Natural antibody-mediated clearing of senescent cells

Abstract

Methods and compositions of useful for removal and / or enhancing removal of senescent cells are provided. Methods of administration of premade antibody mixtures, such as intravenous immunoglobulin (IVIG) to stimulate clearance of senescent cells. IVIG is utilized to enhance efficacy of senolytic agents, whether they be small molecule, nucleic acids, or immunotherapy-based approaches. In another embodiment natural antibody producing cells, such as B1 cells are administered to a patient in which enhance clearance of senescent cells is desired.

Description

NATURAL ANTIBODY-MEDIATED CLEARING OF SENESCENT CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from United States Provisional Application No. 63 / 677,338, filed July 30, 2024, entitled NATURAL ANTIBODY-MEDIATED CLEARING OF SENESCENT CELLS, the entire contents of which are hereby expressly incorporated by reference.FIELD OF INVENTION

[0002] The invention pertains to the fields of longevity and regenerative medicine, more particularly the invention pertains to senolytic therapies for targeting cells related to disease and aging.BACKGROUND

[0003] Natural antibodies are mostly IgM, polyreactive, and are generally encoded by V genes in germline configuration. In contrast to adaptive antibodies, natural antibodies are present in a non-immunized organism from birth, and they do not include anti-Gal antibodies and / or anti -Gal natural antibodies, which are developed as a result of the effect of the a-Gal epitope and physiological flora. Natural antibodies are the first line of the organism's defense before the formation of the immunity created via the stimulation of elements that determine specific and non-specific immunity. This is especially important in the case of infants. Despite the fact that natural antibodies differ in their function from adaptive antibodies, they are polyreactive and they detect autoantigens and new antigenic determinants. Natural antibodies are formed from the subpopulation of B lymphocytes, mainly Bl lymphocytes and B lymphocytes of the marginal zone. This phenomenon is supported by the fact that when the quantity of these cells in the organism decreases, which happens with age, the level of natural antibodies also decreases and the risk of illnesses of old age becomes higher. During ontogenesis, these antibodies participate in many physiological processes, including the "support" of the immune system and homeostasis, the prevention of inflammation, infections and other pathological states, such as autoimmune and cardiovascular diseases, or the process of carcinogenesis. The best known natural antibody is IgM, but the role of IgGs and IgAs is also considered important. Nowadays, many researchers also mention intravenous immunoglobulins, which are used in the treatment of numerous illnesses, and there arediscussions on the possibility of increasing their potential if they were based on natural antibodies.

[0004] The use of natural antibodies in the area of therapeutics has been limited. Some experimental studies have assessed natural antibodies in the therapeutic arena of anti-infectives, however widespread use has not occurred. The current invention provides a novel therapeutic use for natural antibodies in the context of senolysis.SUMMARY

[0005] Embodiments herein are directed to methods of providing senolytic therapy to a subject in need thereof. In certain embodiments, therapy using regenerative cells is provided in combination with natural antibodies having activity in promoting the clearance of senescent cells from a subject with an age-related disease or condition.

[0006] In one embodiment, a method for treating a subject with an age-related disease or condition is provided, the method comprising: a) identifying a subject with an age-related disease or condition; b) administering a bone marrow-derived mononuclear cell population to the subject; wherein the bone marrow-derived mononuclear cells have at least one regenerative effect in the subject; and c) administering natural antibodies to the subject, wherein the natural antibodies reduce the quantities of senescent cells in the subject, and wherein the natural antibodies enhance the regenerative effect of the bone marrow-derived mononuclear cells. In certain embodiments, the regenerative effect of bone marrow mononuclear cells comprises immune modulation. In certain embodiments, immune modulation by bone marrow-derived mononuclear cells is related to expression of one or a plurality of molecules selected from the group comprising IL-10, TGF-P, IL- IRa, hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), HLA-G, and indoleamine 2,3 dioxygenase (IDO). In one embodiment, the bone marrow-derived mononuclear cells are selected from the group comprising hematopoietic stem cells, mesenchymal stem / stromal cells, monocytes, macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells, endothelial progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, and plasma cells. In a specific embodiment, the bone marrow-derived mononuclear cells comprise tissue-specific progenitor cells, wherein the tissue-specific progenitor cells can be induced to differentiate into at least one tissue selected from a 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. In one embodiment, the natural antibodies comprise polyclonal IgM antibodies. In one embodiment, the natural antibodies comprise intravenous immunoglobulin, wherein the intravenous immunoglobulin comprises polyclonal IgG. In one embodiment, the natural antibodies are produced by B-l cells. In other embodiments, the natural antibodies are administered to a subject in the form of antibody-producing cells. Embodiments of the invention provide natural antibodies that target senescent cells, wherein the senescent cells can be identified based on expression of antigens such as pl 6, p21, or both. In one embodiment, the natural antibodies elicit antibody-dependent cell cytotoxicity against senescent cells. In one embodiment, the natural antibodies elicit complement activation against senescent cells.

[0007] In one embodiment, the methods of the invention further comprise administering a senolytic agent to the subject in addition to a regenerative cell population and natural antibodies. In one embodiment, a senolytic agent is selected from the group comprising dasatinib, quercetin, fisetin, navitoclax (ABT-263), UBX1325, UBX0101, FOXO4-DRI, AZD8055, piperlongumine, curcumin analogs, resveratrol, epigallocatechin gallate (EGCG), proxofim, galacto-conjugated prodrugs, senolytic CAR T cells, and dendritic cells expressing one or a plurality of antigens derived from senescent cells.

[0008] In certain embodiments, the methods of the invention are applied for treatment or prevention of a age-related disease or condition selected from the group comprising Alzheimer’s disease, Parkinson’s disease, osteoarthritis, osteoporosis, age- related macular degeneration, cataracts, presbycusis (age-related hearing loss), presbyopia, sarcopenia, atherosclerosis, hypertension, cardiovascular disease, type 2 diabetes, chronic kidney disease, heart failure, stroke, atrial fibrillation, chronic obstructive pulmonary disease, benign prostatic hyperplasia, urinary incontinence, frailty syndrome, immune senescence, anemia of aging, depression, insomnia, glaucoma, degenerative disc disease, and cancer. In one embodiment, the age-related disease or condition involves ischemia in an organ or tissue. In one embodiment, the age-related disease or condition involving ischemia is selected from the group comprising peripheral artery disease, critical limb ischemia, diabetic foot ulcer, and chronic limb-threateningischemia, atherosclerosis-related limb ischemia, Buerger's disease, post-surgical limb ischemia, traumatic limb ischemia, and ischemia-reperfusion injury. In certain embodiments, the disease or condition involving ischemia is reduced or ameliorated by clearance of senescent cells causing occlusion of blood vessels.

[0009] Use of bone marrow-derived mononuclear cells having at least one regenerative effect in combination with natural antibodies from a subject wherein the natural antibodies enhance the regenerative effect of the bone marrow-derived mononuclear cells in the manufacture of a medicament for an age-related disease or condition in said subject.

