germ cell precursors (primordial sex cells). For the purpose of this invention, only male primordial sex cells (PSC) will be discussed, but the qualities and properties of male and female primordial sex cells are equivalent and no limitations are implied. During male primordial sex cell development, the primordial stem cells become closely associated with precursor sertoli cells leading to the beginning of the formation of the seminiferous cords. When the primordial germ cells are enclosed in the seminiferous cords, they differentiate into gonocytes that are mitotically quiescent. These gonocytes divide for a few days followed by arrest at G0 / G1 phase of the cell cycle. In mice and rats these gonocytes resume division within a few days after birth to generate spermatogonial stem cells and eventually undergo differentiation and meiosis related to spermatogenesis. It is known that embryonic stem cells are cells derived from the inner cell mass of the pre- implantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From aImmorta-LivMon-PCT practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural epiblast environment, they only exist transiently during embryogenesis. Manipulation of embryonic stem cells in vitro has led to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage. Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and / or dedifferentiation. In one example of the maturation process, a cell, or group of cells, interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has progressed such that cells and appropriate cellular niches are present for the organism to function and survive post- natally. Developmental processes are highly conserved amongst the different species allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory. During the lifetime of an organism, the cellular composition of the organs and organs systems are exposed to a wide range of intrinsic and extrinsic factors that induce cellular or genomic damage. Ultraviolet light not only has an effect on normal skin cells but also on the skin stem cell population. Chemotherapeutic drugs used to treat cancer have a devastating effect on hematopoietic stem cells. Reactive oxygen species, which are the byproducts of cellular metabolism, are intrinsic factors that compromises the genomic integrity of the cell. In all organs or organ systems, cells are continuously being replaced from stem cell populations. However, as an organism ages, cellular damage accumulates in these stem cell populations. If the damage is inheritable, such as genomic mutations, then all progeny will be affected. A single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged. The body responds to a compromised stem cell by inducing apoptosis thereby removing it from the pool and preventing potentially dysfunctional or tumorigenic properties. Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homingImmorta-LivMon-PCT mechanism of stem cells. Telomeres are the physical ends of chromosomes that contain highly conserved, tandemly repeated DNA sequences. Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre- determined threshold, a signal is activated to initiate cellular senescence. Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress. In one teaching, or embodiment, of the invention, therapeutically reprogrammed cells, in some embodiments mesenchymal stem cells, are provided. Therapeutic reprogramming refers to a maturation process wherein a stem cell is exposed to stimulatory factors according the teachings of the present invention to yield enhanced therapeutic activity. In some embodiments, enhancement of therapeutic activity may increase proliferation, in other embodiments, it may be enhanced chemotaxis. Other therapeutic characteristics include ability to under resistance to apoptosis, ability to overcome senescence, ability to differentiate into a variety of different cell types effectively, and ability to secrete therapeutic growth factors which enhance viability / activity, of endogenous stem cells. In order to induce therapeutic reprogramming of cells, in some cases, as disclosed herein, of wharton’s jelly originating cells, the invention teaches the utilization of stimulatory factors, including without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells. These stimulatory factors induce, among other results, genomic methylation changes in the donor DNA. Embodiments of the present invention include methods for preparing cellular extracts from whole cells, although other types of cellular extracts are contemplated as being within the scope of the present invention. In a non- limiting example, the cellular extracts of the present invention are prepared from stem cells, specifically embryonic stem cells. Donor cells are incubated with the chemicals, biochemicals or cellular extracts for defined periods of time, in a non-limiting example for approximately one hour to approximately two hours, and those reprogrammed cells that express embryonic stem cell markers, such as Oct4, after a culture period are then ready for transplantation, cryopreservation or further maturation. In another embodiment of the present invention, hybrid stem cells are provided which can be used for cellular regenerative / reparative therapy. The hybrid stem cells of the present invention are pluripotent and customized for the intended recipient so that they are immunologically compatible with the recipient. Hybrid stem cells are a fusion product between a donor cell, or nucleus thereof, and a host cell. Typically the fusion occurs between a donorImmorta-LivMon-PCT nucleus and an enucleated host cell. The donor cell can be any diploid cell, including but not limited to, cells from pre-embryos, embryos, fetuses and post-natal organisms. More specifically, the donor cell can be a primordial sex cell, including but not limited to, oogonium or differentiated or undifferentiated spermatogonium, or an embryonic stem cell. Other non-limiting examples of donor cells are therapeutically reprogrammed cells, embryonic stem cells, fetal stem cells and multipotent adult progenitor cells. Preferably the donor cell has the phenotype of the intended recipient. The host cell can be isolated from tissues including, but not limited to, pre-embryos, embryos, fetuses and post-natal organisms and more specifically can include, but is not limited to, embryonic stem cells, fetal stem cells, multipotent adult progenitor cells and adipose-derived stem cells. In a non-limiting example, cultured cell lines can be used as donor cells. The donor and host cells can be from the same individual or different individuals. In one embodiment, lymphocytes are used as donor cells and a two-step method is used to purify the donor cells. After the tissues was disassociated, an adhesion step was performed to remove any possible contaminating adherent cells followed by a density gradient purification step. The majority of lymphocytes are quiescent (in G0 phase) and therefore can have a methylation status than conveys greater plasticity for reprogramming. Multipotent or pluripotent stem cells or cell lines useful as donor cells in embodiments of the present invention are functionally defined as stem cells by their ability to undergo differentiation into a variety of cell types including, but not limited to, adipogenic, neurogenic, osteogenic, chondrogenic and cardiogenic cell.

