Enhancement of regenerative activity by inactivation of senescent cells

Abstract

Methods and compositions of matter for enhancing regenerative activity in a patient by inactivation of senescent cells. Senescent cells are immunologically inactivated allowing for enhancing activity of endogenous and / or exogenous stem cells. Additionally small molecules such as dasatinib and quercetin and / or other means of inactivating senescent cells are used to augment regenerative activity.

Description

ENHANCEMENT OF REGENERATIVE ACTIVITY BY INACTIVATION OFSENESCENT CELLSCROSS REFENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from U.S Provisional Application No. 63 / 660,973, entitled ENHANCEMENT OF REGENERATIVE ACTIVITY BY INACTIVATION OF SENESCENT CELLS, filed on June 17, 2024, the entire contents of which are hereby expressly incorporated by reference.FIELD OF THE INVENTION

[0002] The invention relates to the field of regenerative medicine, more specifically the invention pertains to tissue regeneration and senolytics.BACKGROUND

[0003] As aging progresses, senescent cells accumulate in various tissues and their activities, which include promoting oxidative stress and inflammation, underlie a plethora of different diseases. There remains a need for therapeutic means for addressing the common cellular and molecular changes that underlie the positive correlation between aging and various diseases.

[0004] One means for addressing damaged or inflamed aged tissues involves cellbased therapies leveraging cells with regenerative capabilities, usually stem cells or progenitor cells, that can differentiate into various cell types, stimulate tissue repair mechanisms, and counter inflammation. However, the translation of these therapies into the clinic is challenging due to numerous immunological and physiological barriers encountered by a therapeutic cell population in vivo, including the hostile disease microenvironment that prevents the regenerative cells from surviving or functioning. The present invention addresses the need to improve the tissue microenvironment prior to administration of regenerative cells to a subject. Embodiments of the invention leverage senolytics, drugs or molecules that are designed to selectively eliminate senescent cells, to provide enhanced therapeutic effects from regenerative cell products.SUMMARY

[0005] The invention provides therapeutic compositions and methods of use thereof for treating diseases of aging including cancer and organ-specific diseases that are characterized by inflammation, fibrosis, tissue dysfunction, and immune dysregulation.

[0006] In one embodiment, a method for treating an age-related disease or condition in a subject is provided, the method comprising the following steps: a) identifying a subject with an age-related disease or condition affecting at least one organ or tissue; bjadministering at least one seno lytic agent to the subject, wherein the at least one senolytic agent comprises an agent that induces inactivation or apoptosis of senescent cells; c) administering at least one regenerative cell to the subject, wherein the regenerative cell comprises at least one of a mesenchymal stem cell or a tissue-specific progenitor cell, and wherein the regenerative cell contributes to repairing or replacing the at least one organ or tissue; and d) optionally, administering one or a plurality of soluble factors from at least one regenerative cell to the subject, wherein the one or plurality of soluble factors contribute to repairing or replacing the at least one organ or tissue. In one embodiment, the at least one regenerative cell is derived from an induced pluripotent stem cell. In one embodiment, the induced pluripotent stem cell is derived from a somatic cell from the subject, wherein the somatic cell comprises 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.

[0007] In certain embodiments, at least one senolytic agent is selected from the group comprising dasatinib, quercetin, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-l,4-bis-TPP, a derivative of 2-butene-l,4-bis-TPP, 2-chlorobenzyl-TPP, a derivative of 2-chlorobenzyl-TPP, 3- methylbenzyl-TPP, a derivative of 3-methylbenzyl-TPP, 2,4-dichlorobenzyl-TPP, a derivative of 2,4-dichlorobenzyl-TPP, 1-naphthylmethyl-TPP, a derivative of 1- naphthylmethyl-TPP, p-xylylenebis-TPP, a derivative of p-xylylenebis-TPP, vitamin C, fisetin, berberine, caffeic acid phenyl ester, and silibinin.

[0008] In other embodiments, a senolytic agent comprises a dendritic cell vaccine, wherein the dendritic cell that is pulsed with one or a plurality of senescence-associated antigens. The senescence-associated antigens may comprise pl 6, p21, or both, or other antigens associated with senescence. In one embodiment, the senescence-associated antigens are derived from a lysate of a senescent cell such as a fibroblast. In certain embodiments, the lysate is derived from a cell that is rendered senescent by exposure to one or plurality of stimuli or agents selected from the group comprising ionizing radiation, non-ionizing radiation, genotoxic drug(s), demethylating agent(s), acetylating agent(s), and hypoxia.

[0009] In one embodiment, a regenerative cell comprises a mesenchymal stem cell that is selected from the group comprising a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, a dental pulp mesenchymal stem cell, or an amniotic membrane mesenchymal stem. The mesenchymal stem cell may be autologous or allogeneic.

[0010] In one embodiment, a regenerative cell comprises a tissue-specific progenitor cell that is selected from the group comprising a hematopoietic stem / progenitor cell, a neural progenitor cell, a mesenchymal stem / progenitor cell, a satellite cell, an osteoprogenitor cell, a chondroprogenitor cell, a tenocyte progenitor cell, a basal epithelial progenitor cell, a limbal stem / progenitor cell, an intestinal crypt progenitor cell, a prostate epithelial progenitor cell, a mammary gland progenitor cell, a hepatic progenitor cell, a bronchioalveolar stem / progenitor cell, an alveolar type I progenitor cell, an alveolar type II cell progenitor cell, a pancreatic progenitor cell, a renal progenitor cell, a ureteric bud progenitor cell, a cardiac progenitor cell, an endothelial progenitor cell, a smooth muscle progenitor cell, a hair follicle stem / progenitor cell, a sebaceous gland progenitor cell, a spermatogonial stem / progenitor cell, and an ovarian surface epithelium progenitor cell. The tissue-specific progenitor cell may be autologous or allogeneic.

[0011] In one embodiment, one or a plurality of different types of soluble factors are administered to a subject as sources of regenerative molecules, which may be administered in combination with a regenerative cell therapy. In certain embodiments, the types of soluble factors are selected from the group comprising extracellular vesicles,exosomes, microvesicles, ectosomes, non-vesicular extracellular nanoparticles, apoptotic bodies, proteins, glycoproteins, and glycolipids.

[0012] In certain embodiments, a seno lytic agent is administered to a subject to suppress or reduce the Senescence- Associated Secretory Phenotype (SASP) in an organ or tissue. In certain embodiments, the SASP comprises one or a plurality of cytokines selected from the group comprising IL-la, IL- 1 P, IL-6, IL-8, IL-10, IL-13, IL-15, IL-18, IL-33, TNF-a, GM-CSF, and IFN-y.

[0013] In certain embodiments, a regenerative cell and / or the soluble factors derived thereof, are administered to a subject to increase the concentrations of anti-inflammatory and regenerative molecules in an organ or tissue. In certain embodiments, the antiinflammatory and regenerative molecules are selected from the group comprising IL- 10, IL-13, IL-20, IL-22, IL-35, TGF- , angiopoietin, endoglin, soluble HLA-G, soluble TNF- a receptor p55, soluble TNF- a receptor p75, soluble arginase, indolamine 2,3 dioxygenase, kynurenine, NGF, BDNF, CNTF, PGE-2, bcl-2, livin, survivin, membrane bound TGF-P, membrane bound PD-L1, membrane bound PD-L2, FoxP3, soluble PD-L1, and soluble PD-L2.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a bar graph showing the results of modulating liver damage (aspartate aminotransferase (AST) levels) with senolytic immunotherapy using a dendritic cell vaccine (SENOVAX™) combined with hepatogenic progenitor cells and mesenchymal stem cells.DETAILED DESCRIPTION

[0015] 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.

[0016] Unless defined otherwise, the technical terms used herein have the same meaning as is commonly understood by one of skill in the art.

[0017] As used herein, the terms “subject” or “patient” refers to a human or an animal to whom a composition, method, or therapy of the invention is applied.

[0018] As used herein, “regenerative cell” refers to a cell that contributes to the regeneration, repair, or replacement of a damaged or diseased tissue or organ. A regenerative cell may comprise a cell that is capable of self-renewal, differentiation, and / or tissue repair. Non-limiting examples of regenerative cells include mesenchymal stem cells, hematopoietic stem cells, progenitor cells, tissue-specific cells, neural stem cells, induced-pluripotent stem cells, and lymphocytes. In the context of the invention, a regenerative cell is applied as a cell-based therapeutic that is administered to a subject in need thereof.

[0019] As used herein, “therapeutic cell” refers to a cell that is used in cell therapy for treatment or prevention of a disease, condition, or symptom. A therapeutic cell may include a cell that has been modified to enhance its therapeutic properties. In certain contexts, a therapeutic cell comprises a regenerative cell.

[0020] As used herein, the terms “administration” or "administering" when used in the context of a therapeutic agent such as a senolytic agent or a regenerative cell, or anfother treatment, refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may refer to administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intranodal, intratumoral, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In some embodiments, a treatment or formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0021] As used herein, “induced pluripotent stem cell” (“iPSC”) refers to a type of pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., from skin,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 adult cells under embryonic stem (ES) cell culture conditions. An induced pluripotent stem cell may refer to a stem cell that is amenable to modification or differentiation to provide a therapeutic cell such as a regenerative cell for treating a subject.

[0022] 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 cell type 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.

[0023] 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.

[0024] As used herein, “mesenchymal stem cell” or “MSC” refers to a cell having the following characteristics: (i) adherence to plastic, (ii) expression of CD73, CD90, and CD 105 antigens, while lacking expression of CD 14, CD34, CD45, CD 19, and HLA-DR negative, and (iii) possess ability to differentiate into osteoblasts, adipocytes, and chondroblasts. A mesenchymal stem cell may be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, amniotic tissue, endometrium, trophoblast-derived tissues, umbilical cord blood, Wharton’s jelly, placenta, or derived from pluripotent stem cells. Mesenchymal stem cells may 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. Mesenchymal stem cellsinclude cells described in the literature as mesenchymal stromal cells, bone marrow stromal stem cells, marrow-isolated adult multipotent inducible cells, multipotent adult progenitor cells, mesenchymal adult stem cells, MultiStem®, Prochymal®, remestemcel-L, mesenchymal precursor cells, dental pulp stem cells, 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), and adipose-derived stem and regenerative cells.

[0025] The invention teaches methods for enhancing the therapeutic efficacy of regenerative cells or regenerative cell-derived products using one or a plurality of senolytic agents, wherein therapeutic efficacy refers to the ability of regenerative cells or their products to restore, replace, or repair a damaged or lost tissue. Certain aspects of the invention provide methods for reversing, delaying, or preventing aging of a tissue. Embodiments of the invention provide senolytic agents that can be administered to a subject to overcome or lessen the immunological barriers to effective delivery or function of a therapeutic cell population in vivo. In certain embodiments, one or a plurality of senolytic agents are administered to a subject to restore normal turnover of cells in a tissue and / or to restore tissue homeostasis. In certain embodiments, one or a plurality of senolytic agents are administered to a subject to render a tissue or organ more favorable to migration, engraftment, persistence, or function of a therapeutic cell. In one embodiment, one or a plurality of senolytic agents are administered to a subject to improve the regenerative properties of a therapeutic cell, wherein the regenerative properties are selected from the group comprising self-renewal, multipotency or pluripotency, tissue integration, paracrine signaling, immunomodulation, angiogenesis promotion, anti-apoptotic effects, anti-fibrotic effects, homing and migration, and extracellular matrix remodeling.

[0026] In one embodiment the invention provides methods for augmenting the therapeutic activity of a regenerative cel by inactivation of senescent cells in vivo, wherein said senescent cells are inactivated by immunological, genetic, biochemical, bacterial, viral, or small molecule means. In certain embodiments, senescent cell inactivation is performed to enhance therapeutic activity of exogenously administeredallogeneic and / or autologous progenitor cells. In some cases, said progenitor cells may be endogenous. In certain embodiments, senescent cell inactivation is performed to dampen the production of at least one inflammatory cytokine in the subject, wherein the at least one inflammatory cytokine is selected from the group comprising TNF-a, lymphotoxin, IL-lp, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-17F, IL-18, IL- 21, IL-23, IL-27, IL-33, IFN-a, IFN-p, IFN-y, TRANCE, and HMGB1. Inflammatory cytokines are a hallmark of diseases of aging having widespread effects including but not limited to inducing fibrin production and preventing cellular apoptosis.

[0027] In certain embodiments, at least one seno lytic agent is administered to a subject to reduce dendritic cell maturation. A reduction in dendritic cell maturation in response to the senolytic agent can be measured based on detection of reduced expression of one or plurality of certain costimulatory molecules on isolated dendritic cells (e.g., from peripheral blood mononuclear cells), wherein the costimulatory molecules are selected from the group comprising CD1, CD5, CD 10, CD40, CD80, CD86, and ICAM1.

