Reprogrammed cell modulation of cancer microenvironment by tumor homing personalized regenerative cells

Abstract

Disclosed are methods of augmenting efficacy of oncology therapies by providing reprogrammed cells such as personalized regenerative cells that modulate the tumor microenvironment. In one embodiment, somatic cells are dedifferentiated into OCT-4 expressing cells and endowed with tumor homing properties through culture or gene engineering. Once a stable population of tumor homing cells is established, such cells are made to express immune stimulating agents constitutively, or selectively upon entering the cancer microenvironment. Selective expression of immune stimulatory agents can be induced by exposure to hypoxia, acidosis, immune suppressive signaling or inflammatory signaling.

Description

REPROGRAMMED CELL MODULATION OF CANCER MICROENVIRONMENT BY TUMOR HOMING PERSONALIZED REGENERATIVE CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims the benefit of priority to United States Provisional Application Serial No. 63 / 615,298, filed on December 28, 2023, which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION

[0002] The invention pertains to the fields of oncology and regenerative medicine, and more specifically, regenerative medicine approaches utilizing engineered cells to modify the tumor microenvironment and treat cancer.BACKGROUND

[0003] Therapeutic interventions in cancer have been historically limited by several biological and physical properties of tumors. It is generally accepted that tumors maintain their mass and pathological activities through the function of specialized cells which possess the ability to undergo asymmetric division. These cells, referred to as “cancer stem cells”, have been demonstrated to be essential for failure of chemotherapy, radiotherapy, or immunotherapy. Essentially, tumor stem cells reside in a quiescent state deep in hypoxia areas of the tumor mass and remain in a mitotically inactive state until the overall tumor mass is perturbed. For example, administration of chemotherapy leads to death of rapidly dividing cancer cells, whose death triggers production of soluble mediators which instruct the tumor stem cell to enter cell cycle and produce copies of itself as well as daughter cells that are aggressive tumor cells.

[0004] Currently there are limited means to target the cancer stem cell due to the fact that the majority of these cells reside deep in hypoxic areas of the tumor.

[0005] The current invention provides methods of use of cells with regenerative potential such as mesenchymal stem cells that home into, reside, and proliferate in areas of hypoxia such as tumors. Utilization of mesenchymal stem cells has been relatively limited to use in stimulation of angiogenesis. The current invention therefore providesmesenchymal stem cell-based therapies that are directed toward the production of immunogenic factors once they enter areas of hypoxia.SUMMARY

[0006] Disclosed are methods of augmenting efficacy of antineoplastic therapies through modulation of tumor microenvironment immune suppressive properties by administration of reprogrammed cells such as personalized regenerative cells. In one embodiment somatic cells are dedifferentiated into OCT-4 expressing cells and endowed with tumor homing properties through culture or gene engineering. Once a stable population of tumor homing cells is established, such cells are made to express immune stimulating agents constitutively, or selectively upon entering the cancer microenvironment. Selective expression of immune stimulatory agents can be induced by exposure to hypoxia, acidosis, immune suppressive signaling or inflammatory signaling.

[0007] Embodiments of the invention disclose methods for generating an iPSC cell and subsequent methods for differentiating the iPSC cell into a therapeutic cell type that is suitable for administration to a subject with a disease such as cancer. Embodiments of the invention provide iPSCs that are generated from somatic cells of a subject that are subsequently differentiated into autologous therapeutic cells. In one embodiment, a method of generating a therapeutic cell population for treating cancer is provided, the method comprising: a) identifying a subject in need of treatment for cancer; b) extracting a somatic cell from the subject; c) dedifferentiating the somatic cell to generate an induced pluripotent stem cell; d) inducing the differentiation of the induced pluripotent stem cell into a therapeutic cell; and e) administering the therapeutic cell to the subject. In certain embodiments, a therapeutic cell generated using these methods comprises a mesenchymal stem cell, a monocyte, a fibroblast, a macrophage, an M2 macrophage, a dendritic cell, a regulatory dendritic cell, a fibroblast, a T cell, a regulatory T cell, a B cell, a regulatory B cell, a neutrophil, a natural killer (NK) cell, an NKT cell, an endothelial progenitor cell (EPC), an eosinophil, a mast cell, a neural cell, or a neural progenitor cell.

[0008] Embodiments of the invention provide therapeutic cells that can be administered to a subject to overcome or lessen the immunological and physiological barriers to effective therapy that are imposed by a tumor microenvironment. In oneembodiment, a mesenchymal stem cell is provided that possesses the ability to induce the differentiation of neoplastic cells into cells with reduced metastatic potential. In another embodiment, a mesenchymal stem cell is provided that mediates one or a plurality of the following effects in vitro or in a tumor microenvironment: a) increased activity of tissue inhibitors or metalloproteinases (TIMPs); b) decreased activity of matrix metalloproteinases (MMPs); and c) reduced oxidative stress. In one embodiment, a mesenchymal stem cell is provided that reduces oxidative stress that is measurable on the basis of decreased neutrophil activation, increased neutrophil apoptosis, decreased activation of macrophages (in particular, M2 -type macrophages), decreased myeloid suppressor cell activation, decreased production of superoxide radicals, decreased presence of free oxygen radicals, decreased presence of hydrogen peroxide, or increased expression of superoxide dismutase.

[0009] In one embodiment, a method for generating a therapeutic cell comprising an autologous mesenchymal stem cell is provided, the method comprising: a) isolating a somatic cell from a subject; b) providing one or a plurality of agents to induce dedifferentiation of the somatic cell into an iPSC, wherein the one or plurality of agents include a bone morphogenetic protein (BMP) ; and c) providing one or a plurality of agents for inducing differentiation of the iPSC into a cell that expresses one or a plurality of the following biomarkers on the cell surface: CD73, CD90, CD103, or CD105. In one embodiment, a bone morphogenetic protein added to the culture comprises BMP2, BMP4, or a combination thereof. In one embodiment, BMP is provided to a culture comprising iPSCs for a duration that is sufficient to induce expression of one or a plurality of markers comprising CD73, CD90, CD103, or CD105.

[0010] In one embodiment, a method for generating a therapeutic cell comprising an autologous mesenchymal stem cell is provided, wherein the mesenchymal stem cell expresses one or a plurality of biomarkers comprising CD73, CD90, CD103, CD105, CXCR4, VEGF-R2, or c-met.

[0011] In one embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) generating an induced pluripotent stem cell from a somatic cell of the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 andCXCR4; and c) infecting the mesenchymal stem cell with an oncolytic virus (e.g., vaccinia virus) in vitro. In certain embodiments, an oncolytic virus has undergone genetic modifications to increase its selectivity for tumors. In one embodiment, an oncolytic virus comprises one or a plurality of the following viruses: HSV-1716, G207, MO32, G47delta, Delta-24-RGD, Onyx-015, NDV-HUJ, Reolysin, PVS-RIPO, H-1PV, MV-CEA, or TOCA511. In one embodiment, an oncolytic virus comprises one or a plurality of the following virus types: herpes simplex virus, adenovirus, Newcastle disease virus, reovirus, poliovirus, parvovirus, measles virus, or reovirus. In these embodiments, a mesenchymal stem cell is used as a delivery vehicle for the virus in the subject.

[0012] In another embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; and c) infecting the mesenchymal stem cell with an oncolytic virus; d) optionally, transfecting the mesenchymal stem cell with a gene for molecule or antigen comprising a chemokine, growth factor, or a cytokine. In some embodiments, the gene encodes a molecule or antigen that is selected from the group consisting of C-X-C chemokine receptor type 4 (CXCR4), CXCR7, vascular endothelial growth factor receptor 2 (VEGF-R2), c-met, leukemia inhibitory factor (LIF), interferon-gamma (IFN-g), IFN-beta, intercellular adhesion molecule- 1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2), vascular cell adhesion molecule-1 (VCAM-1), stromal cell-derived factor-la (SDF-1), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), fas ligand, TNF-related apoptosis-inducing ligand (TRAIL), IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL- 15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN- beta, G-CSF, GM-CSF, or M-CSF.

[0013] An object of the invention is to provide therapeutic cells that can exert anticancer effects under conditions of hypoxia. One embodiment provides a mesenchymal stem cell derived from an iPSC that is engineered to express a hypoxia-inducible promoter, wherein a hypoxia inducible promoter comprises a genetic sequence within the DNA of the mesenchymal stem cell that is specifically activated when the cell is exposed to low oxygen levels.

[0014] In one embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; and c) infecting the mesenchymal stem cell with an oncolytic virus; d) transfecting the mesenchymal stem cell with a hypoxia-inducible promoter to drive expression of genes for cell homing and immune function. In some embodiments, a hypoxia inducible promoter drives expression of genes for cell homing and / or immune function selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN- beta, G-CSF, GM-CSF, or M-CSF. In certain embodiments, step d) is performed prior to, after, or concurrently with step c).

[0015] In one embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; c) infecting the mesenchymal stem cell with an oncolytic virus; d) transfecting the mesenchymal stem cell with a hypoxia-inducible promoter to drive expression of genes for cell homing and / or immune function; and e) transfecting the mesenchymal stem cell with a gene for molecule or antigen comprising a chemokine, growth factor, or a cytokine. In certain embodiments, a hypoxia-inducible promoter drives expression of genes for cell homing and immune function selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, or M- CSF. In certain embodiments, a mesenchymal stem cell is transfected with a gene for a molecule or antigen that is selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL- 23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, or M-CSF. In certain embodiments, step c) is performed prior to, after, or concurrently with step d). In certain embodiments, step c) is performed prior to, after, or concurrently with step e). In certain embodiments, step d) is performed prior to, after, or concurrently with step e).

[0016] Different embodiments with reference to aspect number are provided below:

[0017] 1. A method of treating cancer comprising: a) extracting a somatic cell; b) retrodifferentiating said somatic cells; c) endowing to said retrodifferentiated somatic cell ability to selectively home towards neoplastically transformed tissue; d) endowing to said retrodifferentiated somatic cell ability to selectively secrete and / or express factors that modify the tumor microenvironment to increase amenable to therapeutic intervention.

[0018] 2. The method of aspect 1, wherein said somatic cell possesses one or more markers associated with regenerative activity.

[0019] 3. The method of aspect 2, wherein said marker associated with said regenerative activity is a cytokine or growth factor receptor.

[0020] 4. The method of aspect 3, wherein said cytokine or growth factor receptor is interleukin-3 receptor.

[0021] 5. The method of aspect 3, wherein said cytokine or growth factor receptor is interleukin-6 receptor.

[0022] 6. The method of aspect 3, wherein said cytokine or growth factor receptor is interleukin-35 receptor.

[0023] 7. The method of aspect 3, wherein said cytokine or growth factor receptor is VEGF receptor.

[0024] 8. The method of aspect 3, wherein said cytokine or growth factor receptor is c-met.

[0025] 9. The method of aspect 3, wherein said cytokine or growth factor receptor stem cell factor receptor.

[0026] 10. The method of aspect 3, wherein said cytokine or growth factor receptor is TNF alpha p55 receptor.

[0027] 11. The method of aspect 3, wherein said cytokine or growth factor receptor is TNF alpha p75 receptor.

[0028] 12. The method of aspect 2, wherein said marker associated with said regenerative activity is a transcription factor.

[0029] 13. The method of aspect 12, wherein said transcription factor is HIF-1 alpha.

[0030] 14. The method of aspect 12, wherein said transcription factor is RoR gamma.

[0031] 15. The method of aspect 12, wherein said transcription factor is OCT4.

[0032] 16. The method of aspect 12, wherein said transcription factor is NF-kappaB.

[0033] 17. The method of aspect 12, wherein said transcription factor is HIF-1 alpha.

[0034] 18. The method of aspect 2, wherein said marker associated with said regenerative activity is a cell surface marker.

[0035] 19. The method of aspect 18, wherein said cell surface marker is CD5.

[0036] 20. The method of aspect 18, wherein said cell surface marker is CD33.

[0037] 21. The method of aspect 18, wherein said cell surface marker is CD34.

[0038] 22. The method of aspect 18, wherein said cell surface marker is CD90.

[0039] 23. The method of aspect 18, wherein said cell surface marker is CD 105.

[0040] 24. The method of aspect 18, wherein said cell surface marker is CD73.

[0041] 25. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of OCT4.

[0042] 26. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of Lin28.

[0043] 27. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of Pim-1.

[0044] 28. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of Sox2.

[0045] 29. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of KLF.

[0046] 30. The method of aspect 1, wherein said retrodifferentiation of said somatic cell is accomplished by exposure to one or more agents capable of inducing expression of c-met.

[0047] 31. The method of aspects 25-30, wherein said retrodifferentiation of said somatic cell is accomplished by transfection with dedifferentiation genes.

[0048] 32. The method of aspects 25-30, wherein said retrodifferentiation of said somatic cell is accomplished by transfection with cytoplasm from immature cells.

[0049] 33. The method of aspects 25-30, wherein said retrodifferentiation of said somatic cell is accomplished by treatment with histone deacetylase inhibitors.

[0050] 34. The method of aspects 25-30, wherein said retrodifferentiation of said somatic cell is accomplished by treatment with DNA methyltransferase inhibitors.