[0417] In one embodiment, a hepatogenic cell is administered to a subject in need thereof to provide immunomodulatory functions or immune suppressive effects in the liver. In one embodiment, a hepatogenic cell such as a monocyte, a MSC, or a myeloid- derived suppressor cell, possesses one or a plurality of immunomodulatory effects on T cells. Immune modulation by a hepatogenic cell may be assessed in vitro, in a preclinical animal model, or in vivo upon administration to a subject in need thereof. In certain embodiments, an immune modulatory cell of the invention is administered to a subject, peripheral blood lymphocytes are isolated at a time point post-administration and in vitro assessments are subsequently performed. In one embodiment, flow cytometry is used to measure the frequencies of CD4(+)CD25(+)CD127(-) regulatory T cells and CD4(+)CD8(-)IL-17(+) (Th17) cells among freshly isolated peripheral blood lymphocytes or in vitro-stimulated peripheral blood lymphocytes. In certain embodiments, the frequencies of regulatory T cells are increased upon administration of aImmorta-LivMon-PCT therapeutic cell of the invention. In certain embodiments, the ratios of regulatory T cells to Th17 cells are increased upon administration of a therapeutic cell of the invention. In certain embodiments, the changes in the frequencies and ratios of regulatory T cells and Th17 cells are associated with improvement in liver function in the subject.

[0418] In one embodiment, hepatogenic monocytes / macrophages are produced by a multistep process. The first step may include exposing the iPSC to one or more factors, for example in culture. The iPSC may be exposed to bone morphogenetic protein-4 (BMP-4, GenBank Accession Number Q53XC5) preferably at 50 ng / mL The iPSC may be exposed to vascular endothelial growth factor (VEGF, GenBank Accession Number NP_001165097) preferably at 50 ng / mL. The iPSC may be exposed to stem cell factor (SCF, GenBank Accession Number P21583.1) preferably at 20 ng / mL. The iPSC may be factors simultaneously. The second step may include exposing the EB to a factor, for example in culture. The EB may be exposed to macrophage colony stimulating factor (M- CSF, GenBank Accession Number P09603) preferably at 100 ng / mL. The EB may be exposed to Interleukin-3 (IL-3, GenBank Accession Number AAC08706) preferably at 25 ng / mL. The EB may be exposed to glutamax preferably at 2 mM. The EB may be -mercaptoethanol preferably at 0.055 mM. The EB may be exposed to one or more, such as all of these factors simultaneously. The EB may be exposed to the factor or factors in a medium such as X-VIVO™15 media (Lonza, Basel, Switzerland).