[0028] In certain embodiments, a senolytic agent comprises a compound or a molecule that improves tissue function, reduces chronic inflammation, and / or delays the onset of an age-related disease. In certain embodiments, a senolytic agent is used to treat a medical condition affecting a tissue or organ, wherein the medical condition is characterized by age-related tissue dysfunction, inflammation, cancer, fibrosis, or a metabolic disturbance.

[0029] In certain embodiments, a senolytic agent comprises a Bcl-2 family inhibitor, a tyrosine kinase inhibitor, a flavonoid, a heat shock protein inhibitor, a FOXO4-p53 inhibitor, a proteosome inhibitor, a mitochondrial inhibitor, or an autophagy modulator.

[0030] In one embodiment, at least one senolytic agent is administered to a subject with a disease or condition related to aging, wherein the at least one senolytic agent is selected from the group comprising dasatinib, quercetin, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-l,4-bis-TPP, a derivative of 2-butene-l,4-bis-TPP, 2-chlorobenzyl-TPP, a derivative of 2- chlorobenzyl-TPP, 3-methylbenzyl-TPP, a derivative of 3-methylbenzyl-TPP, 2,4- dichlorobenzyl-TPP, a derivative of 2,4-dichlorobenzyLTPP, 1-naphthylmethyl-TPP, aderivative of 1-naphthylmethyl-TPP, p-xylylenebis-TPP, a derivative of p-xylylenebis- TPP, vitamin C, fisetin, berberine, caffeic acid phenyl ester, and silibinin. In certain embodiments, at least one seno lytic agent is provided to a subject to induce inactivation of a senescent cell.

[0031] In certain embodiments, a seno lytic agent is provided that elicits an immune response against senescence-associated antigens expressed by senescent cells upon administration to a subject. In one embodiment, a seno lytic agent comprises a cellular vaccine. Certain embodiments provide personalized (i.e., autologous) or off-the-shelf (i.e. allogeneic) cell vaccines. Preferred embodiments provide a cell vaccine that presents antigens to the immune system, wherein a T cell response and / or an antibody response against a senescent cell is elicited in the subject.

[0032] Embodiments of the invention provide a senolytic agent comprising antigen presenting cells such as dendritic cells that have functions in targeting cells in a tissue or organ comprising senescent cells such as a tumor. In certain embodiments, a cell vaccine elicits an immune response against one or a plurality of senescence-associated antigens in the tissue microenvironment. In certain embodiments, the immune response against senescent cells is directed against one or a plurality of the following molecules: a) a member of the BCL-2 family of proteins; b) a Heat Shock protein; c) Bromodomain containing 4 (BRD4); d) Na / K ATPase; e) Oxidation resistance 1 (OXR1); f) IL-6; g) IL- 8; h) IL-1; and i) TNF-alpha. In other embodiments, the immune response against senescent cells is directed against at least one cyclin-dependent kinase inhibitor, wherein the cyclin-dependent kinase inhibitor comprises, pl 6, p21, or both.

[0033] The methods of the invention are applicable for targeting or eradicating specific cells in the body of a subject using senolytic agents comprising cell-based vaccines, wherein the cells in the body that are being targeted or eradicated are defined by one or a plurality of the following characteristics: a) irreversible cell-cycle arrest; (b) a senescence-associated secretory phenotype (SASP); (c) macromolecular damage; and (d) an altered metabolism.

[0034] Certain embodiments provide treatments for cancer, a disease that increases significantly with aging and is associated with an increased burden of senescent cells within the tumor microenvironment. In certain embodiments, the invention providescompositions and methods for inducing an immune response against one or a plurality of tumor-associated antigens. In one embodiment, a composition of the invention comprises a cell vaccine that modulates one or a plurality of pathways in the immune system. In certain embodiments, a cell vaccine of the invention targets one or a plurality of molecules selected from the group comprising: a) BCL-2 family of proteins (BCL-XL); b) Heat shock proteins including HSP-90; c) Bromodomain containing 4 (BRD4); d) Na / K ATPase; e) Oxidation resistance 1 (OXR1); f) IL-6; g) IL-8; h) IL-1; and i) TNF-a.

[0035] The invention discloses compositions of antigen presenting cells (APCs) and methods of use thereof as cellular vaccines for inducing immunity against senescent cells in a subject in need thereof. In one embodiment, an antigen presenting cell comprises a dendritic cell (DC). In other embodiments, an APC is selected from the group comprising: a) a B cell; b) a macrophage; c) a monocyte; d) a neutrophil; e) a natural killer (NK) cell; f) an endothelial cell; g) an epithelial cell; h) a fibroblast; i) an eosinophil; and j) a mast cell. In certain embodiments, a vaccine comprises an APC that expresses one or a plurality of senescence-related antigens or biomarkers, wherein a senescence-related antigen or biomarker comprises an antigen or biomarker that is upregulated during cellular senescence (e.g., pl 6, p21, or others). In certain embodiments, a senescence-related biomarker is selected from the group comprising: a) Senescence- associated beta-galactosidase (SA-0gal); b) p21; c) DEC1 / DEC2; d) pl6; e) ARF; f) p 15; g) Senescence-associated heterochromatin foci (SAHF); h) Hl / macroH2A / H3.3 / H3metLys9; i) Asfla / HIRA; j) HP1 / HMGA; k) IL-6; 1) IL-8; m) p53; n) telomere-induced foci / DDR; and o) High Mobility Group Box 1 (HMGB1). One or a plurality of biomarkers may be measured in senescent cells using methods known in the art.

[0036] Methods are provided to induce or modify an APC to express or carry one or a plurality of biomarkers or antigens from senescent cells, for example, by pulsing the APC with a lysate or extract from said senescent cells in a manner that induces uptake of antigens from the cell lysate or extract. Methods of the invention provide an APC composition that is suitable for incorporating antigens from senescent cells. Embodiments of the invention provide lysates derived from autologous and allogeneic senescent cells, from primary cells isolated from the tumor or another tissue of a subject, from primary cells isolated from the tumor microenvironment or a tissue microenvironment of a subject(e.g., fibroblasts), and from cell lines. In certain embodiments, a lysate is derived from a senescent cell that has also undergone expansion or modification in vitro.

[0037] In certain embodiments, a lysate or extract from a senescent cell is provided to an APC such as a DC, wherein the antigen uptake capabilities of the APC allow one or a plurality of antigens present in the lysate to be taken up by the DC. Subsequently, detection methods such as flow cytometry, proteomics methods, or other techniques may be used to identify the one or plurality of antigens within the DC, or expressed on the surface of the DC, or both.

[0038] In certain embodiments, the lysate or extract that is applied to the methods of the invention is derived from a cell that has been rendered senescent by exposure to one or a plurality of stimuli that induce oxidative stress. In certain embodiments, a cell has been rendered senescent by exposure to one or a plurality of stimuli or agents from the group comprising: a) ionizing radiation; b) non-ionizing radiation; c) genotoxic drug(s) (e.g., cisplatin, doxorubicin, bleomycin, methotrexate, etoposide, or others); d) demethylating agent(s) (e.g., gemcitabine, decitabine); e) acetylating agent(s) (e.g., histone acetyltransferases); and f) hypoxia. In one preferred embodiment, the lysate is derived from a cell that has been rendered senescent by treatment with doxorubicin, wherein doxorubicin is provided at a concentration between 100 nM and 200 nM.

[0039] The prevent invention discloses patient-specific cell-based therapies that are useful for treating cancer or other diseases or conditions of aging. In certain embodiments, a senolytic agent such as a cell vaccine is provided to reduce the burden of senescent cells in vivo for the purpose of preventing or slowing the onset or progression of a disease. In certain embodiments, the invention provides an autologous APC such as a DC that induces an immune response against senescent cells upon administration to a subject. In certain embodiments, an APC with these capabilities is generated by engineering said APC to express specific antigens, or by pulsing the APC with said antigens in the form of a cellular lysate, and / or selected proteins, peptides, antigens, or neoantigens. In certain embodiments, an APC is pulsed with one or a plurality of antigens derived from an autologous cell, wherein the autologous cell comprises a senescent cell. In certain embodiments, an APC is pulsed with one or a plurality of antigens derived from an allogeneic cell, wherein the allogeneic cell comprises a senescent cell.

[0040] Aspects of the invention are directed toward inactivating senescent cells or inducing death (i.e., apoptosis) of senescent cells using one or a plurality of senolytic agents. In one embodiment, inactivation of a senescent cell refers to hindering or blocking a particular function of the cell, e.g., preventing the production of one or a plurality of cytokines. In one embodiment, methods are provided for inducing inactivation or death or inactivation of a cell that is in cell cycle arrest (i.e., a nondividing cell). In certain embodiments, the cell cycle arrest in the cell is irreversible. In one embodiment, a method is provided for inducing death or inactivation of a cell that has been exposed to one or a plurality of triggers of cellular stress, wherein the triggers comprise telomere shortening, DNA damage, oxidative stress, chromatin disruption, oncogene activation, loss of tumor suppressor genes, excess growth factor signaling, chemotherapy drugs, DNA damaging agents, epigenetic drugs, reactive oxygen speciesinducing compounds, ionizing radiation, ultraviolet light, heat shock, exposure to hypoxic conditions, exposure to hypoxia followed by reoxygenation, nutrient imbalance, starvation, mitochondrial dysfunction, lysosomal stress, endoplasmic reticulum stress, exposure to proinflammatory cytokines, toll-like receptor, activation, or combinations thereof.

[0041] In one embodiment, methods are provided for inducing inactivation or apoptosis of a cell that comprises one or a plurality of molecules associated with cellular senescence comprising pl6, p21, p53, cyclin DI, and retinoblastoma protein (Rb). In other embodiments, the methods of the invention are suitable for targeting cells that express one or a plurality of cell surface or membrane molecules selected from the group comprising CD26 (or dipeptidyl peptidase 4; DPP4), urokinase plasminogen activator receptor (uPAR / CD87), ICAM-1 (CD54), p2-microglobulin, NOTICH1, annexin A5, and EphA2.

[0042] In another embodiment, methods are provided for inducing inactivation or death of a cell that produces one or a plurality of proinflammatory cytokines, chemokines, and molecules selected from the group comprising IL-6, IL-8, TNF-a, interferon-alpha (IFN- a), interferon-beta (IFN-P), interferon-gamma (IFN-y), interleukin-1 alpha (IL-la), IL-ip, IL-33, CCL2, CXCL1, CXCL10, CCL5, VEGF, GM-CSF, HGF, FGF2, insulin-like growth factor binding proteins (IGFBPs), MMP-1 (Collagenase- 1), MMP-3 (Stromelysin-1), MMP-9 (Gelatinase B), MMP-10 (Stromelysin-2), plasminogen activator inhibitor- 1 (PAI-1), and amphiregulin.

[0043] In a preferred embodiment, the methods of the invention suppress or prevent the Senescence- Associated Secretory Phenotype (SASP), characterized by the production of cytokines, chemokines, growth factors, proteases, and extracellular matrix components by senescent cells in the body. Key cytokines in the SASP that are ameliorated or reduced by the methods of the invention comprise IL-la, IL-ip, IL-6, IL-8, IL-10, IL-13, IL-15, IL- 18, IL-33, TNF-a, GM-CSF, and IFN-y.

[0044] In one embodiment, the methods and compositions provided herein target the signaling pathways or molecules involved in SASP production, wherein the signaling pathways or molecules comprise at least one of the following: NF-kB, C / EBPp, p38 MAPK, mTOR, and GATA4, or others. In certain embodiments, the methods of the invention reduce or eliminate the SASP locally in a tissue or systemically.

[0045] In some embodiments, the production of inflammatory cytokines by senescent cells is targeted by inhibiting the activity of one or a plurality of transcription factors or other regulatory elements that stimulate production of inflammatory cytokines. In one embodiment, the one or plurality of transcription factors are selected from the list comprising NF-kappa B, STAT3, STAT4, STAT5, STAT6, RelA, RelB, and RelC. In one embodiment, a senescent cell is inactivated by blocking production of inflammatory cytokines through administration of one or more agents capable of inducing RNA interference to silence genes encoding the inflammatory cytokines. In one embodiment, gene silencing is accomplished by administration of one or more agents capable of inducing RNA interference such as a short interfering RNA molecule or a short hairpin RNA molecule. In other embodiments, or more ribozymes targeting said inflammatory cytokines are administered (e.g., a hammerhead ribozyme). In one embodiment, a senescent cell is inactivated by blocking production of inflammatory cytokines through administration of one or more antisense oligonucleotides targeting said inflammatory cytokines. In one embodiment, a senescent cell is inactivated by gene editing.