[0051] 35. The method of aspects 25-30, wherein said retrodifferentiation of said somatic cell is accomplished by treatment with a ROCK inhibitor.

[0052] 36. The method of aspect 1, wherein said retrodifferentiated cell is differentiated into a mesenchymal stem cell or a cell possessing similarity to a mesenchymal stem cell.

[0053] 37. The method of aspect 36, wherein said cell possesses surface expression of CXCR4.

[0054] 38. The method of aspect 37, wherein said expression of CXCR4 is 10% higher as compared to CXCR4 expression on a peripheral blood derived monocyte.

[0055] 39. The method of aspect 37, wherein said expression of CXCR4 is 50% higher as compared to CXCR4 expression on a peripheral blood derived monocyte.

[0056] 40. The method of aspect 37, wherein said expression of CXCR4 is 100% higher as compared to CXCR4 expression on a peripheral blood derived monocyte.

[0057] 41. The method of aspect 37, wherein said expression of CXCR4 is 200% higher as compared to CXCR4 expression on a peripheral blood derived monocyte.

[0058] 42. The method of aspect 36, wherein said cell possesses surface expression of VEGF-R2.

[0059] 43. The method of aspect 42, wherein said expression of VEGF-R2 is 20% higher as compared to VEGF-R2 expression on a peripheral blood derived monocyte.

[0060] 44. The method of aspect 42, wherein said expression of VEGF-R2 is 40% higher as compared to VEGF-R2 expression on a peripheral blood derived monocyte.

[0061] 45. The method of aspect 42, wherein said expression of VEGF-R2 is 80% higher as compared to VEGF-R2 expression on a peripheral blood derived monocyte.

[0062] 46. The method of aspect 42, wherein said expression of VEGF-R2 is 160% higher as compared to VEGF-R2 expression on a peripheral blood derived monocyte.

[0063] 47. The method of aspect 36, wherein said cell possesses surface expression of c-met.

[0064] 48. The method of aspect 47, wherein said expression of c-met is 5% higher as compared to c-met expression on a peripheral blood derived monocyte.

[0065] 49. The method of aspect 48, wherein said expression of c-met is 10% higher as compared to c-met expression on a peripheral blood derived monocyte.

[0066] 50. The method of aspect 49, wherein said expression of c-met is 50% higher as compared to c-met expression on a peripheral blood derived monocyte.

[0067] 51. The method of aspect 50, wherein said expression of c-met is 100% higher as compared to c-met expression on a peripheral blood derived monocyte.

[0068] 52. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD90 at a concentration of greater than 10% of said CD90 expression on a peripheral monocyte population.

[0069] 53. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD90 at a concentration of greater than 50% of said CD90 expression on a peripheral monocyte population.

[0070] 54. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD90 at a concentration of greater than 90% of said CD90 expression on a peripheral monocyte population.

[0071] 55. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD90 at a concentration of greater than 10% of said CD90 expression on a peripheral monocyte population.

[0072] 56. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD90 at a concentration of greater than 50% of said CD90 expression on a peripheral monocyte population.

[0073] 57. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of timesufficient to induce expression of CD90 at a concentration of greater than 90% of said CD90 expression on a peripheral monocyte population.

[0074] 58. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 10% of said CD73 expression on a peripheral monocyte population.

[0075] 59. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 50% of said CD73 expression on a peripheral monocyte population.

[0076] 60. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 90% of said CD73 expression on a peripheral monocyte population.

[0077] 61. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 10% of said CD73 expression on a peripheral monocyte population.

[0078] 62. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 50% of said CD73 expression on a peripheral monocyte population.

[0079] 63. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD73 at a concentration of greater than 90% of said CD73 expression on a peripheral monocyte population.

[0080] 64. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of timesufficient to induce expression of CD 105 at a concentration of greater than 10% of said CD 103 expression on a peripheral monocyte population.

[0081] 65. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD 105 at a concentration of greater than 50% of said CD 105 expression on a peripheral monocyte population.

[0082] 66. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP2 for a period of time sufficient to induce expression of CD 105 at a concentration of greater than 90% of said CD 105 expression on a peripheral monocyte population.

[0083] 67. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD 105 at a concentration of greater than 10% of said CD 105 expression on a peripheral monocyte population.

[0084] 68. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD 105 at a concentration of greater than 50% of said CD 105 expression on a peripheral monocyte population.

[0085] 69. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by culture in BMP4 for a period of time sufficient to induce expression of CD 105 at a concentration of greater than 90% of said CD 105 expression on a peripheral monocyte population.

[0086] 70. The method of aspect 36, wherein said mesenchymal stem cell is generated from said retrodifferentiated cell by the steps of: a) plating cells onto a plate; b) addition to the plate of a mediat containing small molecules; c) culturing for 1 hour to 72 hour; and d) adding mesenchymal stem cell growth media.

[0087] 71. The method of aspect 70, wherein said cells are selected for expression of CD31 before plating.

[0088] 72. The method of aspect 70, wherein said cells are selected for expression of CD33 before plating.

[0089] 73. The method of aspect 70, wherein said cells are selected for expression of CD34 before plating.

[0090] 74. The method of aspect 70, wherein said cells are selected for expression of CD 133 before plating.

[0091] 75. The method of aspect 70, wherein said cells are selected for expression of c-met before plating.

[0092] 76. The method of aspect 70, wherein said cells are selected for expression of interleukin 3 receptor before plating.

[0093] 77. The method of aspect 70, wherein said cells are selected for expression of EGF -receptor before plating.

[0094] 78. The method of aspect 70, wherein said cells are selected for expression of thrombopoietin receptor before plating.

[0095] 79. The method of aspect 70, wherein said cells are treated with a histone deacetylase inhibitor before plating.

[0096] 80. The method of aspect 79, wherein said histone deacetylase inhibitor is selected from a group comprising of: a) phenylbutyrate; b) trichostatin A; c) valproic acid; d) sulforaphane; and e) genistein.

[0097] 81. The method of aspect 70, wherein said plates are coated with fibronectin.

[0098] 82. The method of aspect 70, wherein said plates are coated with vitronectin.

[0099] 83. The method of aspect 70, wherein said plates are coated with fibronectin and vitronectin.

[0100] 84. The method of aspect 70, wherein said plates are coated with hyaluronic acid.

[0101] 85. The method of aspect 70, wherein said plates are coated with fibronectin and hyaluronic acid.

[0102] 86. The method of aspect 70, wherein said plates are coated with vitronectin and hyaluronic acid.

[0103] 87. The method of aspect 70, wherein said plates are coated with vitronectin, hyaluronic acid and fibronectin.

[0104] 88. The method of aspect 70, wherein said small molecule is an NF -kappa B inhibitor.

[0105] 89. The method of aspect 70, wherein said small molecule is CHIR99021.

[0106] 90. The method of aspect 70, wherein said small molecule is ascorbic acid.

[0107] 91. The method of aspect 70, wherein said small molecule is all trans retinonic acid (ATRA).

[0108] 92. The method of aspect 70, wherein said small molecule is sodium phenylbutyrate.

[0109] 93. The method of aspect 70, wherein said small molecule is forskolin.

[0110] 94. The method of aspect 70, wherein said small molecule is an ALK inhibitor.

[0111] 95. The method of aspect 94, wherein said ALK inhibitor is SB431542.

[0112] 96. The method of aspect 94, wherein said ALK inhibitor is crizotinib.

[0113] 97. The method of aspect 94, wherein said ALK inhibitor is ceritinib.

[0114] 98. The method of aspect 94, wherein said ALK inhibitor is alectinib.

[0115] 99. The method of aspect 94, wherein said ALK inhibitor is brigatinib.

[0116] 100. The method of aspect 94, wherein said ALK inhibitor is lorlatinib.

[0117] 101. The method of aspect 70, wherein said small molecule is tranylcypromine hydrochloride.

[0118] 102. The method of aspect 70, wherein said small molecule is lithium chloride.

[0119] 103. The method of aspect 36, wherein said mesenchymal stem cell possesses ability to induce differentiation of neoplastic cells into cells with reduced metastatic potential.

[0120] 104. The method of aspect 103, wherein said reduced metastatic potential is associated with increased TIMP activity.

[0121] 105. The method of aspect 103, wherein said reduced metastatic potential is associated with reduced MMP activity.

[0122] 106. The method of aspect 103, wherein said mesenchymal stem cell possesses ability to reduce oxidative stress in the tumor microenvironment.

[0123] 107. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased neutrophil activation.

[0124] 108. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with increased neutrophil apoptosis.

[0125] 109. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased M2 activation.

[0126] 110. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased myeloid derived suppressor cell activation.

[0127] 111. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased presence of superoxide radicals.

[0128] 112. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased presence of free oxygen radicals.

[0129] 113. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with decreased presence of hydrogen peroxide.

[0130] 114. The method of aspect 106, wherein said reduction of oxidative stress in said tumor microenvironment is associated with increased expression of superoxide dismutase.

[0131] 115. The method of aspect 36, wherein said mesenchymal stem cell possesses ability to induce differentiation of neoplastic cells into cells with reduced metastatic potential.

[0132] 116. The method of aspect 115, wherein said mesenchymal stem cells are engineered to induce expression of immune stimulatory cytokines when exposed to hypoxia.

[0133] 117. Them method of aspect 116, wherein hypoxia mediated inducibility of immunogenic cytokine production is mediated by transfection of said mesenchymal stem cells with one or more hypoxia inducible promoters.

[0134] 118. The method of aspect 117, wherein said hypoxia inducible promoter utilizes one or more HIF-1 alpha binding elements.

[0135] 119. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 1 by over 25% when exposed to hypoxia.

[0136] 120. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 1 by over 100% when exposed to hypoxia.

[0137] 121. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-2 by over 25% when exposed to hypoxia.

[0138] 122. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-2 by over 100% when exposed to hypoxia.

[0139] 123. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-6 by over 25% when exposed to hypoxia.

[0140] 124. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-6 by over 100% when exposed to hypoxia.

[0141] 125. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-8 by over 25% when exposed to hypoxia.

[0142] 126. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-8 by over 100% when exposed to hypoxia.

[0143] 127. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 11 by over 25% when exposed to hypoxia.

[0144] 128. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 11 by over 100% when exposed to hypoxia.

[0145] 129. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 12 by over 25% when exposed to hypoxia.

[0146] 130. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 12 by over 100% when exposed to hypoxia.

[0147] 131. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 15 by over 25% when exposed to hypoxia.

[0148] 132. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 15 by over 100% when exposed to hypoxia.

[0149] 133. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 17 by over 25% when exposed to hypoxia.

[0150] 134. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 17 by over 100% when exposed to hypoxia.

[0151] 135. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 18 by over 25% when exposed to hypoxia.

[0152] 136. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin- 18 by over 100% when exposed to hypoxia.

[0153] 137. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-23 by over 25% when exposed to hypoxia.

[0154] 138. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-23 by over 100% when exposed to hypoxia.

[0155] 139. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-27 by over 25% when exposed to hypoxia.

[0156] 140. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interleukin-27 by over 100% when exposed to hypoxia.

[0157] 141. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of TNF-alpha by over 25% when exposed to hypoxia.

[0158] 142. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of TNF-alpha by over 100% when exposed to hypoxia.

[0159] 143. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of lymphotoxin by over 25% when exposed to hypoxia.

[0160] 144. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of lymphotoxin by over 100% when exposed to hypoxia.

[0161] 145. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of G-CSF by over 25% when exposed to hypoxia.

[0162] 146. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of G-CSF by over 100% when exposed to hypoxia.

[0163] 147. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of GM-CSF by over 25% when exposed to hypoxia.

[0164] 148. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of GM-CSF by over 100% when exposed to hypoxia.

[0165] 149. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of M-CSF by over 25% when exposed to hypoxia.

[0166] 150. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of M-CSF by over 100% when exposed to hypoxia.

[0167] 151. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interferon gamma by over 25% when exposed to hypoxia.

[0168] 152. The method of aspect 117, wherein said hypoxia inducible promoter stimulates said mesenchymal stem cell to increase production of interferon gamma by over 100% when exposed to hypoxia.DETAILED DESCRIPTION

[0169] 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.

[0170] The invention provides cell compositions and methods of use thereof that are applicable in oncology for overcoming the limitations to current therapeutic approaches that are imposed by the tumor microenvironment. The invention leverages induced pluripotent stem cells (iPSCs) from a subject that are genetically modified and differentiated into specialized cell types that are useful for treating cancer. Disclosed herein are therapeutic cell populations derived from iPSCs, which are generated byreprogramming adult somatic cells, and methods of use thereof for oncology applications. The present invention describes patient-specific cell-based therapies having utility in cancer treatment, wherein the disclosed therapies improve upon or overcome the limitations of traditional cell therapies that have been attempted using non-reprogrammed cell types. In certain embodiments, the invention provides an iPSC-derived autologous cell composition and methods of use thereof that is therapeutically advantageous for treating cancer in a subject 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 a higher therapeutic potential against cancer. In certain embodiments, the invention provides means of treating cancer in a subject by increasing sensitivity of a subject to cancer treatments (e.g., to chemotherapy, immunotherapy, or radiation) through administration of regenerative cells capable of selectively homing to tumors and producing immunogenic factors once having entered the tumor microenvironment

[0171] In certain embodiments, the invention provides means of treating cancer in a subject by increasing sensitivity of a subject to cancer treatments (e.g., to chemotherapy, immunotherapy, or radiation) through administration of regenerative cells capable of selectively homing to tumors and producing immunogenic factors once having entered the tumor microenvironment. In certain embodiments, a method is provided for generating cells that are administered as biologic therapeutics for treating a subject with cancer, including methods of reprogramming and genetic engineering of said cells. Other embodiments provide cell compositions that are suitable for delivering such therapies.