[0419] In one embodiment, iPSCs are harvested using TrypLE™, centrifuged and the cell pellet was resuspended in stem cell maintenance media mTeSR™1, supplemented with 50 ng / mL bone morphogenetic protein-4, 50 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL stem cell factor (SCF), are seeded at a density of 12,000 cells / well into a round-bottom, low adherence, 96-well plate, which is centrifuged and incubated at 37° C. in a 5% CO2atmosphere for 4 days before harvesting the embryoid bodies (EBs). 75% media change is performed on the second day. At day 4, 12 EBs are harvested and transferred into each well of a 6-well plate and cultured in X-VIVO™15 media (Lonza, Basel, Switzerland), supplemented with 100 ng / mL macrophage colony stimulating factor (M-CSF), 25 ng / mL Interleukin-3, - mercaptoethanol. Two-thirds of the media is changed every five to seven days. Pre- macrophages are generated from the EBs in 3 to 4 weeks. Suspended pre-macrophages are collected from the media weekly. They may be used for further differentiation. TheImmorta-LivMon-PCT starting cell population, iPSC, are morphologically similar to human embryonic stem cells, express typical human ESC-specific cell surface antigens and genes, differentiate into multiple lineages in vitro, and form teratomas containing differentiated derivatives of all three primary germ layers when injected into immunocompromised mice. Human iPSC are derived from somatic cells and are typically produced by expression of Oct-3 / 4, Sox-2, c-Myc, and Klf-4 or by Oct-3 / 4, Sox-2, Nanog, and Lin28.

[0420] Methods of producing induced pluripotent stem cells are known in the art. The term “pluripotent” or “pluripotency” as used herein refers to cells with the ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to many or all tissues of a prenatal, postnatal or adult animal. A standard art-accepted test, such as the ability to form a teratoma in 8-12 week-old SCID mice, can be used to establish the pluripotency of a cell population, however identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells. Cell pluripotency is a continuum, ranging from the completely pluripotent cell that can form every cell of the embryo proper, e.g., embryonic stem cells and iPSCs, to the incompletely or partially pluripotent cell that can form cells of all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells, such as, for example, germline transmission or the ability to generate a whole organism. In one embodiment, the pluripotency of a cell is increased from an incompletely or partially pluripotent cell to a more pluripotent cell or, in certain embodiments, a completely pluripotent cell.

[0421] In certain embodiments, pluripotency of induced pluripotent stem cells can be assessed, for example, by teratoma formation, germ-line transmission, and tetraploid embryo complementation. In some embodiments, expression of pluripotency genes or pluripotency markers as discussed elsewhere herein, can be used to assess the pluripotency of a cell. “Pluripotent stem cell characteristics” refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. Expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics. For example, human pluripotentImmorta-LivMon-PCT stem cells express at least some, and optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E- cadherin, UTF-1, Oct4, Rexl, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics. A “somatic cell” as used herein refers to differentiated, or partially differentiated cells relative to embryonic stem cells. Thus, the term includes, e.g., cells such as fibroblasts that are derived from embryonice stem cells, but are differentiated. The term “embryonic stem cell” is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Illustrative distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, normal karyotype, responsiveness to particular culture conditions, and the like.

[0422] In one embodiment the invention teaches the generation of hepatognic macrophage precursor cell may be exposed to one or more hepatic cues to promote its maturation into an Kupffer Cell. For example, the macrophage precursor cell may be cultured in a cell medium comprising one or more such hepatic cues. The hepatic cue may comprise one or more factors secreted by a hepatocyte. For this purpose, hepatocytes may be cultured in a cell culture medium and the soluble factors released by the hepatocytes may be collected in the form of medium conditioned by hepatocyte cell culture (a primary hepatocyte culture medium, PHCM). This conditioned medium may be collected and added to the premacrophage culture medium to expose them to the cue. The macrophage precursor cell may be exposed to a single factor or a combination of factors. The cell culture medium may comprise Advanced DMEM. The cell medium may therefore be supplemented with conditioned medium from culture of hepatocytes, for example conditioned medium from culture of primary human hepatocytes (primary human hepatocyte conditioned medium—PHCM). The cell culture medium may contain a supplement. The supplement may comprise any of the following, such as at the indicated concentrations: 2.5 mL Penicillin / Streptomycin bovine serum albumin (BSA)—(1.25 g / mL) linoleic aImmorta-LivMon-PCT concentration) HEPES, pH 7.4 (15 mM final concentration) GlutaMAX™ 2 mM final concentration

[0423] According to the methods described here, a macrophage precursor cell is exposed to one or more hepatic cues to generate an iPSC-derived Kupffer cell. The macrophage precursor cell may be cultured in the presence of each hepatic cue. Advanced DMEM The hepatic cue may comprise culture in Advanced DMEM. The culture medium may therefore comprise Advanced DMEM containing conditioned medium from another source, for example primary human hepatocyte conditioned media (PHCM). Advanced DMEM may be obtained commercially, for example from Thermo Fisher Scientific. Primary Human Hepatocyte Conditioned Medium (PHCM)

[0424] The conditioned cell culture medium such as a Primary Human Hepatocyte Conditioned Medium (PHCM) may be obtained by culturing a hepatocyte such as a human hepatocyte, a descendent thereof or a cell line derived therefrom in a cell culture medium; and isolating the cell culture medium.