[0046] One embodiment of the invention provides at least one senolytic agent, at least one therapeutic cell composition, and combinations thereof, that create an antiinflammatory milieu in an organ or tissue or systemically. The presence of an anti-inflammatory milieu can be monitored by measuring the concentrations of antiinflammatory cytokines in a specimen or sample taken from a subject to whom the methods of the invention have been applied, for example, using peripheral blood or a tissue biopsy and using methods known in the art for analyzing cytokine gene or protein expression. In another aspect, the relative abundances of pro-inflammatory cytokines vs. anti-inflammatory cytokines can be quantified in the specimen or sample, wherein an abundance of the latter is indicative of an anti-inflammatory milieu. In one embodiment, the treatments of the invention confer increased concentrations of anti-inflammatory cytokines or molecules selected from the group comprising IL-3, IL-4, IL-1 receptor antagonist, IL-10, IL-13, IL-20, IL-22, IL-35, TGF-P, angiopoietin, endoglin, soluble HLA-G, soluble TNF- a receptor p55, soluble TNF- a receptor p75, soluble arginase, indolamine 2,3 dioxygenase, kynurenine, NGF, BDNF, CNTF, PGE-2, bcl-2, livin, survivin, membrane bound TGF-P, membrane bound PD-L1, membrane bound PD-L2, FoxP3, soluble PD-L1, and soluble PD-L2.

[0047] One embodiment of the invention provides at least one senolytic agent that reduces the concentrations of free radicals in an organ or tissue or systemically, wherein the free radicals comprise superoxide, hydroxyl radical, nitric oxide, peroxyl radical, alkoxyl radical, lipid peroxides, nitrogen dioxide, carbonate radical, or combinations thereof. The affected organ or tissue may also comprise a reduced concentration of antioxidants, wherein the antioxidants comprise glutathione, superoxide dismutase (SOD), catalase, glutathione peroxidase, thioredoxin, peroxiredoxin, uric acid, bilirubin, coenzyme Q10 (ubiquinol), melatonin, alpha-lipoic acid, ferritin, ceruloplasmin, transferrin, or combinations thereof. The methods of the invention, when administered to a subject with a disease or condition related to aging, are provided to increase the concentrations of antioxidants in the body locally and / or systemically.

[0048] In certain embodiments, a method of the invention is provided that eliminates or inactivates a cell that has sustained DNA damage. In one embodiment, the cell is identified on the basis of expression of one or a plurality of DNA damage-associated markers comprising yH2AX (phosphorylated histone H2AX on serine 139), 53BP1 (p53 binding protein 1), ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related protein), SAHF (senescence-associated heterochromatin foci), DNA-SCARS (DNA segments with chromatin alterations reinforcing senescence), HP1 proteins (e.g., HP la),H3K9me3 (trimethylation of histone H3 on lysine 9), HMGB1 (high mobility group box 1).

[0049] In one embodiment, a method of the invention is provided that eliminates or inactivates a cell that is resistant to apoptotic stimuli. By way of example, the cells that can be targeted using the methods of the invention may be resistant to apoptotic stimuli including but not limited to heat / thermal stimulation, hypoxia, radiation, and cytotoxic anticancer drugs. In one embodiment, a cell that is targeted using a method of the invention has increased expression of at least one anti -apoptotic protein selected from the group comprising Bcl-2, Bcl-xL, survivin, and MCL-1.

[0050] In one embodiment, methods are provided for repairing or restoring a diseased or damaged tissue, wherein the diseased or damaged tissue comprises an increased concentration of molecules, and wherein the molecules are selected from the group comprising IL-6, IL-8, TNF-a, interleukin- ip (IL-ip), IL-la, IL-33, CCL2, CXCL1, CXCL10, CCL5, VEGF, GM-CSF, HGF, FGF2, insulin-like growth factor binding proteins (IGFBPs), MMP-1 (Collagenase- 1), MMP-3 (Stromelysin- 1), MMP-9 (Gelatinase B), MMP-10 (Stromelysin-2), plasminogen activator inhibitor- 1 (PAI-1), and amphiregulin. In certain embodiments, an increased concentration of a molecule in a tissue is defined by comparison to the level of the same molecule in a healthy tissue (i.e., in a tissue not affected by a disease).

[0051] In one embodiment, methods are provided for treating a subject with a disease related to aging, wherein the disease is selected from the non-limiting group comprising osteoarthritis, atherosclerosis, autoimmunity, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), age-related macular degeneration, sarcopenia, osteoporosis, Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, chronic kidney disease, frailty syndrome, cardiomyopathy, hypertension, hearing loss, cataracts, skin aging, cancer, therapy-induced cancer relapse, liver fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, inflammatory bowel disease (IBD), chronic wounds, impaired wound healing, obesity-related inflammation, insulin resistance, metabolic syndrome, adipose tissue dysfunction, infertility, ovarian aging, testicular dysfunction, preeclampsia, and premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome).

[0052] In one embodiment, a subject with a disease or condition related to aging is selected for treatment with a method of the invention, wherein the method comprises administration of at least one senolytic agent and at least one regenerative cell type. Preferred aspects of the invention include methods of treating a disease or condition of aging by administration of at least one senolytic agent as the first step, followed by administration of at least one type of regenerative cell to the subject for treating the disease or condition as the second step. In certain embodiments, the second step is performed within 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 1 month after performing the first step. In one embodiment, repeat doses of a senolytic agent are provided to the subject. However, other embodiments of the invention are also contemplated, wherein it may be advantageous to provide a senolytic agent following treatment of a subject with at least one regenerative cell type in order to bolster the physiological effects of the regenerative cell in an ongoing disease condition. In one embodiment, a regenerative cell is administered to a subject in need thereof, followed by administration of one or a plurality of senolytic agents to the subject. Certain embodiments involve simultaneous administration of at least one senolytic agent and at least one regenerative cell population to a subject, for example, at the same time, on the same day, or within 30 minutes, within 1 hour, within 1-3 hours, within 3-6 hours, within 6-12 hours, or longer. Regardless of the sequence of administration of a regenerative cell relative to a senolytic agent, it may be preferable to administer multiple doses of regenerative cells to the subject, wherein the multiple doses of regenerative cells are provided at a specified time interval, for example, every day, every 2 days, every 3 days, every 7 days, every 2 weeks, every 4 weeks, every 6 weeks, every 8 weeks, every 12 weeks, or longer, for a defined period of time. It may also be preferable to administer more than one type or one population of regenerative cells to a subject, for example, a hepatic progenitor cell plus a mesenchymal stem cell may be administered to treat a disorder of the liver, or a cardiac progenitor cell plus a mesenchymal stem cell to treat a cardiovascular condition. In this embodiment, the different cell types may be mixed for administration at the same time, or provided to the subject individually.

[0053] In one embodiment, a subject with a disease or condition related to aging is selected for treatment with at least one therapeutic cell type, wherein the at least one therapeutic cell type is selected from the group comprising a bone marrow-derived mesenchymal stem cell, adipose-derived mesenchymal stem cell, umbilical cord-derivedmesenchymal stem cell, placenta-derived mesenchymal stem cell, dental pulp mesenchymal stem cell, amniotic membrane mesenchymal stem cell, bone marrow hematopoietic stem cell, bone marrow progenitor cell, peripheral blood hematopoietic stem cell, peripheral blood progenitor cell, umbilical cord blood hematopoietic stem cell, embryonic stem cell, induced pluripotent stem cell, cardiac progenitor cell, endothelial cell, epithelial cell, endothelial progenitor cell, neural progenitor cell, hepatic progenitor cell, pancreatic progenitor cell, renal progenitor cell, chondroprogenitor cell, osteoprogenitor cell, T cell, CAR-T cell, tumor-infiltrating lymphocyte, regulatory T cell, NK cell, NKT cell, basophil, neutrophil, mast cell, dendritic cell, macrophage, monocyte, gamma delta (y5) T cell, myeloid-derived suppressor cell, fibroblast, myofibroblast, keratinocyte, Schwann cell, oligodendrocyte progenitor cell, retinal pigment epithelial cell, islet cell, hepatocyte, chondrocyte, pericyte, and tenocyte.

[0054] In one embodiment, a subject with a disease or condition related to aging is selected for treatment with at least one therapeutic cell type comprising a cell with regenerative properties, wherein the at least one regenerative cell type or population is selected from the group comprising an embryonic stem cell, an induced pluripotent stem cell, a mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placental mesenchymal stem cell, a dental pulp mesenchymal stem cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a bone marrow progenitor cell, a peripheral blood progenitor cell, a neural stem cell, an epithelial cell, an epithelial stem cell, a muscle satellite cell, an endothelial cell, an endothelial progenitor cell, a cardiac progenitor cell, a hepatic progenitor cell, a pancreatic progenitor cell, a neural progenitor cell, a renal progenitor cell, a chondroprogenitor cell, an osteoprogenitor cell, a macrophage, a monocyte, a T cell, a B cell, a natural killer (NK) cell, an NKT cell, a dendritic cell, a basophil, a neutrophil, a mast cell, a regulatory T cell, a fibroblast, a myofibroblast, a keratinocyte, a Schwann cell, an oligodendrocyte progenitor cell, a retinal pigment epithelial cell, an islet cell, a hepatocyte, a chondrocyte, a tenocyte, and a pericyte. In some embodiments, a regenerative cell has been exposed to a culture system that induces cell stimulation to enhance its regenerative properties such as cytokine production or expression of immune modulatory molecules. In some embodiments, a regenerative cell has been exposed to a culture system that induces cell expansion.

[0055] Certain preferred embodiments provide a regenerative cell comprising a tissuespecific progenitor cell, wherein the tissue-specific progenitor cell is selected from the group comprising a hematopoietic stem / progenitor cell, a neural progenitor cell, a mesenchymal stem / progenitor cell, a satellite cell, an osteoprogenitor cell, a chondroprogenitor cell, a tenocyte progenitor cell, a basal epithelial progenitor cell, a limbal stem / progenitor cell, an intestinal crypt progenitor cell, a prostate epithelial progenitor cell, a mammary gland progenitor cell, a hepatic progenitor cell, a bronchioalveolar stem / progenitor cell, an alveolar type I progenitor cell, an alveolar type II cell progenitor cell, a pancreatic progenitor cell, a renal progenitor cell, a ureteric bud progenitor cell, a cardiac progenitor cell, an endothelial progenitor cell, a smooth muscle progenitor cell, a hair follicle stem / progenitor cell, a sebaceous gland progenitor cell, a spermatogonial stem / progenitor cell, and an ovarian surface epithelium progenitor cell. In some embodiments, selection of a particular tissue-specific progenitor for administration to a subject is tailored to the tissues and organs of the subject that are affected with an age-related disease or condition. By way of example, an alveolar progenitor cell may be selected for treating a subject with a lung disease. In one embodiment, a hepatic progenitor is selected for administration to a subject who is afflicted with a disease of the liver.

[0056] In one embodiment, a subject with a disease or condition related to aging is selected for treatment with at least one soluble factor derived from a therapeutic cell type such comprising a regenerative cell. In certain embodiments, a soluble factor is secreted by a therapeutic cell type into a culture medium in vitro, and the soluble factor is subsequently isolated, purified, or enriched from the culture medium. In certain embodiments, a soluble factor comprises a microparticle, an extracellular vesicle, an exosome, a microvesicle, an ectosome, a non-vesicular extracellular nanoparticle (e.g., an exomere or a supermere), an apoptotic body, a protein, a glycoprotein, and a glycolipid. In certain embodiments, a soluble factor comprises an antigen or molecule (e.g., a free- floating protein or glycoprotein in circulation). In other embodiments, a soluble factor comprises a plurality of antigens (e.g., antigens found on the surface or within the membranes of an exosome).

[0057] In certain embodiments, a therapeutic cell is generated derived from a pluripotent stem cell such as an induced pluripotent stem cell or a parthenogenically derivedpluripotent stem cell. Embodiments of the invention disclose methods for generating an induced pluripotent stem cell and subsequent methods for differentiating the induced pluripotent stem cell into a regenerative cell for administration to a subject in need thereof. Embodiments of the invention provide induced pluripotent stem cells 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 a disease of aging is provided, the method comprising: a) identifying a subject in need of treatment for aging; 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 regenerative cell; and e) administering the regenerative cell to the subject. In certain embodiments, a regenerative cell generated using these methods comprises a mesenchymal stem cell, a progenitor 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, an eosinophil, a mast cell, a neural cell, a neural progenitor cell, or another cell type disclosed herein.

[0058] Certain embodiments provide methods for isolating or selecting a somatic cell type from a subject for subsequent differentiation into an induced pluripotent stem cell. In one embodiment, a somatic cell comprises a peripheral blood cell, a monocyte, a macrophage, a dendritic cell, a fibroblast, a T cell, or a mesenchymal stem cell that is isolated from an organ or tissue of the subject although other adult somatic cell types may be used.