[0172] Unless defined otherwise, the technical terms used herein have the same meaning as is commonly understood by one of skill in the art.

[0173] As used herein, the terms “subject” or “patient” refers to a human or an animal to whom a method or therapy of the invention is applied.

[0174] As used herein, “cancer” is used to describe an uncontrolled growth of cells. The term “anti -cancer” is intended to encompass not only the treatment of a cancer disease, that is, inhibition of proliferation of cancer cells or cancer stem cells, or elimination of cancer cells or cancer stem cells, but also prevention of a cancer disease, that is, improvement of resistance to cancer prior to the onset of cancer. Thus, the term“prevention or treatment of cancer” or “inhibition of cancer” and “anti-cancer” are used interchangeably.

[0175] As used herein, the terms “marker" and "biomarker" are used interchangeably to refer to a gene expression product that is differentially present in a sample taken from two different subjects, e.g., from a test subject or patient having an ischemic event, compared to a comparable sample taken from a control subject (e.g., a subject not having an ischemic event such as a normal or healthy subject). In some contexts, these terms refer to a gene expression product that is differentially present in a subject, cell, tissue, or organ relative to another subject, cell, tissue, or organ.

[0176] As used herein, “inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibiting, activating, or modulating cells, respectively, and are identified using in vitro and in vivo assays for expression or activity. The term “modulator” includes inhibitors and activators. A modulator can be an antibody or a soluble ligand which binds a protein of interest. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Preferred modulators according to the invention, inhibit or suppress immune responses to an antigen or alloantigen. Assays to identify inhibitors and activators include, e.g., applying putative modulators to immune cells and then determining the functional effects of the cell on the immune response. Inhibitors or modulators are compared to control samples without the inhibitor or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control sample is about 80%, optionally 50% or 25 to 1%, or less. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control sample is 110%, optionally 150%, optionally 200-500%, or 1000-3000%, or higher.

[0177] As used herein, the term “isolated” when used with regard to a population of cells refers to a cell population which either has no naturally occurring counterpart or has been separated or purified from other components, including other cell types, which naturally accompany it, e.g., in normal or diseased tissues such as lung, kidney, orplacenta, tumor tissue such as colon cancer tissue, or body fluids such as blood, serum, or urine. Typically, an isolated cell population is enriched at least two-fold, at least fourfold, at least eight-fold, or more for a specified cell type when compared to the natural source from which the population was obtained.

[0178] As used herein, "differentially present" or “differentially expressed” refers to differences in the quantity or frequency (incidence of occurrence) of a biomarker present in a sample taken from a test subject as compared to a control subject. For example, a biomarker can be a gene expression product that is present at an elevated level or at a decreased level in blood samples of a risk subjects compared to samples from control subjects. Alternatively, a biomarker can be a gene expression product that is detected at a higher frequency or at a lower frequency in samples of blood from risk subjects compared to samples from control subjects. In certain contexts, a gene expression product is differentially present between two samples, specimens, or cells if the amount of the gene expression product in one sample, specimen, or cell is statistically significantly different from the amount of the gene expression product in the other sample, specimen, or cell. For example, a gene expression product is differentially present between two samples, specimens, or cells if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, specimen, or cell, or if it is detectable in one sample, specimen, or cell and not detectable in the other.

[0179] As used herein, “antibody” may refer to a monoclonal antibody, a multispecific antibody, a synthetic antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, an F(ab') fragment, a disulfide-linked Fv (sdFv), an anti-idiotypic (anti-Id) antibody, or an epitope-binding fragment of any of the above. In particular, an antibody may include an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that binds to a polypeptide antigen encoded by a gene comprised in the genomic regions or affected by genetic transformation(s). An immunoglobulin molecule may any antibody type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG.sub. l, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub. l and IgA.sub.2) or a subclass of an immunoglobulin molecule.

[0180] As used herein, "immunoassay" refers to an assay that uses an antibody or another type of reagent to specifically bind an antigen (e.g., a marker). For example, an immunoassay may leverage the specific binding properties of a particular antibody to identify, isolate and / or quantify an antigen. A variety of immunoassay formats and conditions may be used to identify an antigen with an appropriate level of selectivity and specificity, as may be defined using methods known in the art.

[0181] As used herein, "specifically” or “selectively” may be used in certain contexts to refer to a binding reaction that is determinative of the presence of the protein, antigen, epitope or molecule in a heterogeneous population of proteins, antigens, epitopes, or molecules. For example, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.

[0182] The terms "affecting the expression" and "modulating the expression" of a protein or gene, as used herein, should be understood as regulating, controlling, blocking, inhibiting, stimulating, enhancing, activating, mimicking, bypassing, correcting, modifying removing, and / or substituting said expression, in more general terms, intervening in said expression, for instance by affecting the expression of a gene encoding that protein.

[0183] As used herein, “induced pluripotent stem cell” (“iPSC”) or “personalized regenerative cell” refers to a type of pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., from skin, from peripheral blood cells, from bone marrow, or from another tissue source). For example, an induced pluripotent stem cell may be reprogrammed by the introduction of specific genes encoding transcription factors OCT- 3 / 4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under embryonic stem (ES) cell culture conditions. A personalized regenerative cell may refer to a stem cell that is amenable to modification or differentiation to provide a therapeutic cell for treating the same subject (i.e., an autologous cell).

[0184] As used herein, “reprogramming” when used in the context of a cell refers to a process that uses reprogramming factors such as transcription factors to convert one celltype into another cell type, for example, the conversion of a somatic cell into a pluripotent cell. Reprogramming of a cell may involve introducing reprogramming factors into a cell type through genetic manipulations or using chemical compounds.

[0185] As used herein, “dedifferentiation” (or “retrodifferentiation”) refers to a process whereby a cell reverts to a less specialized or less differentiated state or an earlier stage of development. This process may involve the loss of one or a plurality of characteristics or functions by a differentiated cell. In certain embodiments, the process of dedifferentiation involves reprogramming a terminally differentiated cell such as a somatic cell into an induced pluripotent stem cell that has self-renewal capabilities. The process of dedifferentiation may involve changes in a cell’s shape, gene expression, protein expression, proliferative potential, and / or function. In certain embodiments, dedifferentiation may be used to create stem cells that are genetically compatible with a subject.

[0186] As used herein, “oncolytic virus” refers to a virus having the ability to infect and destroy a tumor cell. An oncolytic virus may occur naturally, or it may be a modified or engineered version of a virus, for example, a virus comprising a curative transgene or a virus comprising a toxic payload. These engineered oncolytic viruses with enhanced tumor targeting ability, oncolytic activity, or generating potent anti-tumor immune responses are tested in preclinical animal models and cancer patients in clinical trials. An oncolytic virus has one or a plurality of functions with therapeutic value in oncology including but not limited to killing of tumor cells, triggering an immune response against a tumor cell, or enhancing the effects of a drug therapy (e.g., of a chemotherapeutic or immunotherapeutic drug agent, or a radiation treatment. An oncolytic virus may be an RNA virus or a DNA virus. Non-limiting examples of oncolytic viruses including Adenovirus, Herpes simplex virus (e.g., HSV-1 and HSV-2), Maraba virus, Parvovirus, Poxvirus, Newcastle disease virus, Reovirus, Vaccinia virus, Vesicular stomatitis virus, Coxsackie virus, Poliovirus, Flavivirus (e.g., Zika virus), Measles virus, Seneca valley virus, Semliki Forest virus, and Sindbis virus. Specific examples of oncolytic viruses include Rigvir (a picomavirus), Oncorine (adenovirus serotype 5), T-VEC (HSV-1), and DELYTACT (HSV-1).

[0187] As used herein, “mesenchymal stem cell” or “MSC” refers to a cell having the following characteristics: (1) adherent to plastic, (2) express CD73, CD90, andCD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) possess ability to differentiate to osteogenic, chondrogenic and adipogenic lineage. A mesenchymal stem cell may be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and tooth. As used herein, mesenchymal stem cells may include cells that are CD34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally. As used herein, mesenchymal stem cells include cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof and satisfy the ISCT criteria either before or after expansion. Mesenchymal stem cells include cells described in the literature as mesenchymal stromal cells, 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.

[0188] An object of the invention is the generation of a rejuvenated or “younger” source of autologous cells for treatment of cancer. The present disclosure is based on the inventors’ observations that a therapeutic cell population derived from iPSCs possesses specific therapeutic capabilities for treating cancer that exceed the therapeutic capabilities of primary sourced cells of the same lineage. By way of example, in certain embodiments, the invention provides mesenchymal stem cells differentiated from iPSCs generated from a subject that possess enhanced anti-cancer activities in vitro as compared to primary or naturally occurring mesenchymal stem cells isolated directly from an organ or tissue of the subject. The invention also provides a therapeutic cell derived from iPSCs that is more amenable as a vehicle for replication of an oncolytic virus or provides enhanced delivery of an oncolytic virus to a tumor as compared to a primary tissue- sourced mesenchymal stem cell. In certain embodiments, a therapeutic cell derived froman iPSC using the methods of the invention provides a cell that is more resistant to senescence than a primary-sourced cell of the same type or lineage isolated directly from a tissue. In certain embodiments, a therapeutic cell derived from an iPSC comprises a gene signature that is associated with cellular rejuvenation or longevity unlike the corresponding cell of the same lineage or type that is isolated directly from a tissue or organ. Non-limiting examples of rejuvenation genes include INHBE (member of TGF- beta pathway), TP53 (cell cycle control / cell self-renewal), CDKN1C (cell cycle control), CDK10, (cell cycle progression) ELAVL1 (growth and proliferation), DNMT3B (de novo methylation), and others. In one embodiment, the expression of one or a plurality of genes associated with pluripotent stem cells are retained in an iPSC-derived therapeutic cell type. In certain embodiments, a therapeutic cell derived from an iPSC demonstrates telomere elongation as compared to the corresponding cell of the same lineage or type that is isolated directly from a tissue or organ. Additional embodiments comprise a therapeutic cell comprising a mesenchymal stem cell that exhibits expression of rejuvenation- associated genes but retains certain phenotypic characteristics and trilineage differentiation potential into adipogenic, chondrogenic, and osteogenic lineages as a primary mesenchymal stem cell that is isolated directly from an adult tissue. Embodiments of the invention disclose therapeutic cells that have been reprogrammed through the pluripotent state that possess advantageous characteristics for downstream modifications / gene engineering or loading with cargo and, ultimately for administration to a subject to treat disease.

[0189] In certain embodiments, a therapeutic cell generated from an iPSC using the methods of the invention comprises a cell selected from the group consisting of a mesenchymal stem cell, a monocyte, a fibroblast, a macrophage, an M2 macrophage, a dendritic cell, a regulatory dendritic cell, a fibroblast, a T cell, a regulatory T cell (e.g., a CD4+ FoxP3+ cell, a CD8+ FoxP3+ cell, a CD8+ HLA-DR+ cell), a B cell, a regulatory B cell, a neutrophil, a natural killer (NK) cell, an natural killer T (NKT) cell, an endothelial progenitor cell (EPC), an eosinophil, a mast cell, a neural cell, or a neural progenitor cell. In certain embodiments, a therapeutic cell disclosed herein possesses immune stimulatory activities upon administration to the subject. In other embodiments, a therapeutic cell disclosed herein possesses homing or migration characteristics for reaching a tumor. Embodiments of the invention provide specific cell lineages or types that are generated from iPSC that are useful as autologous therapeutics in oncology.

[0190] Accordingly, embodiments of the invention disclose methods for generating an iPSC cell and subsequent methods for differentiating the iPSC cell into a therapeutic cell type that is suitable for administration to a subject with a disease such as cancer. Embodiments of the invention provide iPSCs that are generated from somatic cells of a subject that are subsequently differentiated into autologous therapeutic cells. In one embodiment, a method of generating a therapeutic cell population for treating cancer is provided, the method comprising: a) identifying a subject in need of treatment for cancer; b) extracting a somatic cell from the subject; c) dedifferentiating the somatic cell to generate an induced pluripotent stem cell; d) inducing the differentiation of the induced pluripotent stem cell into a therapeutic cell; and e) administering the therapeutic cell to the subject. In certain embodiments, a therapeutic cell generated using these methods comprises a mesenchymal stem cell, a monocyte, a fibroblast, a macrophage, an M2 macrophage, a dendritic cell, a regulatory dendritic cell, a fibroblast, a T cell, a regulatory T cell, a B cell, a regulatory B cell, a neutrophil, a natural killer (NK) cell, an NKT cell, an endothelial progenitor cell (EPC), an eosinophil, a mast cell, a neural cell, or a neural progenitor cell.