[0425] Methods of culturing primary hepatocytes are well known in the art. The conditioned medium may be filtered or concentrated or both during, prior to or subsequent to use. For example, it may be filtered through a membrane, for example one with a size or molecular weight cut-off. It may be subject to tangential force filtration or ultrafiltration.

[0426] For example, filtration with a membrane of a suitable molecular weight or size cutoff, may be used. The conditioned medium, optionally filtered or concentrated or both, may be subject to further separation means, such as column chromatography. For example, high performance liquid chromatography (HPLC) with various columns may be used. The columns may be size exclusion columns or binding columns.

[0427] The iPSC-derived hepatogenic Kupffer cell prepared according to the methods described here may exhibit a property of a Kupffer cell. The property of a Kupffer Cell may comprise a biological property, such as a biological activity.

[0428] The iPSC-derived hepatogenic Kupffer cell may exhibit any one or more of the biological activities of a Kupffer cell, such as a human Kupffer cell. The iPSC-derived Kupffer cell may for example have a diagnostic, therapeutic or restorative activity of a Kupffer cell. The Kupffer cell may comprise a native Kupffer cell. The native Kupffer cell may comprise a Kupffer cell from a liver of an individual. The native Kupffer cell may comprise a primary Kupffer cell. The native Kupffer cell may comprise a primary adult human KC (pKC). The Examples show that iPSC-derived Kupffer cells compriseImmorta-LivMon-PCT biological activities of Kupffer cells and are capable of substituting for the Kupffer cells themselves. The biological property or biological activity of an iPSC-derived Kupffer cell may therefore correspond to a biological property or activity of a Kupffer cell. The property may comprise a biological property such as a biological activity. Examples of biological activities of Kupffer cells include expression of a macrophage marker, phagocytosis, release of an inflammatory cytokine, growth factor or reactive oxygen species upon activation; and secretion of IL- with LPS. The iPSC-derived Kupffer cells may exhibit one or more such activities. The iPSC-derived Kupffer cells may display each of these activities. The iPSC-derived Kupffer cell may exhibit a biological property of a Kupffer cell comprising expression of a macrophage marker such as a macrophage specific marker. The macrophage marker may comprise CD11, CD14, CD68, CD163, or CD32. Alternatively, or in addition, the macrophage marker may comprise CLEC-4F, ID1, or ID3. Assays for expression of these markers are well known in the art. EXAMPLES

[0429] The following examples are not intended as limitations. Rather they demonstrate illustrative embodiments of the present invention. Example 1: Generation of a Monocyte Population from Induced Pluripotent Stem Cells for Treating Liver Failure

[0430] A population of therapeutic cells was generated for treating liver failure. The therapeutic cell population comprised hepatogenic monocytes that were generated from induced pluripotent stem cells (iPSCs) and exposed to a simulated environment of liver injury to endow the cells with hepatocyte markers and functions.

[0431] Methods for generating monocytes: Monocytes were differentiated from iPSCs using the STEMdiff Monocyte Kit (STEMCELL Technologies, #05320). Approximately 120 iPSC aggregates (50– of human embryonic stem cell (hESC)-qualified Matrigel coated six-well tissue culture plates. The next day, the plates were examined under a light microscope to ensure around 60–80 iPSC aggregates were seeded within each well of the six-well plate. By day 10 of the protocol, monocytes were observed lifting off the basal hematopoietic progenitor cell layer and remained in the media as suspension cells. Starting at day 16 post iPSCImmorta-LivMon-PCT differentiation, a fixed schedule was followed to harvest monocytes from the cell culture supernatant. Upon harvest, the monocytes were seeded onto tissue culture-treated plates to allow the monocytes to adhere to the bottom of the plates. Each well started with 2 mL of monocyte differentiation medium [StemSpan SFEM II (STEMCELL Technologies, #09605) + 1× STEMdiff monocyte differentiation supplement (STEMCELL Technologies, #05324)] and incubated for 2 days. After two days incubation, the cell culture media was topped off with 2 mL of additional monocyte differentiation medium and incubated for another 2–3 days. This method enabled greater cell numbers of monocytes to be harvested, since they were allowed to accumulate in the culture dish for 4–5 days. Monocytes harvested from cell culture supernatants were collected via centrifugation at 300xg for 5 minutes and then transitioned to culture in ImmunoCult-SF macrophage medium (STEMCELL Technologies, #10961) over a period of several days. Specifically, a medium ratio of 1:3 for monocyte differentiation medium : ImmunoCult- SF macrophage medium was used to initially seed freshly isolated monocytes onto 6-well tissue culture plates (2 × 106cells per well plated). In the following days, a daily media change was performed on the adherent cells following the monocyte differentiation medium : ImmunoCult-SF macrophage medium ratio of 1:1 (1 day after harvest) and 3:1 (2 days after harvest). These media transition steps were necessary as we found direct transition of monocytes from the monocyte differentiation media to the ImmunoCult-SF macrophage medium led to significantly reduced cell viability.