[0059] For example, in one embodiment, an adult somatic cell comprising a monocyte is dedifferentiated into an induced pluripotent stem cell, wherein dedifferentiation is induced by exposure to conditioned medium, and wherein the conditioned medium is generated by culture of induced pluripotent stem cells in the form of embryoid bodies. In one embodiment, the medium comprises Iscove’s media, DMEM media, OptiMEM media, EMEM media, RPMI-1640 media, or AIM-V media. In one embodiment, the embryoid body is routinely disaggregated during the culture period of dedifferentiation, for example, once every 2 days, once every 5 days, once every 10 days, or at a different frequency depending on the specific culture densities and compositions. In certainembodiments, the conditioned medium comprises factors or molecules from a cell that has been exposed to stressors, wherein the stressors be selected from the group comprising hypoxia, hypertonicity, hypotonicity, hyperthermia, serum starvation, mTOR inhibition, AMPK activation, and activation of inflammatory pathways. In one embodiment, an inflammatory pathway comprises MAP kinase, Janus Activated Kinase, Signal Transducer and Activator of Transcription-3 (STAT3), Signal Transducer and Activator of Transcription-5 (STAT5), Signal Transducer and Activator of Transcription- 6 (STAT6), TLR2, TLR3, and TLR4.

[0060] In one embodiment, generation of induced pluripotent stem cells is performed by reprogramming the somatic cells, wherein the reprogramming is performed by transfection of genes encoding one or a plurality of pluripotency-inducing factors, wherein the pluripotency-inducing factors are selected from the group comprising OCT4, PIM-1, NANOG, c-met, hTERT, KLF4, RAS, NOTCH, BMP2, BMP4, and AIRE. In one embodiment, the genes encoding pluripotency-inducing 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. In another embodiment, a somatic cell is transfected with cytoplasm from an immature, multipotent, or pluripotent stem cell. In one embodiment, a pluripotent stem cell is generated by transfecting a somatic cell with cytoplasm from an oocyte using a method and reagents such as electroporation, cell fusion, or streptolysin O or cell penetrating peptides to generate transient holes in the cytoplasm of the cell.

[0061] Certain embodiments disclose treating an isolated somatic cell with the appropriate agents or compounds that induces expression or upregulation of one or a plurality of biomarkers including but not limited to 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, a somatic cell is treated with one or a plurality of agents or chemicals toinduce dedifferentiation, wherein the agents or chemicals are selected from the group comprising a histone deacetylase (HD AC) inhibitor, a DNA methyltransferase inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor or an inhibitor of glycogen synthase kinase 3 (GSK-3). 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. In certain embodiments, an induced pluripotent stem cell is engineered to express a cytokine or a cytokine inhibitor. By way of example, an induced pluripotent stem cell may be engineered to express an inhibitor of TGF-P, wherein the cells that then differentiated from the induced pluripotent stem cell are deficient in TGF- P production, which is a cytokine that induces fibrosis. In certain embodiments, an inhibitor of TGF-P comprises antibody molecule, a cameloid antibody, microbody, an aptamer, a molecule capable of inducing RNA interference, a short hairpin RNA, a short interfering RNA, a microRNA, and a long noncoding RNA.

[0062] In certain embodiments, induced pluripotent stem cells generated using these methods are cultured to form embryoid bodies under the appropriate culture conditions. In certain embodiments, a differentiated cell type is generated by exposure of embryoid bodies derived from induced pluripotent stem cells to a decellularized matrix and / or to other growth factors and stimuli in culture. 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.

[0063] In certain embodiments, a regenerative cell that is generated from an induced pluripotent stem cell is selected from the group consisting of a mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placental mesenchymal stem cell, a dental pulp mesenchymal stem cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a bone marrow progenitor cell, a peripheral blood progenitor cell, a neural stem cell, an epithelial cell, an epithelial stem cell, a muscle satellite cell, an endothelial cell, an endothelial progenitor cell, a cardiac progenitor cell, a liver progenitor cell, a pancreatic progenitor cell, a neural progenitor cell, a renal progenitor cell, a chondroprogenitor cell, an osteoprogenitor cell, a macrophage, a monocyte, a T cell, a Bcell, a natural killer (NK) cell, an NKT cell, a dendritic cell, a basophil, a neutrophil, a mast cell, a regulatory T cell, a fibroblast, a myofibroblast, a keratinocyte, a Schwann cell, an oligodendrocyte progenitor cell, a retinal pigment epithelial cell, an islet cell, a hepatocyte, a chondrocyte, a tenocyte, and a pericyte. In certain embodiments, a therapeutic cell disclosed herein possesses immune suppressive activities upon administration to the subject. In other embodiments, a therapeutic cell disclosed herein possesses homing or migration characteristics for reaching a diseased or damaged tissue. Embodiments of the invention provide specific cell lineages or types generated from induced pluripotent stem cells that are useful as therapeutics in oncology.

[0064] 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 time that is sufficient to induce expression of one or a plurality of markers comprising CD73, CD90, and CD105.

[0065] 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 growthmedium, 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 a commercially 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.

[0066] In certain embodiments, a regenerative cell comprises an adult cell type or lineage that possesses or retains proliferation or differentiation ability. In one embodiment, a regenerative cell comprises an adult somatic 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.

[0067] In one embodiment, a regenerative cell comprises a genetically engineered adult somatic cell. In certain instances, a regenerative cell is generated by transient transfectionwith one or a plurality of transcription factors to direct a specific differentiation pathway in the cell without first reprogramming the cell into an induced pluripotent stem cell. Certain adult somatic cell types that may be particularly amenable to this type of manipulation include keratinocytes and fibroblasts. By way of example, in one embodiment, a regenerative cell comprises an adult somatic cell that has been transfected with the gene for OCT4 and then exposed to specific differentiation conditions. By way of example, the cells may be cultured in medium comprising agents or molecules that promote differentiation into the neuronal lineage, for example, Noggin, SB431542, CHIR99021, Shh (sonic hedgehog), retinoic acid, FGF, combinations thereof, or additional molecules. In certain embodiments, gene transfection is accomplished with a retrovirus, i.e., retroviral transduction. A retroviral expression vector may be used such as pMXs vector, lentiviral vector, adenoviral vector, pMXs adeno- associated vector, or herpesvirus vector. In certain embodiments, a plasmid-based vector, an RNA-based vector, an mRNA-based vector, a microRNA-based vector is used. In other embodiments, one or a plurality of stimulators of specific signaling pathways comprising the RAS pathway, the myc pathway, the PIM-1 pathway, and the JAK pathway are also provided to the cultures.

[0068] Embodiments of the invention provide methods of pre-treating a subject with senolytic agents to unmask or augment the regenerative activities of cell types that are not classically considered to be regenerative cells, for example, regulatory T cells, certain myeloid lineage cells, monocytes, and monocyte-derived dendritic cells. In some embodiments, it is also advantageous to administer regenerative cells comprising more than one cell type to a subject.

[0069] In one embodiment of the invention, senolytic agents are utilized to enhance activity of T regulatory cells. The invention discloses augmentation of T regulatory cell activity by senolytic agents, regardless of whether the regulatory T cells are endogenously derived or are exogenous. In some cases, regulatory T cells are generated by treatment of the subject with agents or molecules that expand T regulatory cells in vivo.

[0070] In one embodiment, a regenerative cell comprises a monocyte that is isolated from peripheral blood, menstrual blood, umbilical cord blood, Wharton’s jelly, adipose tissue, bone marrow, or omental tissue. In one embodiment, a monocyte is isolated from mobilized peripheral blood of a subject, wherein mobilization of peripheral blood isachieved by administering one or more agents comprising G-CSF, GM-CSF, M-CSF, IL- 3, IL-10, beta glucan, or IFN-y to the subject from whom the monocyte will be isolated. In certain embodiments, a regenerative cell that is administered to a subject comprises a monocyte. In one embodiment a monocyte is pretreated with one or a plurality of agents to induce a tolerogenic phenotype, wherein the one or plurality of agents are selected from the group comprising prostaglandin E2 (PGE2), genistein, quercetin, hypertonic saline, VEGF, PDGF-BB, IGF-1, salinomycin, erythropoietin, NKkappaB decoy oligonucleotides, Ikk beta decoy oligonucleotides, IL-35, TGF-P, ascorbic acid, N- acetylcysteine, alpha lipoic acid, methylene blue, and valproic acid. In certain embodiments, a monocyte is pretreated with one or a plurality of agents to induce a tolerogenic phenotype that is characterized by production of IL- 10 by monocytes. In one embodiment, one or plurality of agents are provided to cultures of monocytes to induce the production of at least 10 pg / mL, at least 20 pg / mL, or at least 40 pg / mL of IL- 10 per 1 million monocytes. In certain embodiments, a monocyte is pretreated with one or a plurality of agents to induce the secretion of exosomes by the monocytes, wherein the exosomes comprise IL-10. In other embodiments, the monocyte is differentiated into a dendritic cell in vitro, wherein the dendritic cell is subsequently administered to a subject as a therapeutic cell. In a specific embodiment, the dendritic cell comprises an immature dendritic cell. The dendritic cells that are preferably used as regenerative cells in the context of the invention possess regenerative and / or immune modulatory characteristics. In one embodiment, the dendritic cells express one or a plurality of molecules from the group comprising IL-10, IL-4, IL-13, IL-20, IL-22, IL-35, IL-37, IL-38, TGF-beta, endoglin, VEGF, HLA-G, and IL- 12 p40 homodimer. Certain embodiments of the invention provide a regenerative cell comprising an immature dendritic cell, wherein the immature dendritic cell is distinguishable from a mature dendritic cell based on its increased phagocytic activity, increased migratory activity toward a chemotactic gradient, decreased HL A class I and HL A class II expression, decreased CD la expression, decreased CD40 expression, decreased CD80 expression, decreased CD86 expression, decreased IL-15 receptor expression, decreased IL-18 receptor expression, decreased progesterone receptor expression, and decreased c-kit expression. In certain embodiments, an immature dendritic cell that is a suitable therapeutic cell for the disclosed treatments induces the generation of FoxP3+ regulatory T cells in vitro and / or in vivo. Certain embodiments provide methods for selecting an immature dendritic cell that is suitable for administration to a subject by performing in vitro testing of said cell. Inone such embodiment, an immature dendritic cell induces upregulated expression of autoimmune regulator (AIRE) in T cells, wherein AIRE expression induces differentiation of a conventional or naive T cell into a regulatory T cell. In certain embodiments, upregulated expression of AIRE in T cells is dependent on one or a plurality of molecules synthesized or secreted by the immature dendritic cell, wherein the one or plurality of molecules are selected from the group comprising IL- 10, TGF-P, soluble HLA-G, endoglin, FGF-1, FGF-2 FGF-5, and NOTCH.

[0071] In certain embodiments, a regenerative cell that is administered to a subject comprises a myeloid lineage cell, wherein the myeloid lineage cell further comprises an immature neutrophil. In certain embodiments, an immature neutrophil comprises a neutrophil progenitor that retains the ability to differentiate into either a neutrophil or a monocyte under the appropriate culture conditions. In one embodiment, a regenerative cell comprising an immature neutrophil expresses one or a plurality of markers selected from the group comprising PU.l, G-CSF receptor, stem cell factor receptor, c-Met, M- CSF receptor, and GM-CSF receptor. In one embodiment, an immature neutrophil produces IL- 10 in response to stimulation with a toll-like receptor agonist, for example, a TLR4 agonist comprising beta glucan, HMGB1, histones, or hyaluronic acid degradation products.

[0072] In certain embodiments, a regenerative cell that is administered to a subject comprises a myeloid lineage cell, wherein the myeloid lineage cell further comprises a myeloid-derived suppressor cell. The characteristics of the myeloid-derived suppressor cell may include the ability to differentiate into monocytes under the appropriate stimulation conditions, for example, when exposed to all-trans retinoic acid, vitamin D3, M-CSF, GM-CSF, or combinations thereof. In one embodiment, a regenerative cell comprising a myeloid-derived suppressor cell is capable of inhibiting T cell proliferation in vitro and / or in vivo, wherein the inhibition of T cell proliferation is mediated by one or a plurality of molecules selected from the group comprising nitric oxide, reactive oxygen species, superoxide, hydrogen peroxide, arginase, IL-10, soluble PD-L1, soluble VISTA, LAG-3, TIM3, prostaglandin E2, secreted vimentin, and secreted calreticulin.

[0073] In one embodiment, a regenerative cell comprises a tissue-specific progenitor cell that has the capacity to differentiate into a specialized cell type of a particular tissue. In one embodiment, a tissue-specific progenitor cell is selected from the group consisting ofa neural progenitor cell, hematopoietic progenitor cell, mesenchymal progenitor cell, epithelial progenitor cell, endothelial progenitor cell, muscle satellite cell, hepatic progenitor cell, pancreatic progenitor cell, cardiac progenitor cell, lung progenitor cell, renal progenitor cell, olfactory progenitor cell, retinal progenitor cell, thymic epithelial progenitor cell, intestinal stem / progenitor cell, and hair follicle progenitor cell. In certain embodiments, a tissue-specific progenitor cell is differentiated from an induced pluripotent stem cell.