[0191] In one embodiment, generation of iPSCs is performed by reprogramming somatic cells, wherein the reprogramming is performed by introducing genes encoding for one or a plurality of factors including but not limited to PIM1, PIM3, sox-2, c-myc, k- ras, NF-kappa B, NANOG, KLF4, and OCT-4. 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 into the cells by co-inj ection 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 or reprogramming are introduced to the somatic cells to induce cellular dedifferentiation or reprogramming, wherein the RNA nanoparticles are selected from the group consisting of messenger RNA nanoparticles for expressing transcription factors which allow somatic cells or adult stem cells to be dedifferentiated into induced pluripotent stem cells, micro RNA nanoparticles facilitating the dedifferentiation, small interfering RNA nanoparticles facilitating the dedifferentiation, complex RNAnanoparticles which include micro RNA facilitating the dedifferentiation and small interfering RNA facilitating the dedifferentiation, complex RNA nanoparticles which include messenger RNA for expressing a transcription factor and micro RNA facilitating the dedifferentiation, complex RNA nanoparticles which include messenger RNA for expressing a transcription factor and small interfering RNA facilitating the dedifferentiation, and complex RNA nanoparticles which include messenger RNA for expressing a transcription factor, micro RNA facilitating the dedifferentiation, and small interfering RNA facilitating the dedifferentiation is used as the RNA nanoparticles for cell transformation. In one embodiment, the RNA nanoparticles for cell transformation include at least one RNA selected from the group consisting of messenger RNA for expressing transcription factors which allow somatic cells or adult stem cells to be dedifferentiated into induced pluripotent stem cells, microRNA facilitating the dedifferentiation, and small interfering RNA. In one embodiment, at least one RNA nanoparticle is selected from the group consisting of messenger RNA nanoparticles for expressing transcription factors and / or adjuvant genes associated with dedifferentiation which allow somatic cells or adult stem cells to be dedifferentiated into induced pluripotent stem cells. In one embodiment, the RNA nanoparticles capable of inducing cellular dedifferentiation and / or reprograming comprise of a spherical shape and have a diameter of 50 to 200 nm. In another embodiment, one or a plurality of chemicals associated with dedifferentiation are added to cultures of iPSCs to augment the dedifferentiation process of somatic cells, and wherein the one or plurality of chemicals are selected from the following group: an inhibitor of histone deacetylase(s), an inhibitor of DNA methyltransferase(s), a ROCK inhibitor, and an inhibitor of glycogen synthase kinase 3 (GSK-3).

[0192] In certain embodiments, iPSCs generated using these methods are cultured to form embryoid bodies under the appropriate culture conditions. In certain embodiments, an iPSC-derived differentiated cell type is generated by exposure of embryoid bodies derived from iPSCs to a decellularized matrix. In some embodiments, embryoid bodies are disaggregated prior to being subjected to the cellular differentiation protocols of the invention. In some embodiments, embryoid bodies are disaggregated prior to addition or seeding of a decellularized matrix in the cell culture system.

[0193] Certain embodiments disclose isolating or selecting a somatic cell type from a subject for subsequent differentiation into an iPSC. In one embodiment, a somatic cell comprises a peripheral blood cell, a monocyte, a fibroblast, a T cell, or a mesenchymal stem cell that is isolated from an organ or tissue of the subject. In certain embodiments, it may be advantageous to select a somatic cell comprises an adult cell type or lineage that possesses or retains proliferation or differentiation ability. In one embodiment, a somatic cell comprises a cell with regenerative activity, e.g., a multipotent cell in bone marrow, muscle, or another tissue that can be induced to differentiate into a specialized cell type to repair damaged tissue. In one embodiment, a somatic cell that is selected for carrying out the methods of the invention possesses one or a plurality of biomarkers associated with regenerative activity, wherein the one or plurality of biomarkers comprise cytokines or growth factor receptors, and wherein the cytokines or growth factor receptors comprise interleukin-3 receptor, interleukin-6 receptor, interleukin-35 receptor, vascular endothelial growth factor receptor, c-met, stem cell factor receptor, tumor necrosis factor receptor type 1 (p55), or tumor necrosis factor receptor type 2 (p75). In one embodiment, a somatic cell possesses one or a plurality of transcription factors associated with regenerative activity, wherein the one or plurality of transcription factors comprise hypoxia-inducible factor 1 -alpha (HIF-1 alpha), RAR-related orphan receptor gamma (RoR gamma), octamer-binding transcription factor 4 (OCT-4), or nuclear factor-kappa B (NF-kappB). In one embodiment, a somatic cell possesses one or a plurality of cell surface markers, wherein the cell surface markers comprise CD5, CD33, CD34, CD90, CD 105, or CD73.

[0194] Certain embodiments disclose treating an isolated somatic cell with the appropriate agents or compounds to induce dedifferentiation of the somatic cell into an iPSC. In one embodiment, an isolated somatic cell is cultured or treated with an agent or compound that induces expression or upregulation of one or a plurality of biomarkers comprising OCT-4, Lin28, PIM-1, PIM-3, Sox2, Kruppel-like factor (KLF), MYC, I- MYC, k-ras, NANOG, NF-kappaB, and c-met. In one embodiment, an isolated somatic cell is transfected with genes to reprogram or dedifferentiate the somatic cell. In another embodiment, a somatic cell is transfected with cytoplasm from an immature, multipotent, or pluripotent stem cell. In another embodiment, a somatic cell is treated with one or a plurality of the following agents: a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, or a Rho-associated, coiled-coil containing protein kinase(ROCK) inhibitor. In certain embodiments, the histone deacetylase inhibitor is selected from the group consisting of: sulforaphane, valproic acid, phenylbutyrate, sodium phenylbutyrate, trichostatin A, or a combination thereof.

[0195] Other embodiments disclose methods for applying agents or compounds for differentiation of an iPSC into a particular type or lineage of therapeutic cell. One embodiment provides a method for producing a differentiated therapeutic cell from an iPSC that is amenable for subsequent modifications such as genetic engineering or transfection with one or a plurality of genes, loading with a toxic payload (e.g., an anticancer drug such as a chemotherapeutic agent), or loading with an oncolytic virus, or combinations thereof. In certain embodiments, an iPSC is subjected to a treatment condition in culture to generate a cell selected from the group consisting of a mesenchymal stem cell, a monocyte, a fibroblast, a macrophage, an M2 macrophage, a dendritic cell, a regulatory dendritic cell, a fibroblast, a T cell, a regulatory T cell (e.g., a CD4+ FoxP3+ cell, a CD8+ FoxP3+ cell, or a CD8+ HLA-DR+ cell), a B cell, a regulatory B cell, a neutrophil, a natural killer (NK) cell, an natural killer T (NKT) cell, an endothelial progenitor cell (EPC), an eosinophil, a mast cell, a neural cell, or a neural progenitor cell.

[0196] In one embodiment, a method for generating a therapeutic cell comprising an autologous mesenchymal stem cell is provided, the method comprising: a) isolating a somatic cell from a subject; b) providing one or a plurality of agents to induce dedifferentiation of the somatic cell into an iPSC, wherein the one or plurality of agents include a bone morphogenetic protein (BMP) ; and c) providing one or a plurality of agents for inducing differentiation of the iPSC into a cell that expresses one or a plurality of the following biomarkers on the cell surface: CD73, CD90, or CD105. In one embodiment, a bone morphogenetic protein added to the culture comprises BMP2, BMP4, or a combination thereof. In one embodiment, BMP is provided to a culture comprising iPSCs for a period of time that is sufficient to induce expression of one or a plurality of markers comprising CD73, CD90, and CD105.

[0197] In one embodiment, a method for generating a therapeutic cell comprising an autologous mesenchymal stem cell is provided, wherein the mesenchymal stem cell expresses one or a plurality of biomarkers comprising CD73, CD90, CD103, CD105, CXCR4, VEGF-R2, or c-met.

[0198] In certain embodiments, a therapeutic cell generated according to a method of the invention is evaluated phenotypically and functionally in comparison to a primary- sourced cell of the same type or lineage that has been isolated from a tissue of the same subject or from a different subject. In one embodiment, a mesenchymal stem cell generated using a method of the invention exhibits expression of a biomarker that is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 80% higher, at least 100% higher, at least 150% higher, or at least 200% higher than the expression of the same biomarker on a primary mesenchymal stem cell isolated from a tissue, wherein the biomarker comprises CD73, CD90, CD103, CD 105, CXCR4, VEGF-R2, or c-met.

[0199] Specific methods for generating a mesenchymal stem cell from an iPSC are provided. In certain embodiments, an iPSC is selected for a differentiation protocol based on its expression of one or a plurality of biomarkers comprising CD31, CD33, CD34, CD133, c-met, IL-3 receptor, EGF-receptor, or thrombopoietin receptor. In one embodiment, an IPSC is first cultured with one or a plurality of agents or compounds prior to culture in a mesenchymal cell growth medium. In one embodiment, an iPSC is first cultured with one or a plurality of small molecules or compounds selected from the group comprising an NFkappaB inhibitor, CHIR99021, ascorbic acid, all trans retinoic acid (ATRA), sodium phenylbutyrate, forskolin, tranylcypromine hydrochloride, lithium chloride, or an ALK inhibitor, wherein an ALK inhibitor comprises SB431542, crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, or combinations thereof. An iPSC may be treated with a histone deacetylase inhibitor prior to additional of a mesenchymal cell growth medium, wherein a histone deacetylase inhibitor comprises phenylbutyrate, trichostatin, valproic acid, sulforaphane, genistein, or combinations thereof. In certain embodiments, an iPSC is cultured with one or a plurality of small molecules for at least one hour, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours. For generating mesenchymal stem cells from iPSCs, iPSCs may be plated on coated plates comprising one or a plurality of factors comprising fibronectin, vitronectin, hyaluronic acid, or combinations thereof. For example, in one embodiment, low concentrations of iPSCs (e.g., < 10% confluency) are seeded into vitronectin-coated tissue culture plates and cultivated in an appropriate medium such as E8 medium without passaging for a period of time (e.g., 10 days) to stimulate spontaneous differentiation. After this period, the cells may be detached using acommercially available reagent (e.g., Accutase) and transferred into vitronectin-coated plates containing an appropriate medium such as E8 medium and ROCK inhibitor Y27632 (10 pM). Subsequently, such as on the next day, the medium can be changed (e.g., comprising hPL5 + 150 pM L-ascorbic acid 2-phosphate) and replaced regularly such as every other day. After reaching 80% confluency, cells can be passaged (split ratio 1 :3). ROCK inhibitor may then be added to the medium for 24 h after passaging. Cells may then be passaged without further addition of ROCK inhibitor until the morphology of the cells assumes a spindle-shaped mesenchymal cell-like appearance (e.g., after 3-5 passages or an appropriate passage number depending on the culture conditions). Cells exhibiting mesenchymal stem cell morphology may be expanded prior to performing modifications to the cells or prior to evaluating their phenotype and / or function. One of ordinary skill in the art may choose to modify the timing, reagents, media, or other parameters for generating mesenchymal stem cells according to the desired characteristics of the final therapeutic cell product.

[0200] Embodiments of the invention also include evaluating the therapeutic cells that are generated from iPSCs according to the methods of the invention. In one embodiment, methods for characterization of mesenchymal stem cells generated from iPSCs are provided. Specific embodiments disclose methods for assessing differentiation of a mesenchymal stem cell, wherein the mesenchymal stem cells are cultivated in an osteogenic medium, an adipogenic medium, or a chondrogenic medium to evaluate development of the corresponding cell lineages from mesenchymal stem cells. In another embodiment, a senescence assay is performed to evaluate a therapeutic cell population generated using the methods of the invention. Specifically, a commercially available quantitative Senescence Assay kit may be utilized for these purposes, for example, a kit that measures pH-dependent beta-galactosidase activity. In yet other embodiments, a therapeutic cell generated according to the methods of the invention is assessed for telomere length, which may be performed using human length quantification qRT-PCR assays or equivalent methods. In one embodiment, gene expression analysis of mesenchymal cells is performed by quantification of mRNA expression levels using methods known in the art including commercially available kits or reagents. In one embodiment, phenotypic evaluation of a therapeutic cell is performed using methods known in the art such as flow cytometry or immunoassays for monitoring cell surface or intracellular expression of biomarkers. In one embodiment, evaluations of the phenotypeand function of a therapeutic cell generated using methods of invention are compared to the phenotype and function of a mesenchymal stem cell isolated directly from a tissue (i.e., a naturally occurring mesenchymal cell from the subject that is not iP SC -derived). In another embodiment, evaluations of the phenotype and function of a therapeutic cell generated using methods of invention are compared to the phenotype and function of another cell type or lineage isolated directly from a tissue (e.g., to a monocyte or a fibroblast). In certain embodiments, the methods of the invention provide iPSC-derived mesenchymal stem cells having one or a plurality of the following characteristics: a) Trilineage differentiation capacity into adipogenic, chondrogenic, and osteogenic lineages; b) Longer telomeres compared to primary isolated mesenchymal stem cells from a tissue; c) Reduced senescence-associated beta-galactosidase activity of iPSC-derived mesenchymal stem cell compared to a primary mesenchymal stem cell isolated from a tissue, and d) An altered gene and protein expression pattern of iPSC-derived mesenchymal stem cells compared to primary mesenchymal stem cells isolated from a tissue.