[0010] Various aspects of the invention are enumerated in the following paragraphs:

[0011] Aspect 1 : A method of reducing the amount and / or activity of senescent cells in a subject comprising administering a concentration of natural antibody capable of stimulating clearance of said senescent cells.

[0012] Aspect 2: The method of Aspect 1, wherein said natural antibody comprises an IgM isotype or an IgG isotype.

[0013] Aspect 3: The method of Aspect 1, wherein said natural antibody is produced by a Bl cell.

[0014] Aspect 4: The method of Aspect 3, wherein said Bl cell expresses at least one marker selected from the group comprising CD5, CXCR4, autocrine interleukin- 10, IL-7 receptor, and IL-3 receptor.

[0015] Aspect 5: The method of Aspect 1, wherein said natural antibody is a component of intravenous immunoglobulin.

[0016] Aspect 6: The method of Aspect 1, wherein said natural antibody comprises purified IgM antibody.

[0017] Aspect 7: The method of Aspect 1, wherein said natural antibody is administered in the form of an antibody-producing cell.

[0018] Aspect 8: The method of Aspect 7, wherein said antibody-producing cell is isolated and expanded from natural sources.

[0019] Aspect 9: The method of Aspect 7, wherein said antibody-producing cell is artificially generated to produce said natural antibody.

[0020] Aspect 10: The method of Aspect 1, wherein the senescent cells are reduced by natural antibodies that are capable of activating complement.

[0021] Aspect 11 : The method of Aspect 1, wherein the senescent cells are reduced by natural antibodies that are capable of activating antibody-mediated cellular cytotoxicity (ADCC).

[0022] Aspect 12: The method of Aspect 11, wherein ADCC is mediated by monocytes or macrophages.

[0023] Aspect 13: The method of Aspect 12, wherein the monocytes comprise Ml monocytes, or classically activated monocytes that are associated with proinflammatory immune responses.

[0024] Aspect 14: The method of Aspect 12, wherein the macrophages comprise Ml macrophages, or classically activated macrophages that are associated with proinflammatory immune responses.

[0025] Aspect 15: The method of Aspect 12, wherein the monocytes or macrophages produce one or a plurality of factors or molecules selected from the group comprising nitric oxide, IL-1, IL-12, IL-15, IL-18, IL-21, IL-23, and IL-27.

[0026] Aspect 16: The method of 15, wherein the monocytes or macrophages produce higher levels or concentrations of the one or plurality of factors or molecules in comparison to naturally occurring monocytes or macrophages.

[0027] Aspect 17: The method of Aspect 11, wherein ADCC is mediated by neutrophils.

[0028] Aspect 18: The method of Aspect 17, wherein the neutrophils comprise N1 neutrophils, comprising a pro-inflammatory and / or anti-tumor neutrophil.

[0029] Aspect 19: The method of Aspect 17, wherein the neutrophils derive from peripheral blood.

[0030] Aspect 20: The method of Aspect 19, wherein the peripheral blood neutrophils are derived from mobilization of the neutrophils or neutrophil progenitors.

[0031] Aspect 21 : The method of Aspect 20, wherein mobilization is accomplished by one or a plurality of agents that evoke exit of neutrophil progenitors from an isolated niche.

[0032] Aspect 22: The method of Aspect 21, wherein the isolated niche comprises bone marrow.

[0033] Aspect 23 : The method of Aspect 21, wherein the mobilization agent is selected from the group comprising G-CSF, GM-CSF, M-CSF, FLT-3 ligand, poly(I:C), beta glucan, HMGB1, angiopoietin, and a CXCR4 blocking agent.

[0034] Aspect 24: The method of Aspect 11, wherein ADCC is mediated by natural killer (NK) cells

[0035] Aspect 25: The method of Aspect 24, wherein the NK cells are derived from a tissue or source selected from the group comprising peripheral blood, mobilized peripheral blood, bone marrow, menstrual blood, lymphatic tissue, umbilical cord blood, tonsillar tissue, omental tissue, pluripotent stem cells, or induced pluripotent stem cells.

[0036] Aspect 26: The method of Aspect 25, wherein the induced pluripotent stem cells comprise personalized regenerative cells.

[0037] Aspect 27: The method of Aspect 24, wherein the NK cells express one or a plurality of markers selected from the group comprising CD 16, CD56, CD57, NKPR1, a killer inhibitory receptor (KIR), perforin, granzyme, interferon gamma. IL-7, IL- 12, IL- 15, IL-17, IL-18, TIM-3, IL-12 receptor, IL-15 receptor, IL-18 receptor, and TRANCE.

[0038] Aspect 28: The method of Aspect 1, wherein said natural antibodies are administered to the subject in combination with one or a plurality of senolytic agents or agents capable of inactivating senescent cells.

[0039] Aspect 29: The method of Aspect 28, wherein the one or plurality of senolytic agents are selected from the group comprising dasatinib, quercetin, bcl-2, bcl- xL, and tetracycline.

[0040] Aspect 30: The method of Aspect 28, wherein said senescent cell is inactivated by treatment with one or a plurality of the following agents comprising 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 derivate of p-xylylenebis-TPP, Vitamin C, fisetin, berberine, caffeic acid phenyl ester, silibinin, and brutieridin.

[0041] Aspect 31 : A method of enhancing regenerative processes in a subject, said method comprising administration of natural antibodies at a concentration and frequency sufficient to reduce concentration of senescent cells, alternatively said method including administration of a regenerative cell together with administration of said natural antibodies.

[0042] Aspect 32: The method of Aspect 31, wherein the natural antibodies are collected from pooled human plasma.

[0043] Aspect 33 : The method of Aspect 31, wherein the natural antibodies are collected from pooled cord blood plasma.

[0044] Aspect 34: The method of Aspect 31, wherein the natural antibodies are collected from pooled menstrual blood plasma.

[0045] Aspect 35: The method of Aspect 31, wherein the natural antibodies are collected from IVIG.

[0046] Aspect 36: The method of Aspect 31, wherein the natural antibodies are screened in a manner to identify which epitopes on said senescent cells are being recognized by the natural antibodies.

[0047] Aspect 37: The method of Aspect 36, wherein the screening is performed by using existing natural antibodies to pull down antigens found on senescent cells.

[0048] Aspect 38: The method of Aspect 37, wherein the antigens found on the senescent cells are sequenced.

[0049] Aspect 39: The method of Aspect 36, wherein the epitopes are utilized to generate senescence-specific immunotherapy.

[0050] Aspect 40: The method of Aspect 39, wherein the senescence-specific immunotherapy is comprised of one or a plurality of senescence cell-associated epitopes and an immunological adjuvant.

[0051] Aspect 41 : The method of Aspect 40, wherein the immunological adjuvant is selected from the group comprising a toll-like receptor (TLR) agonist, a STING agonist, or a MDA5 agonist.

[0052] Aspect 42: The method of Aspect 41, wherein the STING agonist comprises 8803.

[0053] Aspect 43 : The method of Aspect 41, wherein the TLR agonist is selected from the group comprising poly I:C, poly L:C, poly ICIC, beta Glucan, unmethylated CpG DNA, free histones, dinitrochlorobenzene, tri nitrochlorobenzene, double- stranded RNA, lipopolysaccharide, HMGB1, ozonized plasma, cytoplasmic extract from heat stressed cells, and an agonistic antibody.