[0074] In one embodiment, a tissue-specific regenerative cell is immortalized. Methods for immortalizing a cell may comprise means known in the art including but not limited to the use of certain chemicals, using CRISPR or shRNA knockdown of tumor suppressors, using methods for telomerase activation, or others. In one embodiment, transfection of the regenerative cell with an immortalization factor is performed, wherein the immortalizing factor comprises one or a plurality of oncogenes selected from the group comprising ABCB1, ABCG2, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1B, ACVR2A, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH1A1, ALDH2, ALK, AMER1, ANGPT1, ANGPT2, ANKRD23, APC, AR, ARAF, AREG, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, AR.ID I B, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BBC3, BCL10, BCL11 A, BCL1 IB, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRIM, BRINP3, BRIP1, BTG1, BTG2, BTK, BUB1B, Cl lorfiO, C15orf65, C2orf44, CA6, CACNA1D, CALR, CAMTAI, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD19, CD22, CD274, CD38, CD4, CD70, CD74, CD79A, CD79B, CD83, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDK7, CDK8, CDK9, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDX2, CEBPA, CHCHD7, CHD2, CHD4, CHEK1, CHEK2, CHIC2, CHN1, CHORDCI, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL, COL1A1, COPB1, COX6C, CRBN, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CUL3, CXCR4, CYLD, CYP17A1, CYP2D6, DAXX, DDB2, DDIT3, DDR1, DDR2, DDX10, DDX3X, DDX5, DDX6, DEK, DICER1, DIS3, DLL4, DNM2, DNMT1, DNMT3A, DOT1L, DP YD, DUSP4, DUSP6, EBF1, ECT2L, EDNRB, EED, EGFR, EIF4A2, ELF4, ELK4,ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHA7, EPHA8, EPHB1, EPHB2, EPHB4, EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, EREG, ERG, ERN1, ERRFI1, ESRI, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FAF1, FAIM3, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FAT1, FBX011, FBXW7, FCRL4, FEV, FGF10, FGF14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FKBP1A, FLCN, FLU, FLT1, FLT3, FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FRS2, FSTL3, FUBP1, FUS, GABRA6, GAS7, GATA1, GATA2, GATA3, GATA4, GATA6, GID4, GLI1, GMPS, GNA11, GNA12, GNA13, GNAQ, GNAS, GNRH1, GOLGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GRM3, GSK3B, GUCY2C, H3F3A, H3F3B, HCK, HDAC1, HERPUD1, HEY1, HGF, HIP1, HIST1H1E, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2, HMGN2P46, HNF1A, HNMT, HNRNPA2B1, HNRNPK, H00K3, H0XA11, H0XA13, H0XA9, HOXC11, HOXC13, H0XD11, H0XD13, HRAS, HSD3B1, HSP90AA1, HSP90AB1, IAPP, ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL2, IL21R, IL3RA, IL6, IL6ST, IL7R, INHBA, INPP4B, IRF2, IRF4, IRS2, ITGAV, ITGB1, ITK, ITPKB, JAK1, JAK2, JAK3, JAZF1, JUN, KAT6A, KAT6B, KCNJ5, KDM1A, KDM5A, KDM5C, KDM6A, KDR, KDSR, KEAP1, KEL, KIAA1549, KIF5B, KIR3DL1, KIT, KLF4, KLHL6, KLK2, KMT2A, KMT2C, KMT2D, KRAS, KTN1, LASPI, LCK, LCP1, LGALS3, LGR5, LHFP, LIFR, LM01, LM02, LOXL2, LPP, LRIG3, LRP1B, LUC7L2, LYL1, LYN, LZTR1, MAF, MAFB, MAGED1, MAGI2, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAPK1, MAPK11, MAX, MCL1, MDM2, MDM4, MDS2, MECOM, MED12, MEF2B, MEN1, MET, MITF, MKI67, MKL1, MLF1, MLH1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MLLT6, MMP9, MN1, MNX1, MPL, MRE11 A, MS4A1, MSH2, MSH6, MSI2, MSN, MST1R, MTCP1, MTF2, MTOR, MUC1, MUC16, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, MYH11, MYH9, NACA, NAE1, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NOTCH3, NPM1, NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUMA1, NUP214, NUP93, NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PARK2, PARP1, PATZ1, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PDK1, PEC AMI, PERI, PHF6, PHOX2B, PICALM, PIK3C2B, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1,PLCG2, PML, PMS1, PMS2, POLDI, POLE, POTI, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PREX2, PRF1, PRKAR1A, PRKCI, PRKDC, PRLR, PRPF40B, PRRT2, PRRX1, PRSS8, PSIP1, PSMD4, PTBP1, PTCHI, PTEN, PTK2, PTPN11, PTPRC, PTPRD, QKI, RABEP1, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAFI, RALGDS, RANBP17, RANBP2, RAP1GDS1, RARA, R131, RBM10, RBM15, RCOR1, RECQL4, REL, RELN, RET, RHOA, RHOH, RICTOR, RIPK1, RMI2, RNF213, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RPS6KB1, RPTOR, RUNX1, RUNX1T1, S1PR2, SAMHD1, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP 1, SETD2, SF1, SF3A1, SF3B1, SF3B2, SFPQ, SGK1, SH2B3, SH3GL1, SLAMF7, SLC34A2, SLC45A3, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMC1A, SMC3, SMO, SNCAIP, SNX29, SOCS1, SOXIO, SOX11, SOX2, SOX9, SPECC1, SPEN, SPOP, SPTA1, SRC, SRGAP3, SRSF2, SRSF3, SS18, SS18L1, SSX1, STAG2, STAT3, STAT4, STAT5B, STEAP1, STIL, STK11, SUFU, SUZ12, SYK, TAF1, TAF15, TALI, TAL2, TBL1XR1, TBX3, TCEA1, TCF12, TCF3, TCF7L2, TCL1A, TEK, TERC, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC, TGFB1, TGFBR2, THRAP3, TIMP1, TJP1, TLX1, TLX3, TM7SF2, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF9, TNFSF11, TOPI, TOP2A, TP53, TP63, TPBG, TPM3, TPM4, TPR, TRAF2, TRAF3, TRAF3IP3, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTK, TTL, TYMS, U2AF1, U2AF2, UBA1, UBR5, USP6, VEGFA, VEGFB, VHL, VPS51, VTI1A, WAS, WEE1, WHSCI, WHSC1L1, WIFI, WISP3, WNT11, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT6, WNT7B, WRN, WT1, WWTR1, XBP1, XPA, XPC, XPO1, YWHAE, YWHAZ, ZAK, ZBTB16, ZBTB2, ZMYM2, ZMYM3, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 and ZRSR2.

[0075] In certain contexts, a tissue-specific progenitor cell may be an advantageous therapeutic cell type for regenerating lost or damaged tissue in vivo or may be amenable to further in vitro modifications or genetic engineering to enhance its regenerative capabilities prior to administration to a subject. In one embodiment, a tissue-specific progenitor is capable of differentiating into one or a plurality of cell types from the group comprising pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue,Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.

[0076] In some embodiments, a regenerative cell is provided that produces or secretes one or a plurality of soluble factors in a culture system, wherein the one or plurality of soluble factors are isolated or enriched from the culture system and administered to a subject with a disease or condition related to aging. In certain embodiments, a regenerative cell is cultured in a liquid medium and the supernatant from the liquid medium is collected as a source of soluble factors, wherein the soluble factors are selected from the group comprising a microparticle, an extracellular vesicle, an exosome, a microvesicle, an ectosome, a non-vesicular extracellular nanoparticle (e.g., an exomere or a supermere), an apoptotic body, a protein, a glycoprotein, and a glycolipid. In one embodiment, a soluble factor comprises one or a plurality of antigens or molecules having regenerative functions, wherein the one or plurality of antigens or molecules comprise growth factors, cytokines, chemokines, adhesion molecules, angiogenic factors, signaling molecules, apoptosis-inducing molecules, and the like. In one embodiment, the one or plurality of soluble factors are provided from at least one regenerative cell type, however, in certain embodiments, soluble factors are derived from more than one regenerative cell type. In one embodiment, a soluble factor comprises an exosome, wherein the exosome comprises one or a plurality of antigens that possess regenerative functions. In one embodiment, a regenerative cell and / or the one or plurality of soluble factors from the regenerative cell are administered to a subject in need of treatment for an age-related disease or condition, or to prevent or delay the onset of an age-related disease or condition in a subject.

[0077] In one embodiment, one or a plurality of soluble factors are isolated or enriched from a regenerative cell comprising a regulatory T cell. The soluble factors and their relative concentrations in a cell culture supernatant can be modified or optimized by manipulation of the regulatory T cell. In one embodiment, a regulatory T cell is treated in vitro with at least one HD AC inhibitor, wherein the at least one HD AC inhibitor isselected from the group comprising CXD101, HDAClO-IN-1, Tubastatin A TFA, ACY- 775, Panobinostat, Trichostatin A, Vorinostat, Entinostat, BML-210, Abexinostat, Dacinostat, Quisinostat, Mocetinostat, Valproic Acid, CUDC-101, GSK3117391, Droxinostat, MCI 568, Pracinostat, Divalproex Sodium, Diferuloylmethane, Sodium butyrate, PCI-34051, SR-4370, Givinostat, Tubacin, AR-42, (-)-Parthenolide, Tubastatin A HC1, Resminostat, CUDC-907, M344, Tacedinaline, Romidepsin, 4-Phenylbutyric acid, Sinapinic acid, Sulforaphane, UF010, Suberohydroxamic acid, NKL 22, ITSA-1, KA2507, Isoguanosine, Raddeanin A, BRD3308, TH34, and Tinostamustine. In another embodiment, a regulatory T cell is treated with an inflammatory stimulus, for example, a TLR agonist such as beta glucan, poly IC, poly LC, CpG DNA, BCG, flagellin, lipopolysaccharide, low molecular weight hyaluronic acid, free histone, Isoxazolo[5,4- d]pyrimidine, imiquimod, poly A:U, MPL, poly G3, poly GIO, and hyperthermia exposed cells. In one embodiment, the one or plurality of soluble factors produced or secreted by a regulatory T cell are selected from the group comprising exosomes, exosomes expressing FoxP3, exosomes expressing TGF-P, exosomes expressing phosphatidylserine, apoptotic bodies, ferroptotic bodies, conditioned media, conditioned media from stressed cells, and conditioned media from cells exposed to inflammatory stimuli.

[0078] In one embodiment, one or a plurality of soluble factors are isolated or enriched from a regenerative cell comprising a mesenchymal stem cell. In one embodiment, one or a plurality of soluble factors are isolated or enriched from a mesenchymal stem cell that has been transfected with a growth factor, wherein the growth factor is selected from the group comprising HGF-1, FGF-1, EGF-1, angiopoietin, placental growth factor, adiponectin, vasoactive intestinal peptide precursor, endoglin, myostatin, TGF-beta, vascular endothelial growth factor, GDF-11, GDF-15, hyaluronic acid synthase, interleukin-33, osteosarcoma-derived growth factor, midkine, PDGF-BB, IGF-1, nerve growth factor. In one embodiment, exosomes are isolated from culture supernatant of a mesenchymal stem cell, wherein the exosomes are enriched for one or a plurality of growth factors selected from the group comprising HGF-1, FGF-1, EGF-1, angiopoietin, placental growth factor, adiponectin, vasoactive intestinal peptide precursor, endoglin, myostatin, TGF-beta, vascular endothelial growth factor, GDF-11, GDF-15, hyaluronic acid synthase, interleukin-33, osteosarcoma-derived growth factor, midkine, PDGF-BB, IGF-1, nerve growth factor.