[0201] Embodiments of the invention provide certain molecular and mechanistic assessments of the therapeutic cells prior to administration of the cells to a subject, which may be performed in vitro or in an animal model that simulates or recapitulates a tumor microenvironment. In one embodiment, a mesenchymal stem cell is provided that possesses the ability to induce the differentiation of neoplastic cells into cells with reduced metastatic potential, wherein the reduced metastatic potential is associated with one or a plurality of the following effects: a) increased activity of tissue inhibitors of metalloproteinases (TIMPs); b) decreased activity of matrix metalloproteinases (MMPs); and c) reduced oxidative stress in a tumor microenvironment. In one embodiment, a mesenchymal stem cell is provided that reduces oxidative stress that is measurable based on decreased neutrophil activation, increased neutrophil apoptosis, decreased activation of macrophages (in particular, M2 -type macrophages), decreased myeloid suppressor cell activation, decreased production of superoxide radicals, decreased presence of free oxygen radicals, decreased presence of hydrogen peroxide, or increased expression of superoxide dismutase.

[0202] Disclosed herein are cell compositions and methods of treatment for cancer or for the eradication of cancer cells. In certain embodiments, cancer cells compriseglioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung cancer, cervical carcinoma, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, colorectal cancer, endometrial cancer, ovarian cancer, leukemia, prostate cancer, kidney cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, testicular tumor, Wilm's tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endothelial sarcoma, adenocarcinoma, hidradenoma, sebaceous carcinoma, papillary carcinoma, papillary sarcoma, cystic sarcoma, bronchial carcinoma, medullary carcinoma, mast cell tumor, mesothelioma, synovioma, melanoma, leiomyoma, rhabdomyoma, neuroblastoma, retinoblastoma, oligodendroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, basal cell carcinoma, fibrosarcoma, myxoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, cancer stem cells, breast cancer, or pancreatic cancer cells, but the present invention is not limited thereto.

[0203] In one embodiment, methods are provided to prevent, treat, or lessen the severity of cancer in a subject with cancer or to promote improved immune function in the subject. In one embodiment, methods are provided to lessen the numbers of cancer cells in a subject. In one embodiment, methods are provided to improve the anti -cancer effects of a drug therapy, e.g., an immunotherapy or chemotherapy drug, or of a radiation therapy. Therefore, the methods of the invention may be applied in combination with one or plurality of drug therapies or radiation therapy for treating a subject with cancer.

[0204] In one embodiment, the invention provides a therapeutic cell that delivers cargo to a hypoxic site in the body (i.e., a site of depressed oxygen tension such as within a tumor). In one embodiment, a therapeutic cell is provided that exerts functional activities at a site of hypoxia. Cancer cells in hypoxic areas may demonstrate an increase in glucose uptake and contribute to increased acidity in the tissue. Such cancer cells typically have undergone proteomic and genomic transformations that are regulated by hypoxia-inducible factors (HIFs), which are associated with resistance of cancer cells to treatment with agents such as chemotherapy. To identify a hypoxic area within a tumor, common methods may be implemented such as using polarographic electrodes, positron emission tomography (PET) scans with specific radiolabeled tracers that bind to hypoxic areas within a tumor, magnetic resonance imaging (MRI) techniques, or by identificationof biomarkers to identify hypoxic areas in tumor tissue through immunohistochemical staining, wherein the marker is found to accumulate in hypoxic regions of the tumor tissue. In certain embodiments, the invention provides a therapeutic cell for treatment of a subject having a tumor with hypoxia, wherein hypoxia is defined by low partial pressure of oxygen (pCh), a measurement of the pressure of oxygen dissolved in blood. In certain embodiments, hypoxia in a tumor is defined by pCh of less than 30 mmHg (millimeters of mercury), less than 25 mmHg, less than 20 mmHg, less than 15 mmHg, or less than 10 mmHg. In certain embodiments, a tumor in a subject is presumed to be hypoxic without performing a measurement of the degree of hypoxia, wherein a solid tumor is presumed to exhibit oxygenation levels that are measurably lower than their tissue of origin. In certain embodiments, the invention provides methods of treatment that can be used in conjunction with therapies directed toward overcoming tumor hypoxia including but not limited to nanoparticle carriers to increase oxygen concentrations in the tumor, hypoxia- activated prodrugs, drugs targeting hypoxia-inducible factors, proton therapy, and combination therapies, e.g., radiation and hypoxia targeting drugs.

[0205] An object of the invention is to provide therapeutic cells that can exert anticancer effects under conditions of hypoxia. One embodiment provides a mesenchymal stem cell derived from an iPSC that is engineered to express a hypoxia inducible promoter, wherein a hypoxia inducible promoter comprises a genetic sequence within the DNA of the mesenchymal stem cell that is specifically activated when the cell is exposed to low oxygen levels. In certain embodiments, this aspect of the invention provides a mesenchymal stem cell in which specific genes are turned on within a tumor microenvironment where the oxygen levels are low. In one embodiment, a mesenchymal stem cell derived from an iPSC is transfected with a hypoxia inducible gene expression system comprising a hypoxia inducible promoter. In one embodiment, a mesenchymal stem cell is transfected with one or a plurality of hypoxia inducible promoters, wherein the one or plurality of hypoxia inducible promoters utilize one or a plurality of HIF-lapha binding elements. In one embodiment, a mesenchymal stem cell is transfected with a hypoxia inducible promoter and genes encoding immunogenic or immunostimulatory cytokines, In one embodiment, a hypoxia inducible promoter induces expression of one or a plurality of immunogenic cytokines in the cell, wherein the one or plurality of immunogenic cytokines are selected from the group comprising IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, lymphotoxin, TNF-alpha, IFN-alpha,IFN-beta, G-CSF, GM-CSF, or M-CSF. In one embodiment, the production of the one or plurality of immunogenic cytokines is increased by at least 10%, at least 25%, at least 50%, or at least 100% under hypoxic conditions as compared to normoxic conditions. Embodiments of the invention provide iPSC-derived mesenchymal stem cells that are engineered to upregulate one or a plurality of these immunogenic cytokines under conditions of hypoxia (i.e., in a tumor).

[0206] Another embodiment provides methods for generating a therapeutic cell that involve culturing the cell in low oxygen conditions to enhance the therapeutic potential of the cell for treating cancer. In certain embodiments, various hypoxia incubators and chambers, or pharmaceutical or chemical agents may be applied that are known in the art to be suitable for this purpose. In one embodiment, a therapeutic cell is treated with a pharmacological or chemical hypoxia-mimetic agent such as deferoxamine (DFO), dimethyloxaloylglycine (DMOG), 2,4-dinitrophenol (DNP), cobalt chloride (CoCh), and isoflurane (ISO). In one embodiment, a therapeutic cell is treated with an agent that stabilizes or activates HIF-la in a cell.

[0207] One embodiment provides a method for preparing an iPSC-derived therapeutic cell such as a mesenchymal stem cell for administration to a subject with cancer by exposing the iPSC-derived therapeutic cell to hypoxic conditions in a tissue culture vessel or incubator. In certain embodiments, a hypoxic condition for culturing an iPSC-derived therapeutic cell comprises exposing the cell to 1-5% O2the appropriate chamber to modify the molecular pathways within the cell. In certain embodiments, exposure of a therapeutic cell such as a mesenchymal stem cell to a hypoxic environment induces undergoes changes in gene expression, which may involve changes in genes related to energy metabolism, “sternness” (i.e., the ability to self-renew and differentiate into different cell types), proliferation, and survival. In one embodiment, a therapeutic cell that is cultured in hypoxic conditions displays upregulated expression of glucose transporter 1 (GLUT-1), pyruvate dehydrogenase kinase 1 (PDK-1), and lactate dehydrogenase (LDH). In one embodiment, a therapeutic cell that is exposed to hypoxic culture conditions experiences alterations in gene expression and cellular functions comprising upregulation of one or more antigens or molecules comprising matrix metalloproteinase-9 (MMP-9), placental growth factor (PGF), VEGF, basic fibroblast growth factor, Notch, Notch ligands (such as Delta-like ligands and Jagged 1), epidermalgrowth factors, ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF). In one embodiment, the expression of HIF-1 alpha, which drives expression of hypoxia- regulated genes, is regulated by culture of a cell of the invention in hypoxic conditions. In yet another embodiment, a cell is subsequently cultured under conditions of phasic oxygen availability with varying concentrations of oxygen. In one embodiment, a cell is exposed to normoxic conditions following short hypoxia priming, where normoxia in a tissue culture flask or vessel may typically be defined as approximately 20-21% oxygen. For example, a cell may be grown for a duration of time (e.g., 10 min to 2 hours) in hypoxic conditions (e.g., 1-5% O2) followed by reoxygenation (i.e., normoxia) for a period of culture, possibly followed by one or a plurality of additional cycles of hypoxia and normoxia. In one embodiment, these methods are applied to provide a therapeutic cell population having enhanced pro-survival gene expression. One specific embodiment provides a therapeutic cell population comprising an oncolytic virus that is grown in hypoxic conditions to enhance intracellular replication of the oncolytic virus. Accordingly, one embodiment provides an iPSC-derived therapeutic cell that is cultured in hypoxic conditions prior to, concurrently, or following loading of the cell with an oncolytic virus, wherein the cell is subsequently administered to subject to serve as a delivery vehicle for the oncolytic virus. In one embodiment, an iSPC-derived therapeutic cell is cultured under hypoxic conditions during loading of an oncolytic virus into the cell.

[0208] Yet other embodiments of the invention provide a mesenchymal stem cell derived from an iPSC that is genetically engineered using methods to delete one or a plurality of immunosuppressive or angiogenesis-related (i.e.., pro-angiogenic) genes, wherein the one or plurality of immunosuppressive or angiogenic genes are selected from the group consisting of VEGF, indoleamine 2-3- dioxygenase (IDO), IL-10, TGF-beta, LIF, HLA-G, and prostaglandin E2 (PGE2). The appropriate and most efficient method for gene deletion or replacement can be selected by one of ordinary skill in the art, for example, using gene silencing with small interfering RNA (siRNA) or CRISPR. The invention provides a mesenchymal stem cell that promotes anti-tumor immunity rather than suppressing anti-tumor immunity. Specific embodiments of the invention provide an iPSC-derived mesenchymal stem cell that is engineered to express one or a plurality of genes encoding immunogenic cytokines and that is also engineered by deletion of one or a plurality of genes encoding immunosuppressive or angiogenic genes.

[0209] In one embodiment, a therapeutic cell population for treating cancer comprises a mesenchymal stem cell. In one embodiment, a mesenchymal stem cell generated using the methods of the invention is characterized by expression of the antigens CD73, CD90, and CD 105 on the cell surface. In one embodiment, a mesenchymal stem cell generated using the methods of the invention is characterized by low expression or absence of expression of CD34, CD45, CD1 la, CD14, CD19, and human leukocyte antigen-DR isotype (HLA-DR) antigens or molecules. In another embodiment, a mesenchymal stem cell expresses one or a plurality of adhesion molecules and chemokines selected from the group consisting of CD 166 (activated leukocyte adhesion molecule; ALCAM), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2), vascular cell adhesion molecule-1 (VCAM-1), stromal cell-derived factor- la (SDF-1), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), C-X-C chemokine receptor type 4 (CXCR4), CXCR1, CXCR2, C-C motif chemokine ligand 2 (CCL2), or macrophage inflammatory protein 1 alpha (MIP-lalpha).

[0210] One embodiment of the invention provides a population of mesenchymal stems cells that retains one or a plurality of physical or functional characteristics of more primitive or earlier cells as compared to a primary mesenchymal stem cell that is isolated directly from a tissue source (e.g., from adipose tissue, bone marrow, synovial membranes, trabecular and cortical bone, skeletal muscle, or other tissues). In certain embodiments, an iPSC-derived mesenchymal stem cell of the invention expresses one or a plurality of the following proteins or biomarkers from the group consisting of SRY-box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT-4), homeobox protein NANOG, reduced expression- 1 (REX-1), T cell receptor alpha locus 1-60 (TRA- 1-60), TRA-1-81, GATA-4, TERT, stage-specific mouse embryonic antigen-3 (SSEA-3), and S SEA-4.

[0211] In certain embodiments, the invention provides therapeutic cell comprising a mesenchymal stem cell that is suitable as a vehicle for delivery of a toxic payload to cancer cells, wherein a toxic payload comprises a chemotherapy drug. In certain embodiments, methods are provided for modifying or engineering a mesenchymal stem cell of the invention to carry a toxic payload, for example, by encapsulating a drug into the mesenchymal stem cell cytoplasm, by using nanoparticles to encapsule the drug andloading the nanoparticles into the mesenchymal stem cell, or by genetic modification of the mesenchymal stem cell to produce the drug itself. It will be understood by one of ordinary skill in the art that achieving high payload capacity of a drug inside a cell is critically important in achieving therapeutic drug concentrations upon administration of the cell to a subject. The present invention provides a therapeutic cell comprising a mesenchymal stem cell that is amenable to uptake of therapeutically relevant drug concentrations without compromising the viability and tumor tropism of the mesenchymal stem cell.