[0054] Aspect 44: The method of Aspect 31, wherein the regenerative cell comprises or is derived from a pluripotent stem cell.

[0055] Aspect 45: The method of Aspect 44, wherein the pluripotent stem cell comprises an induced pluripotent stem cell or a parthenogenic derived pluripotent stem cell.

[0056] Aspect 46: The method of Aspect 44, wherein the pluripotent stem cell comprises a dedifferentiated somatic cell.

[0057] Aspect 47: The method of Aspect 31, wherein the regenerative cell comprises a tissue-specific progenitor cell.

[0058] Aspect 48: The method of Aspect 47, wherein the tissue-specific progenitor cell is capable of generating endoderm, ectoderm, and / or mesoderm tissue.

[0059] Aspect 49: The method of Aspect 47, wherein the tissue-specific progenitor cell is capable of differentiating into one or a plurality of tissues selected 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.

[0060] Aspect 50: The method of Aspect 46, wherein the dedifferentiated somatic cell comprises a dedifferentiated monocyte.

[0061] Aspect 51 : The method of Aspect 50, wherein the monocyte is dedifferentiated by exposure to conditioned media from induced pluripotent stem cells.

[0062] Aspect 52: The method of Aspect 51, wherein the conditioned media is generated by culture of induced pluripotent stem cells in the form of embryoid bodies in a liquid media.

[0063] Aspect 53: The method of Aspect 52, wherein the liquid media is selected from the group comprising Iscove’s media, DMEM media, OptiMEM media, EMEM media, RPMI-1640 media, and AIM-V media.

[0064] Aspect 54: The method of Aspect 52, wherein the embryoid body is disassembled once every 2 days, once every 5 days, or once every 10 days.

[0065] Aspect 55: The method of Aspect 52, wherein the embryoid body is comprised of induced pluripotent stem cells together with monocytes.

[0066] Aspect 56: The method of Aspect 55, wherein the monocytes are first treated with a stressor prior to being admixed with induced pluripotent stem cells for generating conditioned media.

[0067] Aspect 57: The method of Aspect 56, wherein the stressor is selected from the group comprising hypoxia, hypertonicity, hypotonicity, hyperthermia, serum starvation, mTOR inhibition, and AMPK activation.

[0068] Aspect 58: The method of Aspect 56, wherein the stressor comprises an agent or molecule that activates one or a plurality of inflammatory pathways.

[0069] Aspect 59: The method of Aspect 58, wherein the inflammatory pathway is selected from the group comprising MAP kinase, Janus Activated Kinase, Signal Transducer and Activator of Transcription-3, Signal Transducer and Activator of Transcription-5, Signal Transducer and Activator of Transcription-6, TLR2, TLR3, and TLR4.

[0070] Aspect 60: The method of Aspect 59, wherein TLR2 is activated by peptidoglycan.

[0071] Aspect 61 : The method of Aspect 59, wherein TLR3 is activated by one or a plurality of agents selected from the group comprising double stranded RNA, poly IC, poly L:C, poly ICIC, or inactivated influenza virus.

[0072] Aspect 62: The method of Aspect 59, wherein TLR4 is activated by one or a plurality of agents selected from the group comprising neutrophil extracellular traps, beta glucan, HMGB1, lipopolysaccharide, yeast cell wall extract, and an anti-TLR4 antibody.

[0073] Aspect 63 : The method of Aspect 51, wherein the induced pluripotent stem cell is engineered to express an inhibitor of TGF-beta.

[0074] Aspect 64: The method of Aspect 63, wherein the inhibitor of TGF-beta is an antibody molecule, a cameloid antibody, a microbody, an aptamer, or a molecule that induces RNA interference.

[0075] Aspect 65: The method of Aspect 64, wherein the molecule that induces RNA interference comprises a short hairpin RNA, a short interfering RNA, a microRNA, or a long noncoding RNA.

[0076] Aspect 66: A method of treating degenerative disc disease in a subject, the method comprising administration of natural antibodies in the periphery of the degenerating disc at a concentration and frequency sufficient to reduce the senescent cell concentration in the periphery of said degenerating disc.

[0077] Aspect 67: The method of Aspect 66, wherein the natural antibodies are administered to remove senescent cells causing occlusion of blood vessels feeding the area surrounding the nucleus pulposus.

[0078] Aspect 68: The method of Aspect 66, wherein the natural antibodies are administered to remove senescent cells causing ischemia in the area surrounding the nucleus pulposus.

[0079] Aspect 69: The method of Aspect 66, wherein the natural antibodies are administered in combination with a regenerative cell population.

[0080] Aspect 70: The method of Aspect 69, wherein the regenerative cell population comprises autologous bone marrow mononuclear cells.

[0081] Aspect 71 : The method of Aspect 70, wherein the autologous bone marrow mononuclear cells are primed with one or a plurality of cytokines selected from the group comprising IL-beta, HGF, IGF, FGF-1, and FGF-2 prior to administration.

[0082] Aspect 72: The method of Aspect 71, wherein the cytokines are administered to the bone marrow mononuclear cells at a concentration and time frame sufficient to induce migration of the mononuclear cells toward an SDF-1 chemotactic gradient, a VEGF-chemotactic gradient, or both.

[0083] Aspect 73 : The method of Aspect 72, wherein the migration of the bone marrow mononuclear cells is at least 25%, at least 50%, or at least 100% increased as compared to migration of non-cytokine-treated bone marrow mononuclear cells.

[0084] Aspect 74: The method of Aspect 70, wherein the autologous bone marrow mononuclear cells are primed with one or a plurality of cytokines selected from the group comprising IL-35, HGF, IGF, FGF, FGF-2, GDF-11, GDF-15, VEGF-C, BlysS, BDNF, NGF, and IGFBP prior to administration.

[0085] Aspect 75: The method of Aspect 74, wherein the cytokines are administered to the bone marrow mononuclear cells at a concentration and time frame sufficient to induce migration of the mononuclear cells toward an SDF-1 chemotactic gradient, a VEGF chemotactic gradient, or both.

[0086] Aspect 76: The method of Aspect 75, wherein the migration of the bone marrow mononuclear cells is at least 25%, at least 50%, or at least 100% increased as compared to migration of non-cytokine-treated bone marrow mononuclear cells.

[0087] Aspect 77: The method of Aspect 69, wherein the regenerative cells comprise autologous very small embryonic like stem cells.

[0088] Aspect 78: The method of Aspect 69, wherein the regenerative cells comprise alternatively activated macrophages.

[0089] Aspect 79: The method of Aspect 78, wherein the alternatively activated macrophages express one or a plurality of molecules selected from the list comprising CD56, YAP1, CD14, CD16, TREM1, IL-4 receptor, IL-3 receptor, IL-10 receptor, IL-13 receptor, TGF-beta receptor, and LIF receptor.

[0090] Aspect 80: The method of Aspect 78, wherein the alternatively activated macrophages are generated by culture in one or a plurality of cytokines selected from the group comprising IL-4, IL-10, IL-13, IL-20, VEGF, and VEGF-C.