[0079] In one embodiment, a regenerative cell is treated with a substance, molecule, or agent that inhibits cellular differentiation while maintaining a pluripotent state of the regenerative cell. In one embodiment, the substance, molecule, or agent comprises growth differentiation factor 11 (GDF-11), GDF-15, bone morphogenetic protein 2 (BMP2), amniotic fluid, or umbilical cord blood, umbilical cord plasma, a histone deacetylase (HD AC) inhibitor, a GSK-3 inhibitor, PIM1, SV40 large T antigen, abll, AFF4, AKT2, AKL, AML1, MTG8, BCL6, MCF2, DCF3, EGFR, MLLT11, ERBB2, ETS1, CSF1R, FOS, FES, GNAS, HER2, FGF3, FGF4, JUN, c-kit, K-SAM, AKAP13, LCK, LM01, LYL1, MASI, MDM2, MOS, MYH11, MYB, MYCN, PAX5, RAF, RAS, REL, ROS1, SKI (PDGF-BB), SET, SRC, TALI, TAN1, TIAN, TSC2, TRK, and an immortalizing oncogene. In one embodiment, the HD AC inhibitor is selected from the group comprising valproic acid, trichostatin A, sodium phenylbutyrate, and butyrate. In one embodiment, the GSK-3 inhibitor comprises lithium. In one embodiment, the substance, molecule, or agent modifies signal transduction in the regenerative cell. In specific embodiments, the biological substance, molecule, or agent suppresses NK-kappaB activation. In other specific embodiments, the substance, molecule, or agent increases the expression or activation of NRF2, heme oxygenase-1, Bcl-2, Bcl-xL, survivin, and livin. In one embodiment, the substance, molecule, or agent can be conditionally inactivated

[0080] In one embodiment, the invention provides the administration of at least one senolytic agent together with at least one regenerative cell type to a subject in need thereof. Regenerative cells may comprise stem cells, progenitor cells, monocytes, T cells, B cells, or other cells capable of directly or indirectly inducing healing of the body, organs, or cells. In one embodiment enhancement of endogenous regenerative activity is achieved through administration of at least one senolytic agent, wherein the at least one senolytic agent is selected from the group comprising tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, a mitoriboscin, a mitoketoscin, a mitoflavoscin, 2 -butene- 1,4- bis-TPP; a derivative of 2-butene-l,4-bis-TPP; 2-chlorobenzyl-TPP; a derivative of 2- chlorobenzyl-TPP; 3-methylbenzyl-TPP; a derivative of 3-methylbenzyl-TPP; 2,4- dichlorobenzyl-TPP; a derivative of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; a derivative of 1-naphthylmethyl-TPP; p-xylylenebis-TPP; a derivative of p-xylylenebis- TPP; Vitamin C, berberine, caffeic acid phenyl ester, silibinin, brutieridin, and melitidin.Examples of endogenous regenerative cells include cells capable of stimulating neurogenesis. In certain embodiments, neurogenic regenerative cells are provided that are suitable for treating pathological states including depression, multiple sclerosis, stroke, and drug addiction. In another embodiment, senolytic agents and / or regenerative cells are used together with approaches that stimulate neurogenesis for example electroconvulsive therapy, transcranial magnetic stimulation, low intensity transcranial ultrasound stimulation, and transcranial direct current stimulation. In certain embodiments, a regenerative cell comprises a mesenchymal stem cell that is selected for release of brain-derived neurotrophic factor upon stimulation with molecular signals associated with tissue injury, wherein at least 10 pg / mL, at least 25 pg / mL, or at least 100 pg / mL of brain-derived neurotrophic factor is produced in response to stimulation [e.g., using poly(I:C)]. In certain embodiments, a regenerative cell comprises a mesenchymal stem cell that is selected for release of ciliary neurotrophic growth factor upon stimulation with molecular signals associated with tissue injury, wherein the at least 4 pg / mL, at least 8 pg / mL, or at least 12 pg / mL of ciliary neurotrophic growth factor is produced in response to stimulation [e.g., using poly(I:C)]. In certain embodiments, a regenerative cell comprises a mesenchymal stem cell that is selected for release of basic fibroblast growth factor upon stimulation with molecular signals associated with tissue injury, wherein the at least 50 pg / mL, at least 100 pg / mL, or at least 200 pg / mL of basic fibroblast growth factor is produced in response to stimulation [e.g., using poly(I:C)].

[0081] In another embodiment, at least one senolytic agent and / or at least one regenerative cell is administered to a subject in combination with a fibrinolytic enzyme, wherein the fibrinolytic enzymes comprises a matrix metalloproteinase (MMP), and wherein the matrix metalloproteinase is selected from the group comprising MMP-3, MMP-5, MMP-6, MMP-9, and MMP-12. In certain embodiments, the fibrinolytic enzymes are administered to a subject prior to administration of a senolytic agent or a regenerative cell, or both.

[0082] In another embodiment, at least one senolytic agent and / or at least one regenerative cell is administered to a subject in combination with an inhibitor of NOGO. In one embodiment, an inhibitor of NOGO comprises antisense oligonucleotides, one or more molecules capable of inducing RNA interference, short interfering RNA (siRNA), short hairpin RNA (shRNA), small molecule inhibitors, antibodies, aptamers, somamers,bispecific antibodies, ribozymes, microantibodies, hammerhead ribozymes, or soluble receptors.

[0083] In one embodiment senolytic agents are used together with approaches that reduce the numbers or activity of type 2 macrophages, wherein a type 2 macrophage is defined as a macrophage that is capable of producing one or a plurality of molecules selected from the group comprising IL-10, TGF-P, IL-1 receptor antagonist (IL-IRa), IL-6, CCL17 (TARC), CCL22 (MDC), CCL24 (Eotaxin-2), CCL18, and CXCL13. In one embodiment, administration of a senolytic agent to a subject in need thereof reduces the concentrations of the one or plurality of molecules in a tissue or in the bloodstream.

[0084] In one embodiment senolytic agents are used together with approaches that reduce the numbers or activity of type 2 neutrophils. In one embodiment, a type 2 neutrophil produces one or a plurality of molecules selected from the group comprising IL-10, TGF- P, VEGF, Arginase-1, GM-CSF, IL-8 (CXCL8), CCL2 (MCP-1), CXCL1, CXCL2, CXCL5. In one embodiment, administration of a senolytic agent to a subject in need thereof reduces the concentrations of the one or plurality of molecules in a tissue or in the bloodstream.

[0085] In one embodiment senolytic agents are used together with approaches that reduce the numbers or activity of type 2 astrocytes. In one embodiment, a type 2 astrocyte produces one or a plurality of molecules selected from the group comprising IL-10, TGF- P, VEGF, Arginase-1, GM-CSF, IL-8 (CXCL8), CCL2 (MCP-1), CXCL1, CXCL2, and CXCL5. In one embodiment, administration of a senolytic agent to a subject in need thereof reduces the concentrations of the one or plurality of molecules in a tissue or in the bloodstream.

[0086] In some embodiments agents that suppress activity of senescent cells are combined with suppressors of interleukin- 17, for example, anti-IL-17 antibodies, for treatment of a condition such as liver failure, ischemia-reperfusion injury, autoimmune heart failure, aplastic anemia, or other conditions that are marked by high IL- 17 production by T cells.

[0087] In one embodiment, senolytic agents are utilized to augment efficacy of regenerative stem cell therapies such as adult stem cells, or combinations of cells. Senolytic agents may include, BTSA1, vilazodone, a SGLT2 inhibitor such ascanagliflozin which directly is senolytic but also stimulates immune mediated clearing of senolytic cells, a casein kinase 2 inhibitor such as 4,5,6,7-tetrabromo-2- azabenzamidazole, LY-D6 / 2, PI3K-inhibitors such as wortmannin and its clinical derivative, PX-866, USP7 inhibitors such as P5091, bcl-2 inhibitors such as the Bh3 mimetic ABT-263, ABT-737, piperlongumine, fisetin, EF24, R406, FOXO4-p53 interfering peptide, lorlatinib, and azithromycin.

[0088] In one embodiment, a regenerative cell population comprises adipose-derived mesenchymal stem cells. One such population is the stromal vascular fraction (SVF), which is comprised of adipose-derived mononuclear cells, pericytes, endothelial progenitor cells with several cell types, including mesenchymal stem cells, hematopoietic stem cells, regulatory T cells, and alternatively activated monocytes. This mixture conceptually may be a useful source of cells with both immune modulatory, and regenerative properties. Whereas mesenchymal stem cells are rare in bone marrow, comprising only 0.01-0.001% of bone marrow cells, these cells are found at 100-500-fold higher frequencies in the SVF. Cell quantification of SVF harvested from middle-aged humans revealed a range of approximately 400 000 cells / mL of aspirate of which approximately 20% of the cells are endothelial and hematopoietic cells. Additionally, the cell yield from SVF is highly dependent upon the site of adipose tissue collection and the methods used for enzymatic digestion. Since adipose-derived mesenchymal stem cells are located in the adipose perivascular niche, the extent of vascularization strongly influences the mesenchymal cell numbers that can be recovered from different subcutaneous body sites.

[0089] In some embodiments, a regenerative cell comprises a mesenchymal stem cell, wherein the mesenchymal stem cell comprises an immature cell that is derived from more immature cellular sources. In other embodiments, mesenchymal stem cells are dedifferentiated. Generation of dedifferentiated mesenchymal stem cells may be achieved using an HD AC inhibitor, for example, valproic acid, to endow a “younger” state onto MSC.

[0090] A hallmark of mesenchymal stem cells is their ability to counteract inflammatory responses. MSC are considered to be poorly immunogenic cells, exhibiting low expression levels of HLA class I and negligible expression of HLA class II or costimulatory molecules, although expression of HLA molecules is upregulated uponexposure of MSC to pro-inflammatory stimuli such as the cytokine IFN-y. MSC possess immune modulatory abilities that are mediated by direct contact with immune cells and / or through secretion of soluble factors such as prostaglandin E2 (PGE2), nitric oxide (NO), transforming growth factor (TGF-0), leukemia inhibitory factor (LIF), and indolamine 2.3 -dioxygenase (IDO). BMSCs inhibit the differentiation of dendritic cells from their precursors, inhibit B cell maturation, and stimulate the release of the immune suppressive cytokine IL-10 from tissue macrophages. MSC also demonstrate inhibitory effects against NK cells, which in turn exhibit a reduced ability to kill tumor cells in vitro.

[0091] In certain embodiments, a regenerative cell comprises a tissue mesenchymal stem cell derived form cord blood. Methods of deriving cord tissue mesenchymal stem cells from human umbilical tissue are provided. The cells are capable of self-renewal and expansion in culture, and have the potential to differentiate into cells of other phenotypes. The method comprises (a) obtaining human umbilical tissue; (b) removing substantially all of blood to yield a substantially blood-free umbilical tissue, (c) dissociating the tissue by mechanical or enzymatic treatment, or both, (d) resuspending the tissue in a culture medium, and (e) providing growth conditions which allow for the growth of a human umbilicus-derived cell capable of self-renewal and expansion in culture and having the potential to differentiate into cells of other phenotypes. Tissue can be obtained from any completed pregnancy, term or less than term, whether delivered vaginally, or through other routes, for example surgical Cesarean section. Obtaining tissue from tissue banks is also considered within the scope of the present invention. The tissue is rendered substantially free of blood by any means known in the art. For example, the blood can be physically removed by washing, rinsing, and diluting and the like, before or after bulk blood removal for example by suctioning or draining. Other means of obtaining a tissue substantially free of blood cells might include enzymatic or chemical treatment. Dissociation of the umbilical tissues can be accomplished by any of the various techniques known in the art, including by mechanical disruption, for example, tissue can be aseptically cut with scissors, or a scalpel, or such tissue can be otherwise minced, blended, ground, or homogenized in any manner that is compatible with recovering intact or viable cells from human tissue. In a presently preferred embodiment, the isolation procedure also utilizes an enzymatic digestion process. Many enzymes are known in the art to be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture. A broad range of digestive enzymes for use in cell isolationfrom tissue is available to the skilled artisan. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially. A nonexhaustive list of enzymes compatible herewith includes mucolytic enzyme activities, metalloproteases, neutral proteases, serine proteases (such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases. Presently preferred are enzyme activites selected from metalloproteases, neutral proteases and mucolytic activities. For example, collagenases are known to be useful for isolating various cells from tissues. Deoxyribonucleases can digest single-stranded DNA and can minimize cell-clumping during isolation. Enzymes can be used alone or in combination. Serine protease are preferably used in a sequence following the use of other enzymes as they may degrade the other enzymes being used. The temperature and time of contact with serine proteases must be monitored. Serine proteases may be inhibited with alpha 2 microglobulin in serum and therefore the medium used for digestion is preferably serum-free. EDTA and DNase are commonly used and may improve yields or efficiencies. Preferred methods involve enzymatic treatment with for example collagenase and dispase, or collagenase, dispase, and hyaluronidase, and such methods are provided wherein in certain preferred embodiments, a mixture of collagenase and the neutral protease dispase are used in the dissociating step. More preferred are those methods which employ digestion in the presence of at least one collagenase from Clostridium histolyticum, and either of the protease activities, dispase and thermolysin. Still more preferred are methods employing digestion with both collagenase and dispase enzyme activities. Also preferred are methods which include digestion with a hyaluronidase activity in addition to collagenase and dispase activities. The skilled artisan will appreciate that many such enzyme treatments are known in the art for isolating cells from various tissue sources. For example, the LIBERASE BLENDZYME (Roche) series of enzyme combinations of collagenase and neutral protease are very useful and may be used in the instant methods. Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources. The skilled artisan is also well-equipped to assess new, or additional enzymes or enzyme combinations for their utility in isolating the cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer. Diluting the digest may also improve yields of cells as cells may be trapped within a viscous digest. While the use of enzymes is preferred, it is not required for isolation methods as providedherein. Methods based on mechanical separation alone may be successful in isolating the instant cells from the umbilicus as discussed above.