[0212] In certain embodiments, the invention provides a mesenchymal stem cell derived from an induced pluripotent stem cell that is a suitable carrier for an anti -cancer virus, i.e., an oncolytic virus. In one embodiment, a mesenchymal stem cell of the invention comprises an oncolytic virus such as vaccinia virus. In one embodiment, a mesenchymal stem cell comprising an oncolytic virus provides viral distribution to a tumor site. In yet another embodiment, a mesenchymal stem cell carrying an oncolytic virus acts as a biologic manufacturer for viral genome replication, as can be assayed based on viral copy numbers within the cell. Aspects of the invention disclose an iPSC- derived mesenchymal stem cell is a superior manufacturer for viral genome replication as compared to a mesenchymal stem cell isolated from a tissue source (i.e., a non-iPSC- derived cell) from the same subject or from a different subject. Methods of the invention include isolating mesenchymal stem cells from a tissue or blood of an adult subject and comparing the isolated cells to mesenchymal stem cells generated using the methods of the invention. One aspect involves performing viral titer measurements in both mesenchymal stem cell types following loading with an approximately equivalent dose of oncolytic virus particles. The quantity of an infectious virus in supernatant of the cells may be determined using methods known in the art. In one embodiment, a mesenchymal stem cell generated using the methods of the invention produces at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1,000-fold, or more, more viral particles than a mesenchymal stem cell isolated from a tissue source. In other embodiments, an iPSC-derived mesenchymal stem cell provides enhanced delivery of an oncolytic virus to a tumor microenvironment compared to a mesenchymal stem cell isolated from a tissue source

[0213] In certain embodiments, a mesenchymal stem cell generated using the methods of the invention has anti-inflammatory and immunosuppressive functions, wherein the anti-inflammatory and immunosuppressive functions can be ascertained based on production of cytokines or factors, and wherein the one or plurality of cytokines or factors are selected from the group consisting of IL-4, IL-6, IL- 10, transforming growth factor beta (TGF-beta), heme oxygenase- 1 (HO- 1), inducible nitric oxide synthase (iNOS), and indoleamine-2-dioxygenase-3 (IDO). In some embodiments, an antiinflammatory or immune suppressive property of a mesenchymal stem cell is ascertained by performing in vitro assays measuring one or a plurality of functions of the mesenchymal stem cell, wherein the one or plurality of functions are selected from the group consisting of inhibiting B cell maturation, restricting immunoglobulin production, inhibiting cytokine secretion by helper T cells, reducing the actions of cytotoxic T cells, decreasing NK cell proliferation, decreasing NK cell cytotoxicity, decreasing NK cell cytokine production, decreasing the numbers of CD 103+ dendritic cells, decreasing the numbers of CD68+ dendritic cells, inhibiting the differentiation of CD14+ monocytes into dendritic cells, inhibiting the differentiation of CD34+ progenitor cells into dendritic cells, restricting dendritic cell differentiation and / or function, increasing CD4+ CD25+ FoxP3+ regulatory T cells, inhibiting the development of IL-10 producing B cells, inhibiting the development of IL-10-producing dendritic cells, and inhibiting the differentiation of CD8+ CD28- T cells. Certain embodiments provide mesenchymal stem cells that are useful for virotherapy and for permitting an oncolytic virus to replicate and destroy cancer cells in a subject. Yet other embodiments provide a mesenchymal stem cell that is capable of homing to areas of hypoxia (i.e., tumors). Further embodiments provide a mesenchymal stem cell derived from an iPSC that is capable of inhibiting tumor angiogenesis.

[0214] In certain embodiments, the invention provides a mesenchymal stem cell derived from an induced pluripotent stem cell that is genetically engineered to enhance or modify its potential as an anti-cancer agent. Genetic engineering of mesenchymal stem cells has been performed in the prior art and has been achieved using non-viral or viral vectors to induce the expression of different factors, depending on the desired results, such as increasing the survival and proliferation rate of a cell and improving its regenerative capacity. Non-limiting examples of genetic engineering methods utilize retrovirus, lentivirus, adenovirus, adeno-associated virus, non-viral (liposomes orplasmids), or methods for site-specific integration of genes such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR / Cas9), Zinc Finger Nuclease (ZFN), and Transcription Activator-Like Effector Nuclease (TALEN) technologies. For example, viral vectors represent useful genetic modification tools owing to the high efficiency of viral transduction that can be achieved without compromising the immunophenotypic characteristics of the cell. One of ordinary skill in the art would select the appropriate molecular editing tool(s) that are appropriate for the specific application, which may comprise mutation, insertion, replacement, or deletion of a gene. In other embodiments, genetic modification of a mesenchymal stem cell is performed by physical or chemical methods such as electroporation, nucleofection, sonoporation, or using lipidic agents, polymers, or inorganic nanoparticles. For gene insertions, a desirable method achieves stable and long-term transcription of the gene of interest and, consequently, highly efficient genetic modification of cells. Currently, there is extensive clinical experience with several types of vectors that include mainly vaccinia, measles, vesicular stomatitis virus (VSV), polio, reovirus, adenovirus, lentivirus, retrovirus, adeno-associated virus (AAV), and herpes virus simplex (HSV). Among those, the most predominant vectors used for cell transduction and transplantation are the lenti- and retroviral vectors. In certain embodiments, an iPSC-derived cell generated using the methods of the invention is a target for a gene modifying tool described herein.

[0215] In one embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) generating an induced pluripotent stem cell from a somatic cell of the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; and c) infecting the mesenchymal stem cell with an oncolytic virus (e.g., vaccinia virus) in vitro. The differentiated mesenchymal stem cells that are carrying the virus are then implanted or injected into the subject. In certain embodiments, an oncolytic virus has undergone genetic modifications to increase its selectivity for tumors. In one embodiment, an oncolytic virus comprises one or a plurality of the following viruses: HSV-1716, G207, MO32, G47delta, Delta-24-RGD, Onyx-015, NDV-HUJ, Reolysin, PVS-RIPO, H-1PV, MV-CEA, or TOCA511. In one embodiment, an oncolytic virus comprises one or a plurality of the following virus types: herpes simplex virus, adenovirus, Newcastle disease virus, reovirus, poliovirus, parvovirus, measles virus, orreovirus. In these embodiments, a mesenchymal stem cell is used as a delivery vehicle for the virus in the subject.

[0216] In another embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; c) infecting the mesenchymal stem cell with an oncolytic virus; and d) optionally, transfecting the mesenchymal stem cell with a gene for molecule or antigen comprising a chemokine, growth factor, or a cytokine. The mesenchymal stem cells generated using these methods are then administered to a subject with cancer. In some embodiments, a molecule or antigen is selected from the group consisting of C-X-C chemokine receptor type 4 (CXCR4), CXCR7, vascular endothelial growth factor receptor 2 (VEGF-R2), c-met, leukemia inhibitory factor (LIF), interferon-gamma (IFN-g), IFN-beta, intercellular adhesion molecule- 1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2), vascular cell adhesion molecule-1 (VCAM-1), stromal cell-derived factor-la (SDF-1), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), fas ligand, TNF-related apoptosis-inducing ligand (TRAIL), IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL- 15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN- beta, G-CSF, GM-CSF, or M-CSF.

[0217] In another embodiment, a method for providing an autologous mesenchymal stem cell for treating a subject with cancer is disclosed, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; c) infecting the mesenchymal stem cell with an oncolytic virus; d) transfecting the mesenchymal stem cell with a hypoxia-inducible promoter to drive expression of genes for cell homing and immune function; and d) administering the mesenchymal stem cell to the subject. In some embodiments, a hypoxia-inducible promoter drives expression of genes for cell homing and / or immune function selected from the group consisting of CXCR4, CXCR7, VEGF- R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, or M-CSF. In certain embodiments, step d) is performed prior to, after, or concurrently with step c).

[0218] In another embodiment, a method for generating an autologous mesenchymal stem cell for treating a subject with cancer is provided, the method comprising: a) obtaining an induced pluripotent stem cell from the subject; b) differentiating the induced pluripotent stem cell into a mesenchymal stem cell in vitro, wherein the mesenchymal stem cell is defined by expression of CD73, CD90, CD105 and CXCR4; c) infecting the mesenchymal stem cell with an oncolytic virus; d) transfecting the mesenchymal stem cell with a hypoxia-inducible promoter to drive expression of genes for cell homing and / or immune function; e) transfecting the mesenchymal stem cell with a gene for molecule or antigen comprising a chemokine, growth factor, or a cytokine; and e) implanting or injecting the mesenchymal stem cell into the subject. In certain embodiments, a hypoxia inducible promoter drives expression of genes for cell homing and immune function selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, or M- CSF. In certain embodiments, a mesenchymal stem cell is transfected with a gene for a molecule or antigen that is selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL- 23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM- CSF, or M-CSF. In certain embodiments, step c) is performed prior to, after, or concurrently with step d). In certain embodiments, step c) is performed prior to, after, or concurrently with step e). In certain embodiments, step d) is performed prior to, after, or concurrently with step e).

[0219] In a specific embodiment, a method for providing a subject with a cancer therapy comprising autologous mesenchymal stem cells is provided, the method comprising: a) identifying a subject with cancer; b) isolating a somatic cell from the subject; c) dedifferentiating the somatic cell to generate an iPSC; d) establishing a cell culture sytem for differentiating a mesenchymal stem cell, wherein an iPSC is added to amesenchymal differentiation medium, and wherein the differentiated mesenchymal stem cells in the culture are induced to express biomarkers or surface antigens comprising CD73, CD90, CD105, and CXCR4; e) optionally, transfecting the differentiated mesenchymal stem cell with a hypoxia-inducible promoter; f) infecting the differentiated mesenchymal stem cells with an oncolytic virus; and g) administering a therapeutically effective amount of the differentiated mesenchymal stem cells to the subject. In certain embodiments, the somatic cell isolated from the subject is a cell type selected from the group consisting of a peripheral blood cell, a monocyte, a fibroblast, a T cell, or a mesenchymal stem cell. In one embodiment, the iPSC expresses one or a plurality of biomarkers comprising OCT-4, Lin28, PIM-1, PIM-3, Sox2, Kruppel-like factor (KLF), MYC, I-MYC, k-ras, NANOG, NF-kappaB, and c-met. In one embodiment, the hypoxiainducible promoter drives expression of genes for cell homing and / or immune function selected from the group comprising CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL- 1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF-alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, or M-CSF. In one embodiment, expression of a gene for cell homing and / or immune function is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 80% higher, at least 100% higher, at least 150% higher, or at least 200% higher than the expression of the same biomarker on a primary mesenchymal stem cell isolated from a tissue. In one embodiment, the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, Newcastle disease virus, reovirus, poliovirus, parvovirus, measles virus, or reovirus. In one embodiment, the method provides a differentiated mesenchymal stem cell that expresses one or a plurality of the following biomarkers comprising SRY-box transcription factor 2 (SOX2), octamer- binding transcription factor 4 (OCT-4), homeobox protein NANOG, reduced express! on- 1 (REX-1), T cell receptor alpha locus 1-60 (TRA-1-60), TRA-1-81, GATA-4, TERT, stage-specific mouse embryonic antigen-3 (SSEA-3), and SSEA-4. In one embodiment, a mesenchymal differentiation medium comprises a bone morphogenetic protein, wherein a bone morphogenetic protein comprises BMP2, BMP4, or a combination thereof. In another embodiment, a cell culture system for differentiating a mesenchymal stem cell comprises a hypoxic environment, wherein the hypoxic environment induces alterations in gene expression and cellular functions in the mesenchymal stem cell, and wherein an alteration in gene expression induces upregulation of one or more antigens or moleculesin the mesenchymal stem cell comprising matrix metalloproteinase-9 (MMP-9), placental growth factor (PGF), VEGF, basic fibroblast growth factor, Notch, Notch ligands (such as Delta-like ligands and Jagged 1), epidermal growth factors, ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF). In a specific embodiment, a biomarker or an antigen on the differentiated mesenchymal stem cells is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 80% higher, at least 100% higher, at least 150% higher, or at least 200% higher than the expression of the same biomarker or antigen on a primary mesenchymal stem cell isolated from a tissue. In one embodiment, administering a therapeutically effective amount of the differentiated mesenchymal stem cell to the subject mediates one or a plurality of effects comprising increasing activity of tissue inhibitors of metalloproteinases (TIMPs), decreasing activity of matrix metalloproteinases (MMPs), or reducing oxidative stress in the tumor microenvironment, and wherein a reduction in oxidative stress is measurable based on one or more effects comprising decreased neutrophil activation, increased neutrophil apoptosis, decreased activation of macrophages, decreased myeloid suppressor cell activation, decreased production of superoxide radicals, decreased presence of free oxygen radicals, decreased presence of hydrogen peroxide, or increased expression of superoxide dismutase.

[0220] In one embodiment, the invention provides methods for generating mesenchymal stem cells from dedifferentiated cells from an autologous subject. In one embodiment pluripotent stem cells are generated from peripheral blood cells, subsequently said pluripotent stem cells are gene modified to express one or more immunogenic cytokines selectively in the presence of hypoxia. Numerous inflammatory cytokines may be utilized. For examples, in conditions in which increased antigen presentation is desired, cytokines such as interferon gamma are selectively activity. In conditions in which homeostatic proliferation of T cells or NK cells is desired, cytokines such as interleukin-7 or interleukin- 15 are transfected.