[0091] Aspect 81 : The method of Aspect 78, wherein the alternatively activated macrophages are generated by culturing monocytes in the presence of regulatory T cells.

[0092] Aspect 82: The method of Aspect 81, wherein the regulatory T cells express amphiregulin.

[0093] Aspect 83: The method of Aspect 81, wherein the regulatory T cells are treated with one or a plurality of agents comprising anti-CD3 antibody, IL-1, IL-2, IL-35, TGF-beta, endoglin, Klotho, HGF, prolactin, human chorionic gonadotropin, anti-LAP antibody, IL- 10

[0094] Aspect 84: The method of Aspect 81, wherein the regulatory T cells are treated with IVIG.

[0095] Aspect 84: The method of Aspect 78, wherein the alternatively activated macrophages express one or a plurality of molecules or factors selected from the group comprising arginase, IL- 10, IL-1 receptor antagonist, HLA-G, and indoleamine 2,3 dioxygenase (IDO).

[0096] Aspect 85: The method of Aspect 84, wherein the one or plurality of molecules or factors are expressed at higher levels by alternatively activated macrophages in comparisons to naive macrophages.

[0097] Aspect 86: The method of Aspect 78, wherein the alternatively activated macrophages are compared to naive macrophages, and wherein the alternatively activated macrophages express lower levels of one or a plurality of molecules selected from the group comprising nitric oxide synthase, perforin, granzyme, TNF-alpha, lymphotoxin, IL- Ibeta, IL-2, IL-6, IL-8, IL-8, IL-11, IL-12 IL-15, IL-17, IL-21, IL-22, and IL-27.

[0098] Aspect 87: The method of Aspect 86, wherein the alternatively activated macrophages are compared to the naive macrophages upon TRL4 stimulation.

[0099] Aspect 88: The method of Aspect 78, wherein the alternatively activated macrophages comprise M2 macrophages.

[0100] Aspect 89: The method of Aspect 78, wherein the alternatively activated macrophages are generated from cord blood monocytes, peripheral blood monocytes, mobilized peripheral blood monocytes, bone marrow-derived monocytes, monocytic progenitor cells, or hematopoietic stem cells.

[0101] Aspect 90: The method of Aspect 89, wherein the hematopoietic stem cells express one or a plurality of molecules selected from the group comprising CD34, CD33, CD 133, c-kit, CXCR4, c-met, jagged, and NOTCH.

[0102] Aspect 91 : The method of Aspect 69, wherein the regenerative cell population comprises bone marrow mononuclear cells.

[0103] Aspect 92: The method of Aspect 91, wherein the bone marrow mononuclear cell is pretreated under conditions of hypoxia.

[0104] Aspect 93 : The method of Aspect 92, wherein hypoxia is utilized to induce nuclear translocation of HIF-lalpha in the bone marrow mononuclear cells.

[0105] Aspect 94: The method of Aspect 92, wherein hypoxia activates transcription of one or a plurality of the following molecules selected from the group comprising CXCR4, IL- 10, HLA-G, and EGF.

[0106] Aspect 95: The method of Aspect 94, wherein hypoxia activates transcription of the one or plurality of molecules by at least 10%, at least 25%, or at least 50% higher levels than culturing the same cells under normoxic conditions.

[0107] Aspect 96: The method of Aspect 69, wherein the regenerative cell population comprises myeloid derived suppressor cells.

[0108] Aspect 97: The method of Aspect 96, wherein the myeloid-derived suppressor cell is derived from a granulocytic or a monocytic lineage.

[0109] Aspect 98: The method of Aspect 69, wherein the regenerative cell population comprises mesenchymal stem cells.

[0110] Aspect 99: The method of Aspect 98, wherein the mesenchymal stem cells are purified from cord blood mononuclear cells.

[0111] Aspect 100: The method of Aspect 99, wherein the cord blood mononuclear cells are treated with one or a plurality of molecules selected from the group comprising CCL2, MIP-1 alpha, MIP-1 beta, IL-1 beta, IFN-alpha, IFN-beta, HMGB1, and beta glucan.

[0112] Aspect 101 : The method of Aspect 99, wherein the cord blood mononuclear cells are treated with one or a plurality of agents or stimuli selected from the group comprising unmethylated CpG DNA, zymosan, lipopolysaccharide, BCG, heat shock protein, calreticulin, histone, allogenic T cells, and monocyte conditioned media.

[0113] Aspect 102: The method of Aspect 101, wherein monocyte conditioned media is obtained by extracting liquid media in which monocytes have been activated.

[0114] Aspect 103: The method of Aspect 102, wherein the monocytes have been activated by agonism of a danger-associated receptor.

[0115] Aspect 104: The method of Aspect 103, wherein the danger-associated receptor is selected from the group comprising a toll like receptor, a sting receptor, and an MDA5 receptor.

[0116] Aspect 105: The method of Aspect 102, wherein the monocytes are activated by exposure to a molecule stimulating signal transduction via one or a plurality of pathways selected from the group comprising TLR3, TLR4,TLR7, TLR8, and TLR9.

[0117] Aspect 106: The method of Aspect 105, wherein the molecule comprises a small molecule, a peptide, a peptoid, a nucleic acid, a cameloid antibody, a microbody, an aptamer, or a somamer.

[0118] Aspect 107: The method of Aspect 102, wherein the monocytes are activated by exposure to T cells.

[0119] Aspect 108: The method of Aspect 107, wherein the T cells are autologous, allogeneic, or xenogeneic.

[0120] Aspect 109: The method of Aspect 107, wherein the T cells are activated before incubation with the monocytes.

[0121] Aspect 110: The method of Aspect 109, wherein T cell activation is achieved by culture with allogeneic cells selected from the cell types comprising T cells, B cells, antigen presenting cells, endothelial cells, artificial antigen presenting cells, fibroblasts, or cytokine-activated fibroblasts.

[0122] Aspect 111 : The method of Aspect 110, wherein the allogeneic cells are activated using IFN-gamma, TNF-alpha, or both.

[0123] Aspect 112: The method of Aspect 111, wherein the allogeneic cells are induced to upregulate the expression of one or a plurality of antigens or molecules selected from the group comprising HLA I, HLA II, CD80, CD86, ICAM-1, LFA-1,

[0124] Aspect 113: The method of Aspect 109, wherein T cell activation is achieved by stimulation of the T cells with one or a plurality of the following agents selected fromthe group comprising anti-CD3 antibody, anti-CD28 antibody, anti-CD7 antibody, and anti-CD25 antibody.

[0125] Aspect 114: The method of Aspect 98, wherein the mesenchymal stem cells are purified based on expression of a marker associated with therapeutic activity.

[0126] Aspect 115: The method of Aspect 114, wherein the marker associated with therapeutic activity is selected from the group comprising IL-3 receptor, IL-7 receptor, IL- 11 receptor, IL- 12 receptor, IL- 15 receptor, IL- 17 receptor, IL- 18 receptor, IL-20 receptor, IL-22 receptor, IL-23 receptor, IL-25 receptor, IL-27 receptor, IL-35 receptor, leukemia inhibitory factor receptor, c-met, c-kit, aldehyde dehydrogenase, ABC drug efflux pump, extracellular vimentin, Brother of the Regulator of Imprinted Sites, extracellular calreticulin, or extracellular histone or histone proteins.