[0432] Methods for generating hepatogenic monocytes: The cells generated using the abovementioned methods were then induced to undergo differentiation into hepatocyte-like cells Monocytes were cultured in media alone (control cells), hepatocyte growth factor (HGF) (20 ng / ml), plasma from a subject with alcohol induced liver failure (i.e., as a source of growth factors) (5% v / v), or the combination for a period of 2, 4, or 6 days. The differentiation of hepatocyte-like cells was determined by measuring the concentration of albumin in the medium by ELISA, since albumin is a major plasma protein synthesized by hepatocytes. The results are shown in Figure 1.

[0433] Conclusions: Culture of monocytes in medium comprising HGF or in medium supplemented serum from a subject with liver failure induced albumin production in the cultures. The largest increase in albumin production was observed in cultures supplemented with the combination of HGF and serum from the patient. Albumin production in the combination-treated cultures increased with the duration ofImmorta-LivMon-PCT culture. These data indicate that HGF plus serum from a subject with liver failure induces the differentiation of monocytes into hepatocyte-like cells. Example 2: Hepatogenic Activity of Hepatogenic Monocytes In Vivo Nude mice were randomly divided into groups of control, hepatogenic monocytes generated as described above with culture of HGF and liver failure patient sera cultured for 4 or 6 days were injected IV at 500,000 per mouse once a week after CCL4. Mice were injected with 10% (vol / vol) CCl4 at a dose of 10 mL / kg body weight by intraperitoneal injection. AST was assessed at the indicated times. Results are shown in FIG. 2. Example 3 Hepatogenic Activity of iPSC Derived Monocytes is Augmented in c-kit Expressing Monocytes iPSC derived monocytes cultured for 4 days with HGF (20 ng / ml) were administered 3 once weekly in the CCL4 induced model from Example 2. Cells were administered at 500,000 per injection intravenously via tail vein as unseparated cells, c-kit expressing cells, and c-kit negative cells. Cells were purified using magnetic activated cell sorting (MACS) . Results are shown in FIG. 3.

Claims

Immorta-LivMon-PCT WHAT IS CLAIMED IS:

1. A method for treating a subject with liver failure, the method comprising: a) identifying a subject with liver failure; b) isolating a primary somatic cell from a biological fluid or tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce dedifferentiation into an induced pluripotent stem cell; d) providing a second cell culture system; wherein the induced pluripotent stem cell is provided with culture conditions to induce differentiation into a monocyte; e) providing a third cell culture system, wherein the differentiated monocyte is provided with culture conditions to induce the expression of one or a plurality of hepatogenic factors; f) isolating the hepatogenic monocyte from the third culture system; and g) administering the hepatogenic monocyte to the subject, wherein the hepatogenic monocyte exerts regenerative functions, immune modulatory functions, or both.

2. The method of Claim 1, wherein the hepatogenic monocyte is capable of reversing liver failure in the subject.

3. The method of Claim 1, wherein the primary somatic cell is selected from the group consisting of a fibroblast, a lymphocyte, a myeloid cell, a neutrophil, an eosinophil, a basophil, a myeloblast, a monocyte, a macrophage, a mesenchymal stem cell, a keratinocyte, an epithelial cell, a renal epithelial cell, an epithelial progenitor cell, a hematopoietic stem cell, a common myeloid progenitor cell, a natural killer (NK) cell, an NK T cell, a common lymphoid progenitor cell, an endothelial cell, an endothelial progenitor cell, a synovial cell, a mobilized CD34+ peripheral blood mononuclear cell, a blood mononuclear cell, or a tissue-specific stem or progenitor cell.Immorta-LivMon-PCT 4. The method of Claim 1, wherein the hepatogenic monocyte shares one or a plurality of morphological, phenotypic, and functional characteristics in common with a primary hepatocyte from liver.