[0221] Stimulation of inflammatory and / or immunomodulatory cytokine production is selectively induced in the tumor microenvironment, preferably in proximity to cancer stem cells, through the close proximity of said cancer stem cells to hypoxic areas of the tumor. Mesenchymal stem cells of the invention preferentially home to hypoxic areas of the tumor. Once in a hypoxic area, the cells produce immunomodulatory and / orinflammatory cytokines by means of hypoxia inducible promoter stimulation. Essentially, one or more gene constructs are introducing into said mesenchymal stem cells in order to allow for hypoxia inducible stimulation of gene expression. This is performed by using the hypoxia induced factor alpha protein and its associated promoter binding regions. Hypoxia inducible factor- l(HIF-l) is a mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia (Wang, G. L., et al., Proc. Natl. Acad. Sci. USA 92:5510-5514, 1995; Wang, G. L., and Semenza, G. L., J. Biol. Chem. 270: 1230-1237, 1995; U.S. Pat. No. 5,882,914). HIF-1 is a basic helix loop-helix protein that binds to cis-acting hypoxia-responsive elements of genes induced by hypoxia (Wang, G. L., and Semenza, G. L., Curr. Opin. Hematol. 3: 156-162, 1992; Jiang, B. H., et al., J. Biol. Chem. 212: 19253-19260, 1997). The genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase- 1, inducible nitric oxide synthase, and glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1 (Semenza, G. L., et al., Kid. Int. 51 :553-555, 1997). HIF-1 DNA binding activity and HIF-1 protein concentration increase exponentially as cells are subjected to decreasing 02 concentrations (Jiang, B. H., et al., Am J. Physiol. 271 :C 172- C1180, 1996). HIF-1 also activates transcription of the VEGF gene in hypoxic cells (Forsythe et al., 1996; Iyer et al., 1998). When cultured cells are transfected with pCEP4 / HIF-l alpha plasmid under conditions that allow expression of HIF-1 alpha from a cytomegalovirus promoter and a reporter plasmid containing the hypoxia response element from the VEGF gene, reporter gene expression is increased in cells under non- hypoxic conditions and there is a dramatic superinduction under hypoxic conditions that is dependent upon the presence of an intact HIF-1 binding site (Forsythe et al., 1996). In embryonic stem cells from a knockout mouse, which lack HIF-1 alpha expression, there is no expression of VEGF mRNA in response to hypoxia (Iyer et al., 1998). HIF-1 is a heterodimer of two subunits, HIF-lalpha and HIF-lbeta. The HIF-lalpha subunit is unique to HIF-1, whereas HIF-lbeta (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) can dimerize with other proteins. The concentration of HIF-lalpha and HIF-lbeta RNA and HIF-lalpha and HIF-lbeta polypeptide increases in cells exposed to hypoxic conditions (Wiener, C. M., et al., Biochem. Biophys. Res. Commun. 225:485-488, 1996; Yu, A. Y., et al., Am J. Physiol. 275:L818-L826, 1998). Structural analysis of HIF-lalpha revealed that dimerization requires two domains, termed HLH and PAS. DNA binding is mediated by a basic domain (Semenza, G. L., et al., Kid. Int.51 :553-555, 1997). Two transactivation domains are contained in HIF-lalpha, located between amino acids 531 and 826. The minimal transactivation domains are at amino acid residues 531-575 and 786-826 (Jiang, B. H., et al., 1997, supra; Semenza, G. L., et al., 1997, supra). Amino acids 1-390 are required for optimal heterodimerization with HIFlbeta (ARNT) and DNA binding. In addition, deletion of the carboxy terminus of HIF-lalpha (amino acids 391-826) decreased the ability of HIF-1 to activate transcription. However, HIF-lalpha (1-390) was expressed at high levels in both hypoxic and non- hypoxic cells in contrast to full-length HIF-lalpha (1-826) which was expressed at much higher levels in hypoxic relative to non-hypoxic cells (Jiang, B.-H, et al., J. Biol. Chem. 271 : 17771-17778, 1996). Thus, hypoxia has two independent effects on HIF-lalpha activity: (1) hypoxia increases the steady-state levels of HIF-lalpha protein by stabilizing it (i.e. decreasing its degradation); and (2) hypoxia increases the specific transcriptional activity of the protein (i.e. independent of the protein concentration). In one embodiment of the invention, the inventors disclose an isolated nucleic acid sequence encoding a stable HF-lalpha protein that is a chimeric transactivator. This chimeric transactivator includes: a) a nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia inducible factor (e.g., HIF-lalpha, HIF-2alpha, or HIF-3 alpha); and b) a nucleotide sequence encoding a transcriptional activation domain. The preferred hypoxia inducible factor of the invention is HIF-lalpha. The construct is generated so as to allow for stimulation of the target gene(s) and transcription thereof selectively under conditions of hypoxia, such as found in the tumor microenvironment.

[0222] The invention further provides a method for providing constitutive expression of a hypoxia inducible factor in a cell, under hypoxic or non-hypoxic conditions. This may be utilized to stimulate expression of genes that increase immunogenicity such as interferon genes, or downstream genes such as TAP-1 or HLA genes. The method includes contacting the cell with a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-lalpha as described herein, under conditions that allow expression of the nucleic acid sequence, thereby providing constitutive expression of a hypoxia inducible factor. In some embodiments hypoxia associate with tumors is used to drive expression of a suicide gene. In other embodiments necrosis or immunogenic cell death inducers are controlled by expression of hypoxia in mesenchymal stem cells. For example, in one embodiment, hypoxia is utilized to induce transcription of perforin, which induces death of the mesenchymal stemcell and surrounding neoplastic cells. In some embodiments a “two signal” approach is taken in which gene activation requires both hypoxia and a secondary inducer gene.

[0223] The invention also provides a method for increasing expression of a hypoxia inducible gene in a cell in order to drive similarity to neoplastic cells. The method includes contacting the cell with an expression vector containing a polynucleotide encoding a stable HIF-1 alpha of the invention or a chimeric transactivator protein as described herein under conditions that allow expression of the nucleic acid sequence contained in the vector thereby providing for increased expression of hypoxia inducible genes in the cell. Such genes include, for example, VEGF. Further included in the invention is a method for reducing hypoxia or ischemia-related tissue damage in a subject having or at risk of having such damage. The method includes administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-1 alpha as described herein, in a pharmaceutically acceptable carrier, thereby inducing gene expression that will reduce, or prevent, or repair tissue damage. Examples of gene products whose expression is induced by sHIF-1 alpha resulting in a therapeutic effect include VEGF and other mediators of angiogenesis and insulin-like growth factor 2 (IGF-2) and other factors promoting cell survival (Iyer et al., 1998; Feldser, D., et al., Cancer Res. 59:3915, 1999). In another embodiment, the invention provides a method for providing prophylactic therapy for tissue in a subject in need thereof comprising administering to the subject an amount of a polypeptide encoded by a polynucleotide encoding a chimeric transactivator protein as described herein, or a stable HIF-lalpha as described herein, such that angiogenesis is induced at levels that are greater than before administration of the polypeptide, thereby providing prophylactic therapy. The invention provides a substantially pure stable hypoxia-inducible factor- 1 (sHIF-1 alpha) protein, or mutein. Wild-type, full-length HIF- lalpha is expressed at lower levels in nonhypoxic cells as compared to hypoxic cells (Wang, G. L., et al., Proc. Natl. Acad. Sci. USA 92:5510-5514, 1995; Wang, G. L., and Semenza, G. L., J. Biol. Chem. 270: 1230-1237, 1995; Jiang, B. H., et al., J. Biol. Chem. 272: 19253-19260, 1997, herein incorporated by reference) while sHIF-lalpha is stable under nonhypoxic as well as hypoxic conditions. Wild type HIF-lalpha and sHIF-lalpha are characterized as being able to form heterodimers with HIF-lbeta to form a DNA- binding protein, hypoxia inducible factor- 1 (HIF-1), a mammalian transcription factor. HIF-1 activates transcription of multiple genes including those encoding erythropoietin(EPO), vascular endothelial growth factor (VEGF), glucose transporters, and glycolytic enzymes. In some embodiments, HIF-lalpha switch which is used is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify HIF-lalpha using standard techniques for protein purification, such as DNA affinity chromatography (e.g., Wang, G. L., and Semenza, J., J. Biol. Chem. 270: 1230-1237, 1995) and immunoprecipitation (e.g., Jiang, B. H., et al., J. Biol. Chem. 271 : 17771-17778, 1996). The substantially pure polypeptide will yield a single band on a nonreducing polyacrylamide gel. The purity of the HIF- lalpha polypeptide can also be determined by amino-terminal amino acid sequence analysis. HIF-lalpha protein includes functional fragments of the polypeptide, as long as the activity and the stability in nonhypoxic conditions of sHIF-1 alpha remains. Smaller peptides containing the biological activity of sHIF-1 alpha are thus included in the invention.

[0224] In another embodiment, the invention provides polynucleotides encoding sHIF-1 alpha as well as nucleic acid sequences complementary to polynucleotides encoding sHIF-1 alpha. The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotides at least 10 bases in length. By isolated polynucleotide is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA. A complementary sequence may include an antisense nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C, and T in the polynucleotide encoding sHIF-1 alpha are replaced by ribonucleotides A, G, C, and U, respectively, Also included in the invention are fragments of the above-identified nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to nucleic acid that encodes sHIF-1 alpha, but not SEQ ID NO: 1 under physiological conditions. Specifically, the fragments should selectively hybridize to nucleic acid encoding sHIF-lalpha polypeptide. The term “selectivelyhybridize” refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences. In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. An example of progressively higher stringency conditions is as follows:2><SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2><SSC / 0.1% SDS at about room temperature (low stringency conditions); 0.2xSSC / 0.1% SDS at about 42 / C (moderate stringency conditions); and O.l xSSC at about 68 / C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. When using an sHIF-lalpha specific probe, it may be necessary to amplify the nucleic acid prior to binding with an sHIF-1 alpha specific probe. Preferably, polymerase chain reaction (PCR) is used, however, other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The sHIF-1 alpha polynucleotide of the invention can be derived from a mammalian organism, and most preferably from human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequences must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. In a preferred embodiment, the probe can delineate between sHIF-lalpha and wild-type HIF-lalpha. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid nonspecific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1998).

[0225] In one embodiment, a recombinant nucleic acid construct encoding a chimeric transactivator protein of the invention may be placed under the control of or “operatively linked to” a suitable promoter and / or other expression control regulatory sequences. It may be desirable for the transactivator protein to be placed under the control of a constitutively active promoter sequence, although the transactivator protein may also be placed under the control of an inducible promoter, such as the metallothionein promoter or a tissue specific promoter. An inducible promoter allows for controlled increase or decrease of expression of a particular gene, while constitutive expression allows for continual expression of a gene, for example, for producing a gene product in culture, or in a transgenic animal. Other promoter sequences that are useful include, but are not limited to, the SV40 early promoter region; RSV or other retroviral LTRs; herpes thymidine kinase promoter, human cytomegalovinis (CMV) immediate early promoter / enhancer. Other promoters that have been used for this purpose include the elastase 1 gene control region; insulin gene control region; immunoglobulin gene control region; mouse mammary tumor virus control region; albumin gene control region; alphafetoprotein gene control region; alpha 1 -antitrypsin gene control region and beta-globin gene control region.

[0226] For delivery into regenerative cells, Mammalian expression systems which utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the sHIF-1 alpha coding sequence may be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence or a heterologous (e.g., CMV) promoter cloned into a replication-deficient adenovirus (Armentano, D., et al., Hum. Gene Ther. 6: 1343- 1353, 1995; Hehir, K. M., et al., J. Virol. 70:8459-8467, 1996). Alternatively, the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett et al., Proc. Natl. Acad.Sci. USA 79:7415-7419, 1982; Mackett et al., J. Virol. 49:857-864, 1984; Panicali et al., Proc. Natl. Acad. Sci. USA 79:4927-4931, 1982). Vectors based on bovine papilloma virus have the ability to replicate as extrachromosomal elements (Sarver, et al., Mol. Cell. Biol. 1 :486, 1981). Shortly after entry of this nucleic acid into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the sHIF-lalpha gene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA 81 :6349-6353, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionein IIA promoter and heat shock promoters.

[0227] In some embodiments control of inflammatory and / or immunomodulatory gene expression is accomplished by tissue specific promoters. A promoter, in the context of the present specification, refers to a polynucleotide element capable of regulating the transcription of a gene adjacent and downstream (3') of the promoter. The promoter may contain all of, or only a portion of, the complete 5' regulatory sequences of the gene from which it is derived. A sequence in the promoter region is typically recognized by RNA polymerase molecules that start RNA synthesis. A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and / or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the chimeric gene containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult). Promoters useful in the practice of the present invention are preferably tissue-specific— that is, they are capable of driving transcription of a gene in one tissue while remaining largely "silent" in other tissue types. It will be understood, however, that tissue-specific promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue / activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of thepresent invention typically has a selectivity ratio of greater than about 5. Preferably, the selectivity ratio is greater than about 15. It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the present invention. For example, promoters that are active in a variety of central nervous system (CNS) neurons may be therapeutically useful in protecting against damage due to stroke, which may effect any of a number of different regions of the brain. Tissue-specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different tissues. A further desirable characteristic of promoters useful in the present invention is that they possess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissues. One means of achieving this is to select promoters of genes encoding proteins that have a relatively low turnover rate in adult tissue, such as the cancer specific promoters described herein. Another means is to use "silencer" elements, which suppress the activity of a selected promoter in the absence of hypoxia. The level of expression of a gene under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis). Vectors used for such experiments typically contains a reporter gene, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities. This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring hypoxia sensitivity. A number of tissue specific promoters, described below, may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector.