[0127] Aspect 116: The method of Aspect 66, wherein the natural antibodies are administered by the intra-discal route to reduce senescent cell concentrations.

[0128] Aspect 117: The method of Aspect 116, wherein one or a plurality of senolytic agents are administered by the intra-discal route alone or in combination with the natural antibodies.

[0129] Aspect 118: The method of Aspect 66, wherein the regenerative cells are administered by the intra-discal route.

[0130] Aspect 119: The method of Aspect 66, wherein the method further comprises administration of one or a plurality of anti-inflammatory agents.

[0131] Aspect 120: The method of Aspect 119, wherein the one or plurality of antiinflammatory agents are selected from the group comprising a COX-1 inhibitor, a COX-2 inhibitor, an IL-1 receptor antagonist, IL-3, IL-10, IL-35, IL-4, endoglin, TGF-beta, oxytocin, CAMPATH, CNTF, erythropoietin, high molecular weight hyaluronic acid, and GC-MAF.

[0132] Aspect 121 : A method of treating limb ischemia in a subject, the method comprising administration of an angiogenic cell combined with administration of natural antibodies.

[0133] Aspect 122: The method of Aspect 121, wherein the natural antibodies are administered in the form of IVIG.

[0134] Aspect 123 : A method for enhancing the efficacy of immunotherapy by reducing senescent cells through administration of natural antibodies.DETAILED DESCRIPTION

[0135] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0136] The invention provides means of inducing and / or augmenting senolytic activities in a mammal through administration of natural antibodies alone, and / or augmenting agents that increase ability of natural antibodies to inactivate and / or clear senescent cells.

[0137] When describing an absolute value of a characteristic or property of a thing or act described herein, the terms “substantial,” “substantially,” “essentially,” “approximately,” “about” and / or other terms or phrases of degree may be used without the specific recitation of a numerical range. When applied to a characteristic or property of a thing or act described herein, these terms refer to a range of the characteristic or property that is consistent with providing a desired function associated with that characteristic or property.

[0138] As used herein, the terms “subject” or “patient” refers to any mammal, preferably a human.

[0139] As used herein, "administering" or “administration” 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. The phrase "parenteral administration" as used herein means modes of 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, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the 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.

[0140] As used herein, "antibody," as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab).sub.2, as well as single chain antibodies and humanized antibodies. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0141] The term "immunoglobulin" or "Ig," as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0142] As used herein, the term “natural antibody” refers to a germline-encoded antibody that is found in a subject without known prior exposure to an antigen. Natural antibodies are polyreactive. In terms of their composition, natural antibodies may predominantly comprise IgM antibodies but may also comprise IgG and IgA isotypes. In certain embodiments, a natural antibody comprises a purified IgM antibody, a purified IgG antibody, or a purified IgA antibody. Natural antibodies may bind to a variety of antigens including self-antigens, microbial antigens, damaged cells, apoptotic cells, or other foreign antigens. In one embodiment, a natural antibody binds to a senescent cell. Ina specific embodiment, a natural antibody binds to an antigen or an epitope of a senescent cell.

[0143] As used herein, the term “intravenous immunoglobulin” (IVIG) refers to a therapeutic preparation of antibodies. In certain embodiments, IVIG is predominantly IgG. IVIG may be collected and pooled from healthy donors and administered to a subject in need thereof, for example, to a subject in need of immune modulation. In certain embodiments, IVIG comprises a purified preparation. For example, IgG antibodies may be purified to remove other antibody types (e.g., IgA, IgM). IVIG may be formulated in a solution (e.g., comprising stabilizers or other components), and sterilized in vials for clinical use involving intravenous administration to a subject.

[0144] As used herein, the term "antigen" ("Ag") is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full- length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0145] As used herein, the term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0146] As used herein, “cytokine” refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate animmune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL- 15, promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF).

[0147] As used herein, the term “senescence” refers to the cellular state in which a cell permanently stops dividing but remains metabolically active. It is typically a response to stress, damage, or aging. Non-limiting examples of diseases of aging that are associated with cellular senescence include osteoarthritis, atherosclerosis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 2 diabetes, chronic kidney disease, macular degeneration, cancer (tumor progression and therapy resistance), sarcopenia, cardiovascular disease, idiopathic pulmonary fibrosis, obesity-related metabolic disorders, chronic obstructive pulmonary disease (COPD), liver fibrosis, and osteoporosis. Senescent cells have a distinct phenotype including expression of specific markers (antigens) including but not limited to pl 6, p21, upregulated expression levels of inflammatory cytokines and matrix metalloproteinases, epigenetic alterations, and senescence-associated beta-galactosidase activity.

[0148] As used herein, the term “senolytic agent” refers to a compound, drug, molecule or biologic agent that induces the death of senescent cells, i.e., cells that have permanently stopped dividing but remain metabolically active and often secrete pro- inflammatory and tissue-degrading factors known as the senescence-associated secretory phenotype (SASP). Non-limiting examples of senolytic agents include dasatinib, quercetin, fisetin, navitoclax (ABT-263), UBX1325, UBX0101, FOXO4-DRI, AZD8055, piperlongumine, curcumin analogs, resveratrol, epigallocatechin gallate (EGCG), proxofim, galacto-conjugated prodrugs, senolytic CAR T cells, and dendritic cells expressing antigens derived from senescent cells.

[0149] As used herein, the term “senolysis” refers to the process of selectively eliminating senescent cells from the body or from a tissue, typically through the use of one or a plurality of senolytic agents. Senolysis aims to improve tissue function and reduce inflammation by removal or clearance of senescent cells, thereby reducing, delaying or preventing age-associated tissue damage.

[0150] As used herein, the term refers to an amount of an agent, molecule, or compound that provides a therapeutic or prophylactic benefit to a subject.

[0151] In one embodiment the invention provides means of utilizing natural antibodies to treat conditions associated with the presence of senescent cells. Specific embodiments include, for example, the use of natural antibodies to reduce inflammation associated with aging. Such inflammation, defined by some as “inflammaging”, is characterized by presence of chronically elevated underlying production of cytokines such as IL-1 beta, IL-6, IL-11, and TNF-alpha. In one embodiment, patients are assessed for presence of inflammatory mediators and administered IVIG to reduce these. Correlation with senolysis can be made by assessing products of lysed senescent cells such as pl6 or p21 antigens.