[0092] The cells can be resuspended after the tissue is dissociated into any culture medium as discussed herein above. Cells may be resuspended following a centrifugation step to separate out the cells from tissue or other debris. Resuspension may involve mechanical methods of resuspending, or simply the addition of culture medium to the cells.

[0093] In order to properly generate mesenchymal stem cells, it is important that the growth conditions allows for a wide range of options as to culture medium, supplements, atmospheric conditions, and relative humidity for the cells. Presently preferred are methods which provide cells which require no exogenous growth factors, except as are available in the supplemental serum provided with the Growth Medium. Also provided herein are methods of deriving umbilical cells capable of expansion in the absence of particular growth factors. The methods are similar to the method above, however they require that the particular growth factors (for which the cells have no requirement) be absent in the culture medium in which the cells are ultimately resuspended and grown in. In this sense, the method is selective for those cells capable of division in the absence of the particular growth factors. Preferred cells in some embodiments are capable of growth and expansion in chemically-defined growth media with no serum added. In such cases, the cells may require certain growth factors, which can be added to the medium to support and sustain the cells. Presently preferred factors to be added for growth on serum-free media include one or more of FGF, EGF, IGF, and PDGF. In more preferred embodiments, two, three or all four of the factors are add to serum free or chemically defined media. In other embodiments, LIF is added to serum-free medium to support or improve growth of the cells.

[0094] Methods are provided wherein the cells can undergo at least 25, 30, 35, or 40 doublings prior to reaching a senescent state. Methods for deriving cells capable of doubling to reach 10. sup.14 cells or more are provided. Preferred are those methods which derive cells that can double sufficiently to produce at least about 10. sup.14, 10. sup.15, 10. sup.16, or 10. sup.17 or more cells when seeded at from about 10. sup.3 to about 10. sup.6 cells / cm.sup.2 in culture. Preferably these cell numbers are produced within 80, 70, or 60 days or less. In one embodiment, cord tissue mesenchymal stem cellsare isolated and expanded, and possess one or more markers selected from a group comprising of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-alpha, or HLA-A,B,C. In addition, the cells do not produce one or more of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP, DQ.

[0095] In one embodiment, the regenerative cells comprise mesenchymal stem cells that are generated according to protocols known to one of ordinary skill in the art for bone marrow derived mesenchymal stem cells. Specifically, bone marrow is aspirated (10-30 ml) under local anesthesia (with or without sedation) from the posterior iliac crest, collected into sodium heparin containing tubes and transferred to a Good Manufacturing Practices (GMP) clean room. Bone marrow cells are washed with a washing solution such as Dulbecco's phosphate-buffered saline (DPBS), RPMI, or PBS supplemented with autologous patient plasma and layered on to 25 ml of Percoll at a concentration of approximately 1-2 x 107cells / ml. Subsequently the cells are centrifuged at 900 g for approximately 30 min or a time period sufficient to achieve separation of mononuclear cells from debris and erythrocytes. The cells are then washed with PBS and plated at a density of approximately 1 x 106cells per mL in 175 cm2tissue culture flasks in DMEM with 10% FCS with flasks subsequently being loaded with a minimum of 30 million bone marrow mononuclear cells. The mesenchymal stem cells are allowed to adhere for 72 h followed by media changes every 3-4 days. Adherent cells are removed with 0.05% trypsin-EDTA and replated at a density of 1 x 106per 175 cm2. Said bone marrow MSC may be administered intravenously, or in a preferred embodiment, intrathecally in a patient suffering radiation associated neurodegenerative manifestations. Although doses may be determined by one of skill in the art, and are dependent on various patient characteristics, intravenous administration may be performed at concentrations ranging from 1-10 million MSC per kilogram, with a preferred dose of approximately 2-5 million cells per kilogram.

[0096] Exosomes from MSC may be generated from a mesenchymal stem cell conditioned medium (MSC-CM). Said exosomes are used in the context of the invention to reprogram immunocytes for tolerance induction ex vivo or in vivo. Said particle may be isolated for example by being separated from non-associated components based on any property of the particle. For example, the particle may be isolated based on molecular weight, size, shape, composition or biological activity. The conditioned medium may befiltered or concentrated or both during, prior to or subsequent to separation. For example, it may be filtered through a membrane, for example one with a size or molecular weight cut-off. It may be subject to tangential force filtration or ultrafiltration. With regard to a donor tissue, cell, graft or solid organ transplant in a recipient patient, it is believed that the method according to the invention may be effective in preventing acute rejection of such transplant in the recipient and / or for long-term maintenance therapy to prevent rejection of such transplant in the recipient (e.g., inhibiting rejection of insulin-producing islet cell transplant from a donor in the patient recipient suffering from diabetes). Thus, the method of the invention is useful for preventing Host-Versus-Graft-Disease (HVGD) and Graft- Versus-Host-Disease (GVHD). Typically, the method of the present invention is applied to the patient before and / or after transplantation. As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By a “therapeutically effective amount” is meant a sufficient amount of cells generated with the present invention for the treatment of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total usage of these cells will be decided by the attending physicians within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and survival rate of the cells employed; the duration of the treatment; drugs used in combination or coincidental with the administered cells; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of cells at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0097] The invention provides novel stem cell types, methods of manufacture, and therapeutic uses. Provided are means of deriving stem cells possessing regenerative, immune modulatory, anti-inflammatory, and angiogenic / neurogenic activity from umbilical cord tissue such as Wharton’s Jelly. In some embodiments manipulation of stem cell “potency” is disclosed through hypoxic manipulation, growth on non- xenogeneic conditions, as well as addition of epigenetic modulators.

[0098] Mesenchymal stem cells may be encapsulated by membranes, as well as capsules, prior to implantation. It is contemplated that any of the many methods of cell encapsulation available may be employed. In some embodiments, cells are individually encapsulated. In some embodiments, many cells are encapsulated within the same membrane. In embodiments in which the cells are to be removed following implantation, a relatively large size structure encapsulating many cells, such as within a single membrane, may provide a convenient means for retrieval. A wide variety of materials may be used in various embodiments for microencapsulation of reprogrammed immune cells. Such materials include, for example, polymer capsules, alginate-poly-L-lysine- alginate microcapsules, barium poly-L-lysine alginate capsules, barium alginate capsules, polyacrylonitrile / polyvinylchloride (PAN / PVC) hollow fibers, and polyethersulfone (PES) hollow fibers. Techniques for microencapsulation of cells that may be used for administration of reprogrammed immune cells are known to those of skill in the art. All of the foregoing are incorporated herein by reference in parts pertinent to encapsulation of Reprogrammed immune cells. The cells of the invention are cultured under hypoxia, in one embodiment, cultured to induce and / or augment expression of chemokine receptors. One such receptor is CXCR-4. The population of cells, including population of umbilical cord mesenchymal cells, may be enriched for CXCR-4, such as (or such as about) 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the population expressing CXCR-4, CD31, CD34, or any combination thereof. In addition, <1%, <2%, <3%, <4%, <5%, <6%, <7%, <8%, <9%, or <10% of the population of cells may express CD14 and / or CD45. The umbilical cord cells of the invention may further possess markers selected from the group consisting of STRO-1, CD 105, CD54, CD56, CD 106, HLA-I markers, vimentin, ASMA, collagen- 1, fibronectin, LFA-3, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b / CD29, CD49c / CD29, CD49d / CD29, CD61, CD 18, CD29, thrombomodulin, telomerase, CD 10, CD 13, STRO-2, VCAM-1, CD 146, and THY-1, and a combination thereof. In some embodiments said placental cells of theinvention are admixed with endothelial cells. Said endothelial cells may express one or more markers selected from the group consisting of: a) extracellular vimentin; b) CD133; c) c-kit; d) VEGF receptor; e) activated protein C receptor; and f) a combination thereof. In some embodiments, the population of endothelial cells comprises endothelial progenitor cells.

[0099] The population of cells may be allogeneic, autologous, or xenogenic to an individual, including an individual being administered the population of cells. In some embodiments, the population of cells are matched by mixed lymphocyte reaction matching.

[0100] In some embodiments, senolytic agents are utilized to enhance therapeutic activity of regenerative cells. Various regenerative cell types exist. In one embodiment said regenerative cells are stem cells, in a specific embodiment said stem cells are of the mesenchymal lineage. The population of mesenchymal stem cells may be derived from tissue selected from the group consisting of the placental body, placenta, umbilical cord tissue, peripheral blood, hair follicle, cord blood, Wharton's Jelly, menstrual blood, endometrium, skin, omentum, amniotic fluid [257-269], and a combination thereof. In some embodiments, the population of cells, the population of umbilical mesenchymal stem cells, or the population of endothelial cells comprises human umbilical cord derived adherent cells. The human umbilical cord derived adherent cells may express a cytokines selected from the group consisting of FGF-1, FGF-2, HGF, interleukin-1 receptor antagonist, and a combination thereof. In some embodiments, the population of cells, the population of umbilical cord cells express arginase, indoleamine 2,3 deoxygenase, interleukin- 10, and / or interleukin 35. In some embodiments, the population of cells, the population of umbilical cord cells, or the population of endothelial cells express hTERT and Oct-4 but does not express a STRO-1 marker.

[0101] The following preferred embodiments of the invention provides therapeutic compositions and methods of use thereof for treating diseases of aging that are characterized by inflammation, fibrosis, tissue dysfunction, and immune dysregulation.

[0102] In one embodiment, a method for treating an age-related disease or condition in a subject is provided, the method comprising the following steps: a) identifying a subject with an age-related disease or condition affecting at least one organ or tissue;b)administering at least one senolytic agent to the subject, wherein the at least one senolytic agent comprises an agent that induces inactivation or apoptosis of senescent cells; c) administering at least one regenerative cell to the subject, wherein the regenerative cell comprises at least one of a mesenchymal stem cell or a tissue-specific progenitor cell, and wherein the regenerative cell contributes to repairing or replacing the at least one organ or tissue; and d) optionally, administering one or a plurality of soluble factors from at least one regenerative cell to the subject, wherein the one or plurality of soluble factors contribute to repairing or replacing the at least one organ or tissue. In one embodiment, the at least one regenerative cell is derived from an induced pluripotent stem cell. In one embodiment, the induced pluripotent stem cell is derived from a somatic cell from the subject, wherein the somatic cell comprises 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.

[0103] In certain embodiments, at least one senolytic agent is selected from the group comprising dasatinib, quercetin, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-l,4-bis-TPP, a derivative of 2-butene-l,4-bis-TPP, 2-chlorobenzyl-TPP, a derivative of 2-chlorobenzyl-TPP, 3- methylbenzyl-TPP, a derivative of 3-methylbenzyl-TPP, 2,4-dichlorobenzyl-TPP, a derivative of 2,4-dichlorobenzyl-TPP, 1-naphthylmethyl-TPP, a derivative of 1- naphthylmethyl-TPP, p-xylylenebis-TPP, a derivative of p-xylylenebis-TPP, vitamin C, fisetin, berberine, caffeic acid phenyl ester, and silibinin.

[0104] In other embodiments, a senolytic agent comprises a dendritic cell vaccine, wherein the dendritic cell that is pulsed with one or a plurality of senescence-associated antigens. The senescence-associated antigens may comprise pl6, p21, or both, or other antigens associated with senescence. In one embodiment, the senescence-associated antigens are derived from a lysate of a senescent cell such as a fibroblast. In certain embodiments, the lysate is derived from a cell that is rendered senescent by exposure to one or plurality of stimuli or agents selected from the group comprising ionizing radiation, non-ionizing radiation, genotoxic drug(s), demethylating agent(s), acetylating agent(s), and hypoxia.

[0105] In one embodiment, a regenerative cell comprises a mesenchymal stem cell that is selected from the group comprising a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, a dental pulp mesenchymal stem cell, or an amniotic membrane mesenchymal stem. The mesenchymal stem cell may be autologous or allogeneic. In one embodiment, a regenerative cell comprises a tissuespecific progenitor cell that is selected from the group comprising a hematopoietic stem / progenitor cell, a neural progenitor cell, a mesenchymal stem / progenitor cell, a satellite cell, an osteoprogenitor cell, a chondroprogenitor cell, a tenocyte progenitor cell, a basal epithelial progenitor cell, a limbal stem / progenitor cell, an intestinal crypt progenitor cell, a prostate epithelial progenitor cell, a mammary gland progenitor cell, a hepatic progenitor cell, a bronchioalveolar stem / progenitor cell, an alveolar type I progenitor cell, an alveolar type II cell progenitor cell, a pancreatic progenitor cell, a renal progenitor cell, a ureteric bud progenitor cell, a cardiac progenitor cell, an endothelial progenitor cell, a smooth muscle progenitor cell, a hair follicle stem / progenitor cell, a sebaceous gland progenitor cell, a spermatogonial stem / progenitor cell, and an ovarian surface epithelium progenitor cell. The tissue-specific progenitor cell may be autologous or allogeneic.