[0228] In another embodiment, the present invention is directed to a method of treating cancer including administering regenerative cell derived mesenchymal stem cells with improved ability to inhibit proliferation of cancer cells, produced by culturing mesenchymal stem cells in a medium containing aspirin. In another embodiment, thepresent invention is directed to the use of mesenchymal stem cells with improved inhibitory activity against proliferation of cancer cells, produced by culturing mesenchymal stem cells in a medium containing aspirin, for the treatment of cancer.

[0229] In one embodiment of the invention, regenerative cell derived mesenchymal stem cells are utilized to modify the tumor microenvironment by insertion of a suicide gene. Said gene is used in the present invention to encode a prodrug activating enzyme which converts a non-toxic prodrug of an anticancer agent to the toxic anticancer agent. Exemplary suicide genes include the genes encoding herpes simplex type 1 thymidine kinase (HSV-TK) and cytosine deaminase (CD). HSV-TK converts non-toxic gancyclovir (GCV) to toxic phosphorylated metabolite, and CD converts non-toxic 5 -fluorocytosine (5-FC) to toxic 5 -fluorouracil (5-FU). CD gene is preferable for use in the gene therapy because 5-FU exhibits a strong bystander effect.

[0230] The suicide gene may be introduced into a mesenchymal stem cell, or progenitor of said cell by employing a viral vector, preferably, a retroviral vector, comprising the gene in accordance with any known method for introducing a gene into a cell. For instance, the suicide gene can be introduced into the mesenchymal stem cell by introducing it into a retroviral vector to obtain an expression vector, transfecting a packaging cell with the expression vector, culturing the transfected cell under an appropriate culture condition, filtering the culture medium to obtain a retroviral solution, and transfecting mesenchymal stem cells with the retroviral solution. Then, mesenchymal stem cells which continuously express the suicide gene can be obtained by using a selection marker contained in the retroviral vector. The mesenchymal stem cells expressing a suicide gene may be mass produced in vitro by introducing the suicide gene into stem cells and selecting and amplifying the resulting stem cells under proper conditions; or by sufficiently proliferating stem cells, introducing the suicide gene into the proliferated stem cells and harvesting the resulting stem cells.

[0231] The stem cells which may be used in the present invention can be isolated from the bone marrow, peripheral blood or cord blood of any mammal including human, preferably from the human bone marrow. The inventive composition comprising mesenchymal stem cells expressing a suicide gene is useful in the treatment of a cancer. Exemplary cancers include brain cancer, breast cancer, liver cancer, pancreas cancer, colorectal cancer, and lung cancer, but not limited thereto.

[0232] The inventive composition may further comprise pharmaceutically acceptable excipients, carriers, or diluents. Preferably, it may be formulated into an injection formulation suitable for injecting into a tissue or organ. The compositions may additionally include lubricating agents, flavoring agents, emulsifiers, preservatives and the like. The inventive composition can be injected into the patient's body according to the conventional methods well known in the art such as the clinical method disclosed by Bjorklund and Stenevi (Brain Res., 177, 555-560 (1979) and Lindvall et al. (Arch. Neurol., 46, 615-31 (1989)). The unit dose of the mesenchymal stem cells of the present invention to be administered is determined by considering various relevant factors including the disease to be treated, the severity of the subject's symptom, the chosen route of administration, and the age, sex and body weight of the individual subject.

[0233] Moreover, the present invention also provides a kit for treating a cancer comprising an expression vector comprising a suicide gene, a mesenchymal stem cell and a prodrug of an anticancer agent. In the inventive kit, the expression vector comprising the suicide gene and the mesenchymal stem cell may be provided separately or provided in the form of a mesenchymal stem cell transfected with the expression vector comprising the suicide gene or transduced with viruses expressing the suicide gene. The expression vector is preferably prepared by introducing the suicide gene into a viral vector, preferably, a retroviral vector. The present invention also includes within its scope a method for treating a subject suffering from a cancer, which comprises administering a therapeutically effective amount of mesenchymal stem cells expressing a suicide gene to the subject, followed by the administration of a prodrug of an anticancer agent.

[0234] Immune rejections can be minimized in gene therapy using the inventive composition when the mesenchymal stem cells are obtained from the patient's own regenerative cells such as pluripotent cells derived from bone marrow or from the bone marrow of others having the same HLA (human leukocyte antigen) type as the patient. Accordingly, the inventive composition can be repeatedly administered for thoroughly preventing the relapse of the cancer. Further, the use of the mesenchymal stem cells in gene therapy have advantages in that the mesenchymal stem cells can be easily cultured without using an oncogene and obtained in a large amount sufficient for transplantation to the patient. Moreover, the inventive composition can be applied for the treatment of acancer hiding from immune surveillance because the effect thereof does not depend on improvement of immune responses.

[0235] Accordingly, the inventive composition can be used alone or in combination with other therapy for the treatment of a cancer, especially an intractable cancer. Upon injection, the mesenchymal stem cells in the inventive composition exhibit tropism toward cancer tissues. Accordingly, adverse side effects to the normal cells can be minimized by specifically delivering the mesenchymal stem cells expressing a suicide gene to cancer tissues and then administering a prodrug of an anticancer to be activated by the suicide gene so that the anticancer agent is generated only around the cancer tissues. Further, the mesenchymal stem cells expressing a suicide gene exhibits bystander effects on surrounding cancer cells wherein the suicide gene is not directly introduced, thereby increasing its anti -cancer effect.

[0236] Example 1: Generation of Tumor Homing Cells Mesenchymal Stem Cells to Deliver Oncolytic Virus

[0237] Induced pluripotent stem cells were dissociated with the aid of Versene EDTA (Thermo Fisher Scientific Life Sciences, Waltham, MA) and seeded into nonadherent 384-well conical polymerase chain reaction plates, 10,000 cells per well, with Iscove’s modified Dulbecco’s medium (IMDM) (MDM basal media, 17% KnockOut Serum Replacement, 1% minimal essential medium nonessential amino acids, 110 mM 2-mercaptoethanol, and 1% PSA antifungal-antibacterial solution [Thermo Fisher Scientific Life Sciences]). On day 2, the EBs were transferred to nonadherent 2.4- pg / cm2poly-HEMA-coated flasks and cultured for 3 more days. On day 5, the EBs were transferred to 1% gelatin-treated flasks for 3 more days of culture. On day 8, some EBs attached to the surface and cells grew outward from the EBs. The nonattached EBs were transferred to another 1% gelatin-coated flask. During this process, two populations were identified: cells that migrated out of the EB during days 2-5 (attached cells) and cells that grew out of the EB during days 5-8, after the EB had been transferred to another plate (transferred cells). Both cell types were fed with medium supplemented with 10 ng / ml TGF-pi (R&D Systems, Minneapolis, MN, http: / / www.mdsystems.com) on days 8-10, in the presence of hypoxia 1% oxygen for 48 hours after which the medium was switched to standard DMEM culture medium containing 10% fetal bovine serum, 2 mM 1- glutamine, and 100 U / ml penicillin / streptomycin (Thermo Fisher Scientific LifeSciences). The medium was changed twice a week, and the cells were split upon confluence in a 1 :3 ratio. Cells were subsequently infected with Newcastle Disease Virus and administered into mice bearing 4T1 tumors. Reduction of tumor growth was seen only with virus infected mesenchymal stem cells.

Claims

WHAT IS CLAIMED IS:

1. A method for providing a subject with a cancer therapy comprising autologous mesenchymal stem cells, the method comprising: a) identifying a subject with cancer; b) isolating a somatic cell from the subject; c) dedifferentiating the somatic cell to generate an induced pluripotent stem cell (iPSC); d) establishing a cell culture system for differentiating a mesenchymal stem cell, wherein the iPSC is added to a mesenchymal differentiation medium, wherein the differentiated mesenchymal stem cells in the culture express biomarkers or antigens comprising CD73, CD90, CD105, and CXCR4; e) transfecting the differentiated mesenchymal stem cell with a hypoxiainducible promoter; f) infecting the differentiated mesenchymal stem cells with an oncolytic virus; and g) administering a therapeutically effective amount of the differentiated mesenchymal stem cells to the subject.

2. The method of Claim 1, wherein a somatic cell isolated from the subject is a cell type selected from the group consisting of: a peripheral blood cell, a monocyte, a fibroblast, a T cell, and a mesenchymal stem cell.

3. The method of Claim 1, wherein an iPSC expresses a biomarker selected from the group consisting of: OCT-4, Lin28, PIM-1, PIM-3, Sox2, Kruppel-like factor (KLF), MYC, I-MYC, k-ras, NANOG, NF-kappaB, and c-met.

4. The method of Claim 1, wherein the hypoxia-inducible promoter drives expression of genes for cell homing and / or immune function selected from the group consisting of CXCR4, CXCR7, VEGF-R2, c-met, LIF, IFN-g, IFN-beta, ICAM-1, ICAM-2, VCAM-1, SDF-1, VEGF-A, bFGF, fas ligand, TRAIL, IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, IL-27, IL-23 receptor, lymphotoxin, TNF -alpha, IFN-alpha, IFN-beta, G-CSF, GM-CSF, and M-CSF.

5. The method of Claim 4, wherein expression of a gene for cell homing and / or immune function is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 80% higher, at least 100% higher, at least 150% higher, or at least 200% higher than the expression of the same biomarker on a primary mesenchymal stem cell isolated from a tissue.

6. The method of Claim 1, wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, Newcastle disease virus, reovirus, poliovirus, parvovirus, measles virus, and reovirus.

7. The method of Claim 1, wherein the differentiated mesenchymal stem expresses a biomarker selected from the group consisting of: SRY-box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT-4), homeobox protein NANOG, reduced expression- 1 (REX-1), T cell receptor alpha locus 1-60 (TRA- 1-60), TRA-1-81, GATA-4, TERT, stage-specific mouse embryonic antigen-3 (SSEA-3), and SSEA-4.

8. The method of Claim 1, wherein a mesenchymal differentiation medium comprises a bone morphogenetic protein.

9. The method of Claim 8, wherein a bone morphogenetic protein comprises BMP -2, BMP-4, or a combination thereof.

10. The method of Claim 1, wherein the iPSC that is added to a mesenchymal differentiation medium has been cultured in one or a plurality of small molecules or compounds.

11. The method of Claim 10, wherein the one or plurality of small molecules or compounds are selected from the group consisting of an NFkappaB inhibitor, CHIR99021, ascorbic acid, all-trans retinoic acid (ATRA), sodium phenylbutyrate, forskolin, tranylcypromine hydrochloride, lithium chloride, and an ALK inhibitor.

12. The method of Claim 11, wherein an ALK inhibitor is selected from the group consisting of: SB431542, crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib.

13. The method of Claim 1, wherein dedifferentiating the somatic cell is performed using a compound selected from the group consisting of: a histone deacetylase inhibitor, inhibitor of DNA methyltransferase, a ROCK inhibitor, and an inhibitor of glycogen synthase kinase 3.

14. The method of Claim 13, wherein the histone deacetylase inhibitor is selected from the group consisting of: phenylbutyrate, trichostatin, valproic acid, and sulforaphane, genistein.

15. The method of Claim 1, wherein the cell culture system for differentiating a mesenchymal stem cell comprises a hypoxic environment.

16. The method of Claim 15, wherein a hypoxic environment induces alterations in gene expression and cellular functions in the mesenchymal stem cell.

17. The method of Claim 16, wherein an alteration in gene expression mediates upregulation of an antigen or molecule selected from the group consisting of: mesenchymal stem cell comprising matrix metalloproteinase-9 (MMP-9), placental growth factor (PGF), VEGF, basic fibroblast growth factor, Notch, Notch ligands (such as Delta-like ligands and Jagged 1), epidermal growth factors, ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF).

18. The method of Claim 1, wherein a biomarker or an antigen on the differentiated mesenchymal stem cells is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 80% higher, at least 100% higher, at least 150% higher, or at least 200% higher than the expression of the same biomarker or antigen on a primary mesenchymal stem cell isolated from a tissue.

19. The method of Claim 1, wherein administering a therapeutically effective amount of the differentiated mesenchymal stem cell to the subject mediates one or a plurality of effects comprising increasing activity of tissue inhibitors of metalloproteinases (TIMPs), decreasing activity of matrix metalloproteinases (MMPs), or reducing oxidative stress in the tumor microenvironment.

20. The method of Claim 19, wherein a reduction in oxidative stress is measurable based on one or more effects comprising decreased neutrophil activation, increased neutrophil apoptosis, decreased activation of macrophages, decreased myeloid suppressor cell activation, decreased production of superoxide radicals, decreased presence of free oxygen radicals, decreased presence of hydrogen peroxide, or increased expression of superoxide dismutase.