[0152] In one embodiment, natural antibodies are administered to a subject as therapeutic means for addressing senescent cells in the body. In specific embodiments, natural antibodies are administered to a subject to address a disease process, inflammation, or a tissue injury, wherein the disease process, inflammation, or injury typically involves the accumulation of senescent cells at the site or systemically. In the context of the invention, natural antibodies may be administered to a subject in single or multiple doses, and as a component of one or a plurality of treatment sessions for the subject. In one embodiment, the natural antibodies comprise polyclonal IgM preparations, which in certain embodiments, may comprise purified IgM antibodies. In one embodiment, natural antibodies are derived from plasma that is rich in natural IgM antibodies. In certain embodiments, natural antibodies are derived from plasma that is pooled from multiple donors, preferably from donors with no known disease or condition. More preferably, natural antibodies are derived from plasma of donors who are young in terms of chronological age. For example, a donor may be 30 years old or younger, 25 years old or younger, or 20 years old or younger. In one embodiment, natural antibodies comprise a commercial IgM-enriched immunoglobulin preparation, for example, Pentaglobin or Trimodulin. A commercial immunoglobulin preparation that is used to practice the invention may comprise IgM, IgA, or IgG antibodies, or combinationsthereof. In other embodiments, natural antibodies applied to the disclosed methods comprise monoclonal natural antibodies. In some embodiments, monoclonal natural antibodies are derived from B-l cells and may be selected for natural reactivity against one or a plurality of antigens or epitopes. In another embodiment, the natural antibodies applied to the methods of the invention comprise engineered antibodies of a particular isotype. In yet another embodiment, the natural antibodies comprise intravenous immunoglobulin or IVIG preparations.

[0153] In certain embodiments, natural antibodies are administered to a subject to promote the clearance of senescent cells in vivo. In certain embodiments, natural antibodies are administered to a subject to opsonize senescent cells by tagging them for destruction by immune cells. In other embodiments, the natural antibodies that are administered to a subject recognize altered self-antigens present on senescent cells or surface markers that are exposed by cellular senescence or stress. In one embodiment, natural antibodies bind to stress-induced ligands on senescent cells such as MHC Class-1- Related Chain A and / or B. In one embodiment, natural antibodies bind to damage- associated molecular patterns on senescent cells. In one embodiment, natural antibodies modulate the senescence-associated secretory phenotype, the mechanism by which senescent cells promote inflammation.

[0154] Bone marrow mononuclear cells can modulate immune responses through a variety of modalities. For instance, the invention provides the use of senolytic therapies, such as administration of natural antibodies to increase efficacy of cellular therapeutics. In one embodiment, cellular therapeutics whose activity is desired are therapies using bone marrow derived mononuclear cells. The therapeutic use of mononuclear cells from the bone marrow has been demonstrated in heart failure, limb ischemia, liver failure, in one embodiment the invention teaches enhancement of efficacy of such procedures by coadministration of senolytic agents. In one specific embodiment, the invention teaches enhancement of efficacy of bone marrow mononuclear cells or another therapeutic cell population by administration of natural antibodies. In one embodiment, said natural antibodies are administered in the form of IVIG. Certain embodiments provide compositions of natural antibodies that are useful for coadministration to a subject along with regenerative cells such as bone marrow mononuclear cells, wherein the coadministration provides enhanced activity of the regenerative cells.

[0155] In the context of the invention, the term “enhanced bone marrow mononuclear cells” refers to augmentation of therapeutic activity of such cells throughadministration of natural antibodies. These cells can alter the host in positive and beneficial ways. Such direct effects are primarily a matter of direct contact between bone marrow mononuclear cells of the host or graft. The contact may be with structural members of the cells or with constituents in their immediate environment. Such direct mechanisms may involve direct contact, diffusion, uptake, or other processes well known to those skilled in the art. The direct activities and effects of the bone marrow mononuclear may be limited spatially, such as to an area of local deposition or to a bodily compartment accessed by injection.

[0156] The invention teaches administration of natural antibodies leads to enhancement of regenerative activities of bone marrow mononuclear cells. Enhanced bone marrow mononuclear cells also can "home" in response to homing or migration signals, such as those released at sites of injury or disease. Since homing often is mediated by signals whose natural function is to recruit cells to the sites where repairs are needed, the homing behavior can be a powerful tool for concentrating enhanced bone marrow mononuclear cells to therapeutic targets. This effect can be stimulated by specific factors, said factors including PDGF, PDAF, VEGF, PDEGF, PF-4, TGF-B, FGF-A, FGF-B, TGF-A, IGF-1, IGF-2, BTG, TSP, vWF, PAI-1, IgG, IgM, IgA, KGF, EGF, FGF, TNF, IL-1, KGF -2, fibropeptide A, fibrinogen, albumin, osteonectin, gro-alpha, vitronectin, fibrin D-dimer, favtor V, antithrombin III, a2 macroglobulin, angiogenim, Fg- D, and elastase. In further detail, growth factors, cytokines, or the like that can be present and include, without limitation, LIF, anticancer growth factors such as IGFBP3, eicosanoids such as PGs orleukotrienes, IL-1 TNF alpha, INFs, TNF-a, IL-6, IL-l(a / b), prostanoid metabolites, complement components, reactive oxygen intermediates, arachidonic acid metabolites, coagulation factors, nitrates, and chemokines. Human derived growth factors, chemokines, cytokines, and hormones can include alpha defensin, alpha synuclein, beta synuclean, 4-1BBL, 6Ckine, acidic FGF, activin A, avtivin Rib, angiopoietin 2, B-DNF, BAFF, BCA-1, BCA-1, BD-1, BMP-2, BMP-4, BMP-7, BMPRA1, BDNF, CNTF, CTGF, CTLA-4Fc, CXCL1, CXCL2, cardiotrophin-1, Cripto, Cy statin C, Dkk-1, EGF AOF, EGF, EMAP II, ENA-78, EPO, Eotaxin, FGF basic AOF, FGF-10, FGF-16, FGF 17, FGF 18, FGF19, FGF4, FGF6, FGF7, FGF8, FGF8b, FGF9, Flt3, G-CSF, GDNF, GMCSF, HGF, HGH, IFN alpha A, IFN alpha ND, IFN alpha D, IFN alpha a2b, IFN, beta 1A, IFN-gamma, IGF1, IGFII, IGFBP-4, IGFBP6, IL 1 alpha, IL-lBeta, IL10, IL11, IL12, IL13, IL15, IL17, IL17A. IL17F, IL18, IL19, IL2, IL20, IL21, IL23, IL28A, IL28B, IL29, IL3, IL31, IL33, IL4, IL5, IL6, IL7, IL8, IL9, IL10,ITAC, KGF2, Kallikreinl 1, Kallikrein4, Kallikrein7, LEFTY- A, LIF, Leptin, MCSF AOF, MCSF, MCP-1, MCP2, MCP3, MCP4, MDC, MIG, MIPlalpha, MIP1 beta, MIP3 alpha, MIP3 beta, MIP4, MIP5, midkine, NAP2, NT3, NT4, Neurotactin, neurturin, Oncostatin, osteoprotegrerin, PDGF-AA, PDGF-AB, PDGF-BB, PTN, Rank ligand, Rank receptor, RANTES<SCF, SCFAOF, SDF-1 alpha, SDF-lBeta, CD4, CD40L, TNF-RI, TNFRII, TARC, TECK, TGF alpha, TGF1 Betal, TGF Beta2, TGF Beta3, TNF beta / lymphotoxin, TNF-alpha, TPO, TRAIL, TWEAK, and VEGF.