5. The method of Claim 1, wherein the hepatogenic monocyte expresses one or a plurality of transcription factors selected from the group comprising a) signal transducer and activator of transcription 6 (STAT6); b) Peroxisome proliferator- activated receptor gamma (PPAR- l-like factor 4 (KLF4); d) c-Maf,e) MafB; and f) SP1.

6. The method of Claim 1, wherein the hepatogenic monocyte produces one or a plurality of molecules selected from the group comprising a) albumin; b) fibrinogen; c) a clotting factor (e.g., factor V, VII, IX, X, XI, XII, protein C, protein S, antithrombin, or a combination thereof); d) transferrin; e) plasminogen; f) ceruloplasmin; g) a complement protein; h) a bactericidal protein; i) an opsonin; j) an iron-sequestering protein; k) bile; l) bicarbonate; m) fetuin-B; and n) an aminotransferase (i.e., ALT and / or AST).

7. The method of Claim 1, wherein the hepatogenic monocyte produces one or a plurality of cytokines and chemokines selected from the group comprising a) hepatocyte growth factor (HGF); b) insulin-like growth factor-1 (IGF-1); c) fibroblast growth factor (FGF); d) platelet-derived growth factor (PDGF); e) transforming growth factor beta (TGF-beta); f) nerve growth factor (NGF); g) Interleukin-4 (IL-4); h) IL-13; h) IL-10; i) colony-stimulating factor 1 (CSF1); j) Vascular endothelial growth factor (VEGF); k) Stromal cell-derived factor-1 (SDF-1); l) CXCR4; m) CCR2; n) CX3CR1; o) CXCL12; p) CC chemokine ligand 1 (CCL1); q) CXCR7; r) intercellular adhesion molecule 1 (ICAM-1); s) tumor necrosis factor alpha (TNF- ); t) IL-22; u) Oncostatin M (OSM); and v)Stem cell factor (SCF).

8. The method of Claim 1, wherein the hepatogenic monocyte expresses one or a plurality of molecules selected from the group comprising a) CD14; b) CD16; c) CD11b; d) CD45; e) CD33; f) CD163; g) CD80; h) CD206; i) 25F9; j) CD1d; k) CD9; l) CD36; m) CD99; n) CD163; o) CLEC5A; and p) CD15.

9. The method of Claim 1, wherein the second culture system comprises monocyte conditioned medium.Immorta-LivMon-PCT 10. The method of Claim 9, wherein the monocyte conditioned medium is derived from culture of monocytes that have been stimulated with a toll-like receptor agonist.

11. The method of Claim 1, wherein the second culture system generates monocytes of the M2-type .

12. The method of Claim 11, wherein the M2-type monocytes are characterized by production of IL-10, transforming growth factor-beta (TGF- ), or both.

13. The method of Claim 11, wherein the M2-type monocytes express signal transducer and activator of transcription 6 (STAT6).

14. The method of Claim 1, wherein the second culture system comprises one or a plurality of factors selected from the group comprising hepatocyte growth factor (HGF), fibroblast growth factor-1 (FGF-1), FGF-2, bone morphogenetic protein-2 (BMP-2), BMP-4, macrophage colony-stimulating factor (M-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF).

15. The method of Claim 1, wherein the third culture system comprises HGF.

16. The method of Claim 1, wherein the third culture system comprises one or a plurality of factors selected from the group comprising FGF-1, FGF-2, BMP-2, BMP-4, M-CSF GM-CSF, and LIF.

17. The method of Claim 1, wherein the induction of expression of one or a plurality of hepatogenic factors into the differentiated monocyte is performed by gene transfection.

18. The method of Claim 17, wherein the transfected genes are selected from the group comprising a) HNF4A; b) FOXA2; c) FOXA1; and d) C / EBP alpha.

19. The method of Claim 1, wherein the third cell culture system comprises serum harvested from a subject with liver failure.

20. The method of Claim 1, wherein the hepatogenic monocyte produces insulin-like growth factor 1 (IGF-1).

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