[0106] In one embodiment, one or a plurality of different types of soluble factors are administered to a subject as sources of regenerative molecules, which may be administered in combination with a regenerative cell therapy or alone. In certain embodiments, the types of soluble factors are selected from the group comprising extracellular vesicles, exosomes, microvesicles, ectosomes, non-vesicular extracellular nanoparticles, apoptotic bodies, proteins, glycoproteins, and glycolipids. The soluble factors derived from regenerative cell comprise cargo from their originating cells and are therefore useful therapeutic compositions for diseases of aging.EXAMPLES

[0107] The following examples are not intended as limitations. Rather they demonstrate illustrative embodiments of the present invention.Example 1 : Combined Senolytic Immunotherapy using a Dendritic Cell Vaccine with Mesenchymal Stem Cells and Hepatic Progenitor Cells.

[0108] SENOVAX™ is a trade name for dendritic cells pulsed with senescent fibroblast lysate and were prepared as follows: Bone marrow mononuclear cells were isolated from BALB / c mice. Cells were extracted from femurs and tibia by isolation of bone marrow cords through flushing bones with saline. Cords were dissociated over a sterile mesh to remove debris or aggregates and single cells were diluted in 12 ml of phosphate buffered saline. Cells were spun for 15 minutes at 1000g. The supernatant was decanted and the pellet was resuspended by gentle tapping. 2 ml of lysis buffer (ThermoFisher, Carlsbad, CA) was added to the pellet for 3 minutes after which 10 ml of phosphate buffered saline was added. Cells were washed twice and subsequently mononuclear cells were plated in 20 ml of DMEM media with 10% fetal calf serum and GM-CSF (100 lU / ml) and IL-4 (100 lU / ml). Cells were incubated in a fully humidified atmosphere with 5 % carbon dioxide. Media was changed every second day. This generates CD80 and CD86 expressing dendritic cells. Concentration of cells was 100,000 per ml. Lysate of senescent dermal fibroblasts of BALB / c origin were used. The concentration added was 10 ug of lysate per million cells.

[0109] To generate a lysate comprising senescence-associated antigens for pulsing dendritic cells, BALB / c dermal fibroblasts were obtained by shaving a 1 x 1 centimeter portion of the mouse dermis and surgically removing the skin. The excised skin was subsequently cut into 1 x 1 mm pieces using surgical scissors and incubated in RPMI containing 100 microgram per ml collagenase for 20 minutes. Cells were first isolated by passing through the sterile mesh and subsequently washed in phosphate buffered saline. Adherent cells were growth for 2 weeks in the presence of 5 ug / ml of doxorubicin. Cells expressing more than 80% beta galactosidase were considered senescent and were then lysed by 10 freeze-thaw cycles in liquid nitrogen. Lysate was passed through a 2 micron filtrate to removed debris and used to pulse dendritic cells to provide a source of senescence-associated antigens.

[0110] PPC (personalized progenitor cells of hepatic origin were generated by differentiation of pluripotent stem cells (PSCs), PSCs were first cultured in a defined, feeder-free system using mTeSRl medium on Matrigel-coated plates to maintain pluripotency. Differentiation is initiated by transitioning to a basal medium supplemented with Activin A (100 ng / mL) and Wnt3a (50 ng / mL) for 3 days to induce definitive endoderm, followed by a 5-day culture in medium with BMP4 (10 ng / mL) and FGF2 (20ng / mL) to specify hepatic progenitors. Subsequently, cells are matured for 10-14 days in hepatocyte maturation medium containing HGF (20 ng / mL), Oncostatin M (10 ng / mL), and dexamethasone (100 nM), with daily medium changes. The resulting hepatocyte-like cells are characterized for functionality via albumin secretion, cytochrome P450 activity, and glycogen storage. To generate pMSC, PSCs were cultured in mTeSRl medium on Matrigel-coated plates to maintain pluripotency under feeder-free conditions.Differentiation was initiated by transitioning to a mesoderm induction medium containing DMEM / F12 supplemented with 10 pM SB431542, 20 ng / mL BMP4, and 30 ng / mL Activin A for 2-3 days. Subsequently, cells were cultured in MSC specification medium with 10 ng / mL FGF2, 10 ng / mL PDGF, and 2% FBS for 7-10 days to promote mesenchymal lineage commitment. The cells were then passaged and maintained in MSC maintenance medium (DMEM low glucose, 10% FBS, 1% penicillin-streptomycin) for an additional 7-14 days, with medium changes every 2-3 days. The resulting MSC-like cells were characterized for surface markers (CD73, CD90, CD105 positivity; CD34, CD45 negativity), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and plastic adherence, with all steps conducted at 37°C in a 5% CO2 incubator under sterile conditions.

[0111] A model of liver injury was used to test the effects of administration of a senolytic agent and regenerative cells in an animal model. Carbon tetrachloride (CCL4) was purchased from Sigma Aldrich (ThermoFisher) and dissolved in vegetable oil at 10% concentration and administered to animals by gavage at a dose of 0.28 mL / kg.

[0112] Non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice on the BALB / c genetic background were divided into the groups, with each group consisting of 12 mice per group. For the group that received the senolytic dendritic cell vaccine, NOD-SCID mice received purified IgG antibodies (2 micrograms per mouse) from wild-type BALB / c mice adoptively transferred.

[0113] The following treatment groups were evaluated:1. Control saline2. CCL43. CCL4 + PPC (“personalized progenitor cell” comprising a hepatic progenitor cell)4. CCL4 + pMSC (“personalized” mesenchymal stem cell)5. CCL4 + SENOVAX™ (Senolytic agent; dendritic cell vaccine against senescent cells)6. CCL4 + PPC + pMSC7. CCL4 + PPC + SENOVAX™8. CCL4 + pMSC + SENOVAX™9. CCL4 + pMSC + PPC + SENOVAX™

[0114] Animals received the experimental interventions comprising the SenoVax dendritic cell vaccine and the regenerative cell 24 hours after CCL4 administration. Cells were administered at a concentration of 500,000 cells per mouse. After the cellular transplantation, blood samples were collected at 24, 48, and 72 hours following administration of the cells, and liver function was assessed based on the levels of aspartate aminotransferase (AST) in blood, wherein higher concentrations of AST are associated with liver damage. The results are shown in Figure 1.

[0115] The results verify the liver damage incurred in the CLL4 model, based on elevated AST levels in the animals compared to controls. Both populations of regenerative cells, the hepatic progenitor cells and the mesenchymal stem cells, individually reduced the AST levels, but had a strong effect in reducing AST levels when the two cell types were administered to the mice in combination. Modulation of liver injury was evident in animals that received senolytic immunotherapy with SenoVax and one of the regenerative cell types. However, the most potent reversal and / or cessation of liver injury was observed by administration of the combination of senolytic immunotherapy with SenoVax combined with both mesenchymal stem cells and hepatogenic progenitor cells.

Claims

WHAT IS CLAIMED IS:

1. A method for treating an age-related disease or condition in a subject, the method comprising: a) identifying a subject with an age-related disease or condition affecting at least one organ or tissue; b) administering at least one senolytic agent to the subject, wherein the at least one senolytic agent comprises an agent that induces inactivation or apoptosis of senescent cells; c) administering at least one regenerative cell to the subject, wherein the regenerative cell comprises at least one of a mesenchymal stem cell or a tissue-specific progenitor cell, and wherein the regenerative cell contributes to repairing or replacing the at least one organ or tissue; and d) administering one or a plurality of soluble factors from at least one regenerative cell to the subject, wherein the one or plurality of soluble factors contribute to repairing or replacing the at least one organ or tissue.

2. The method of, Claim 1, wherein the at least one regenerative cell is derived from an induced pluripotent stem cell.

3. The method of Claim 2, wherein the induced pluripotent stem cell is derived from a somatic cell from the subject.

4. The method of Claim 3, wherein 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.

5. The method of Claim 1, wherein the at least one senolytic agent is selected from the group consisting of: dasatinib, quercetin, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-l,4-bis-TPP, a derivative of 2-butene-l,4-bis-TPP, 2- chlorobenzyl-TPP, a derivative of 2-chlorobenzyl-TPP, 3-methylbenzyl-TPP, a derivative of 3-methylbenzyl-TPP, 2,4-dichlorobenzyl-TPP, a derivative of 2,4- dichlorobenzyl-TPP, 1 -naphthylmethyl -TPP, a derivative of 1-naphthylmethyl- TPP, p-xylylenebis-TPP, a derivative of p-xylylenebis-TPP, vitamin C, fisetin, berberine, caffeic acid phenyl ester, and silibinin.

6. The method of Claim 1, wherein the senolytic agent comprises a dendritic cell vaccine.

7. The method of Claim 6, wherein the dendritic cell that is pulsed with one or a plurality of senescence-associated antigens.

8. The method of Claim 7, wherein the senescence-associated antigens comprise pl 6, p21, or both.

9. The method of Claim 7, wherein the senescence-associated antigens derived from a lysate of a senescent cell.

10. The method of Claim 9, wherein the cell is rendered senescent by exposure to one or plurality of stimuli or agents selected from the group consisting of: ionizing radiation, non-ionizing radiation, genotoxic drug(s), demethylating agent(s), acetylating agent(s), and hypoxia.

11. The method of Claim 9, wherein the senescent cell comprises a fibroblast.

12. The method of Claim 1, wherein the mesenchymal stem cell is selected from the group consisting of: a bone marrow-derived mesenchymal stem cell, adipose- derived mesenchymal stem cell, umbilical cord-derived mesenchymal stem cell, placenta-derived mesenchymal stem cell, dental pulp mesenchymal stem cell, and an amniotic membrane mesenchymal stem.

13. The method of Claim 1, wherein the mesenchymal stem cell is autologous.

14. The method of Claim 1, wherein the tissue-specific progenitor cell is selected from the group consisting of: a hematopoietic stem / progenitor cell, a neural progenitor cell, a mesenchymal stem / progenitor cell, a satellite cell, an osteoprogenitor cell, a chondroprogenitor cell, a tenocyte progenitor cell, a basal epithelial progenitor cell, a limbal stem / progenitor cell, an intestinal crypt progenitor cell, a prostate epithelial progenitor cell, a mammary gland progenitor cell, a hepatic progenitor cell, a bronchioalveolar stem / progenitor cell, an alveolar type I progenitor cell, an alveolar type II cell progenitor cell, a pancreatic progenitor cell, a renal progenitor cell, a ureteric bud progenitor cell, a cardiac progenitor cell, an endothelial progenitor cell, a smooth muscle progenitor cell, a hair follicle stem / progenitor cell, a sebaceous gland progenitor cell, a spermatogonial stem / progenitor cell, and an ovarian surface epithelium progenitor cell.

15. The method of Claim 1, wherein the tissue-specific progenitor cell is autologous.

16. The method of Claim 1, wherein the one or plurality of soluble factors are selected from the group consisting of: an extracellular vesicle, an exosome, a microvesicle, an ectosome, a non-vesicular extracellular nanoparticle, an apoptotic body, a protein, a glycoprotein, and a glycolipid.

17. The method of Claim 1, wherein the senolytic agent is provided to suppress or reduce the Senescence- Associated Secretory Phenotype (SASP) in the organ or tissue.

18. The method of Claim 17, wherein the SASP comprises one or a plurality of cytokines selected from the group consisting of: IL-la, IL-ip, IL-6, IL-8, IL-10, IL-13, IL-15, IL-18, IL-33, TNF-a, GM-CSF, and IFN-y.

19. The method of Claim 1, wherein the regenerative cell is provided to increase the concentrations of anti-inflammatory and regenerative molecules in the organ or tissue.

20. The method of Claim 19, wherein the anti-inflammatory and regenerative molecules are selected from the group consisting of: IL-10, IL-13, IL-20, IL-22, IL-35, TGF-P, angiopoietin, endoglin, soluble HLA-G, soluble TNF- a receptor p55, soluble TNF- a receptor p75, soluble arginase, indolamine 2,3 dioxygenase, kynurenine, NGF, BDNF, CNTF, PGE-2, bcl-2, livin, survivin, membrane bound TGF-P, membrane bound PD-L1, membrane bound PD-L2, FoxP3, soluble PD- Ll, and soluble PD-L2.

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