[0157] Enhanced bone marrow mononuclear cells may also modulate immune processes by their response to factors. This may occur additionally or alternatively to direct modulation. Such factors may include homing factors, mitogens, and other stimulatory factors. They may also include differentiation factors, and factors that trigger particular cellular processes. Among the latter are factors that cause the secretion by cells of other specific factors, such as those that are involved in recruiting cells, such as stem cells (including Enhanced bone marrow mononuclear cells), to a site of injury or disease.

[0158] Enhanced bone marrow mononuclear cells may, in addition to the foregoing or alternatively thereto, secrete factors that act on endogenous cells, such as stem cells or progenitor cells. The factors may act on other cells to engender, enhance, decrease, or suppress their activities, enhanced bone marrow mononuclear cells may secrete factors that act on stem, progenitor, or differentiated cells causing those cells to divide and / or differentiate. Exemplary factors produced by enhanced bone marrow mononuclear cells are microvesicles or exosomes. Enhanced bone marrow mononuclear cells that home to a site where repair is needed may secrete trophic factors that attract other cells to the site. In this way, Enhanced bone marrow mononuclear cells may attract stem, progenitor, or differentiated cells to a site where they are needed. Enhanced bone marrow mononuclear cells also may secrete factors that cause such cells to divide or differentiate. Secretion of such factors, including trophic factors, can contribute to the efficacy of enhanced bone marrow mononuclear cells in, for instance, limiting inflammatory damage, limiting vascular permeability, improving cell survival, and engendering and / or augmenting homing of repair cells to sites of damage. Such factors also may affect T-cell proliferation directly. Such factors also may affect dendritic cells, by decreasing their phagocytic and antigen presenting activities, which also may affect T-cell activity. Furthermore, such factors, or Enhanced bone marrow mononuclear cells themselves, may be capable of modulating T regulatory cell numbers.

[0159] In one embodiment of the invention, immortalized stem cells, such as mesenchymal stem cells are utilized to stimulate regeneration, while natural antibodies are used to provide senolysis. Immortalization of cells is known in the art and can be performed by administration of one or more agents. Administration of agents can be performed via the procedure of cellular transfect. Agents, or genes, useful for immortalization can be selected from a group comprising of: 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, ARID1B, 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 lorf30, 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, DPYD, 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, FBXO11, 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, G0LGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GRAB, 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, HOXD11, HOXD13, 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, MRE1 1 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, SETBP1, 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.

[0160] By these and other mechanisms, enhanced bone marrow mononuclear cells can provide beneficial immunomodulatory effects, including, but not limited to, suppression of undesirable and / or deleterious immune reactions, responses, functions, diseases, and the like. Enhanced MSC in various embodiments of the invention provide beneficial immunomodulatory properties and effects that are useful by themselves or in adjunctive therapy for precluding, preventing, lessening, decreasing, ameliorating, mitigating, treating, eliminating and / or curing deleterious immune processes and / or conditions. Such processes and conditions include, for instance, autoimmune diseases, anemias, neoplasms, HVG, GVHD, and certain inflammatory disorders. In one particular embodiment, said enhanced MSC are useful for treatment of Neurological disease, inflammatory conditions, psychiatric disorders, inborn errors of metabolisms, vascular disease, cardiac disease, renal disease, hepatic disease, pulmonary disease, ocular conditions such as uveitis, gastrointestinal disorders, orthopedic disorders, dermal disorders, neoplasia, prevention of neoplasia, hematopoietic disorders, reproductive disorders, gynecological disorders, urological disorders, immunological disorders, olfactory disorders, and auricular disorders.

[0161] In one embodiment of the invention, treatment of peripheral artery disease and other conditions affecting peripheral and / or small blood vessels can also benefit fromangiogenesis. All blood vessels that are surrounded by smooth muscle cells that can dilate in response to changes in nitric oxide. However, in general, the large blood vessels respond strongly to nitric oxide as compared to smaller ones. As one moves into arterioles, the vessels are more closely linked with tissue beds, these vessels are influenced to dilate not only in response to increased nitric oxide production by endothelial cells, but is also in response to regional changes in the levels of other vasodilators, compounds such as adenosine and prostaglandin E2 (these compounds act directly on smooth muscle cells to induce the nitric oxide -independent relaxation). Moreover, when endothelium is damaged or so compromised that nitric oxide is not enough to sufficiently relax the vascular system, other vasodilating agents need to be used. In one embodiment, the invention teaches the use of autologous bone marrow mononuclear cells that are injected with oxytoxicin in order to increase nitric oxide producing ability of endothelium. In another aspect of the invention, bone marrow mononuclear cells are administered together with natural antibodies in order to enhance sensitivity to nitric oxide production.

[0162] For the purpose of the invention, bone marrow mononuclear cells may be used either freshly isolated, purified, or subsequent to ex vivo culture. Concurrent with administration of bone marrow cells, one or more senolytic agents is added. In one embodiment addition of senolytic agents is administration of natural antibodies. In another embodiment, the source of natural antibodies is IVIG. A typical bone marrow harvest for collecting starting material for practicing one embodiment of the invention involves a bone marrow harvest with the goal of acquiring approximately 5-700 mL of bone marrow aspirate. Numerous techniques for the aspiration of marrow are described in the art and part of standard medical practice. One particular methodology that may be attractive due to decreased invasiveness is the "mini -bone marrow harvest". In one specific embodiment bone marrow mononuclear cells are isolated by pheresis or gradient centrifugation. Numerous methods of separating mononuclear cells from bone marrow are known in the art and include density gradients such as Ficoll Histopaque at a density of approximately 1.077 g / mL or Percoll gradient. Separation of cells by density gradients is usually performed by centrifugation at approximately 450 g for approximately 25-60 minutes. Cells may subsequently be washed to remove debris and unwanted materials. Said washing step may be performed in phosphate buffered saline at physiological pH. An alternative method for purification of mononuclear cells involves the use of apheresis apparatus such as the CS3000-Plus blood-cell separator (Baxter, Deerfield, USA), theHaemonetics separator (Braintree, Mass.), or the Fresenius AS 104 and the Fresenius AS TEC 104 (Fresenius, Bad Homburg, Germany) separators. In addition to injection of mononuclear cells, purified bone marrow subpopulations may be used. Additionally, ex vivo expansion and / or selection may also be utilized for augmentation of desired biological properties for use in treatment of ischemic conditions, wherein said cells are administered together with natural antibodies.

[0163] In the methods of the present invention, autologous bone-marrow is isolated from the subject usually under general anesthesia by aspiration from the tibia, femur, ilium or sternum with a syringe, preferably containing 1 mL heparin with an 18-gauge needle. Bone-marrow mononuclear cells are isolated using standard techniques with which one of skill is familiar; such techniques may be modified depending upon the species of the subject from which the cells are isolated. The marrow cells are transferred to a sterile tube and mixed with an appropriate amount of medium, e.g., 10 mL culture medium (Iscove's modified Dulbecco medium IMDM with 10% fetal bovine serum, penicillin G [100 U / mL] and streptomycin [100 .mu.g / mL]). The tube is centrifuged to pellet the bone marrow cells, e.g., at 2000 rpm for five minutes, and the cell pellet resuspended in medium, e.g., 5 mL culture medium. Low density bone-marrow mononuclear cells are separated from the suspension, e.g., by density gradient centrifugation over Histopaque-1083.TM. (Sigma). Briefly, the cell suspension is loaded on 20% to 60% gradient, e.g. Histopaque-1083.TM. (Sigma), Ficoll-Hypaque or Percoll according to manufacturer's instructions. For example, the cells are centrifuged at 400 g for 20 minutes for Ficoll-Hypaque or at 2000 rpm for 10 minutes for Percoll. Following centrifugation, the top two-thirds of total volume are transferred into a tube, as these layers contain most of the low-density bone marrow mononuclear cells. The cells are centrifuged, e.g. at 2000 rpm for 10 minutes to remove the Histopaque. This is repeated and the cell pellet of bone marrow mononuclear cells is resuspended in culture medium or buffer, e.g., IMDM, saline, phosphate buffered saline, for transplantation. Preferably, fresh bone marrow mononuclear cell, isolated as described above, are used for transplantation.

[0164] This invention provides a method of treating diseased tissue in a subject, the method comprising: a) isolating autologous bone marrow mononuclear cells from the subject; and b) transplanting locally into the diseased tissue an effective amount of the autologous bone marrow mononuclear cells, thereby treating the diseased tissue in the subject. In a preferred embodiment the diseased tissue is ischemic tissue or tissue in needof repair or regeneration. The invention teaches that augmentation of levels of natural antibodies, locally, or systemically in a patient receiving bone marrow mononuclear cell administration results in increasing angiogenesis in diseased tissue in a subject. Accordingly, the invention provides a method for treating a subject, the method comprising: a) Administering natural antibodies in a subject; b) Isolating autologous bone-marrow mononuclear cells from the subject; and c) transplanting locally into the diseased tissue an effective amount of the autologous bone marrow mononuclear cells, thereby increasing angiogenesis and repair in the diseased tissue in the subject. In a preferred embodiment, the tissue is ischemic tissue or tissue in need of repair or regeneration. In certain embodiments, the bone marrow mononuclear cells are administered systemically to a subject with a diseased tissue, wherein a proportion of the bone marrow

[0165] This invention also provides a method of preventing heart failure in a subject which is treated with natural antibodies and further subjected to a procedure comprising: a) isolating autologous bone marrow mononuclear cells from the subject; and b) transplanting locally into heart tissue an effective amount of the autologous bone marrow mononuclear cells so as to result in formation of new blood vessels in the heart tissue, to increase angiogenesis and repair in the heart tissue in the subject, thereby preventing heart failure in the subject. In a preferred embodiment the heart tissue is ischemic heart tissue or heart tissue in need of repair or regeneration after injury or surgery. In other preferred embodiments, compromised or occluded coronary blood vessels are treated by the abovedescribed methods resulting in formation of new blood vessels.

[0166] The invention provides a method of utilizing natural antibodies administration, either locally, systemically, or in delayed release form for the purpose of augmentation of tissue regeneration in a subject which comprises: a) isolating autologous bone marrow mononuclear cells from the subject; and b) transplanting locally into the tissue an effective amount of the autologous bone marrow mononuclear cells, resulting in formation of new blood vessels in the tissue, i.e. increasing angiogenesis and repair in diseased tissue in the subject. In a preferred embodiment the tissue is diseased tissue. More preferably, the diseased tissue is ischemic tissue or damaged tissue in need of repair or regeneration.

[0167] In some embodiments, the bone marrow mononuclear cells may also be cultured in any complete medium containing up to 10% serum, e.g., IMDM containing 10% fetal bovine serum and antibiotics, as described above, for up to four weeks beforetransplantation. The cells may be cultured with growth factors, e.g., vascular endothelial growth factor. The medium is changed about twice a week. The cultured cells are dissociated from the culture dishes with trypsin, neutralized with culture medium and collected by centrifugation, for example, at 2000 rpm for five minutes at room temperature.

[0168] In some embodiments, it is important to assess the efficacy of augmented responsiveness to vasodilatory agents as a means of assessing endothelial function. It is possible to measure endothelial function by measuring vasodilatation after intra-arterial pharmacologic stimulation with substances that enhance the release of endothelial nitric oxide (such as acetylcholine and bradykinin). Therefore, noninvasive tests of endothelial function have come into existence. One based on ultrasound measures flow-mediated changes in arterial diameter in relatively superficial arteries, such as the brachial, radial or femoral vessels. Thus, this technique measures endothelial function in conduit arteries rather than resistance vessels. Flow-mediated changes in conduit artery diameter are caused by shear-stress induced generation of endothelial derived vasoactive mediators (flow-mediated dilatation). Since the arterial dilator response to shear-stress can be almost completely blocked by pretreatment with nitric oxide synthase inhibitors,, it has been suggested that the phenomenon is predominantly due to endothelial release of nitric oxide. However, endothelial function assessed by this method correlates significantly with invasive testing of coronary endothelial function, as well as with the severity and extent of coronary atherosclerosis. Accordingly, in one embodiment of the invention, noninvasive endothelial function testing has provided valuable insights into vascular changes associated with early atherogenesis and the potential reversibility of arterial disease.

[0169] The invention teaches the use of bone marrow mononuclear cells administered in various forms for stimulation of regeneration in patients in need of treatment. Examples of patients (e.g. a human or a veterinary animal) in need of stimulation of regeneration of peripheral blood vessels are those suffering from, or at risk of suffering from, diminished blood flow in such blood vessels. For example, a subject may suffer and / or be at risk of, peripheral vascular disease, e.g. Raynaud's disease, peripheral artery disease (PAD), intermittent claudication (found in subjects suffering from early stages of PAD, this condition results from decreased blood flow to the legs during periods of exercise, including walking / moving around, and causing pain, fatigue or other discomfort in the affected muscle; the discomfort dissipates with the cessation ofthe activity), vasculitis of small blood vessels, vasospasm, venous thrombosis, venous insufficiency, lymphatic disorders (e.g. lymphatic insufficiency), critical limb ischemia (severe obstruction of the arteries which decreases blood flow to the hands, feet, and legs; one of the symptoms of PAD), acute limb ischemia (an arterial occlusion which suddenly limits blood flow to the arm or leg), atheroembolism (an embolism of lipid debris from an ulcerated atheromatous deposit), and / or lower extremity ischemia (an occlusive disease in arteries supplying blood to lower extremities causing inadequate blood flow).

[0170] In one embodiment the invention teaches the use of bone marrow mononuclear cells administered together with natural antibodies as a method of treating or preventing a condition or conditions selected from the group consisting of Raynaud's disease, peripheral artery disease (PAD), intermittent claudication, vasculitis of small blood vessels, vasospasm, venous thrombosis, venous insufficiency, lymphatic disorders (e.g. lymphatic insufficiency), critical limb ischemia, acute limb ischemia, atheroembolism, and lower extremity ischemia.

[0171] In one embodiment, the invention teaches the treatment of ischemic disease using administration of natural antibodies together with autologous bone marrow mononuclear cells, administration of this combination is used to treat the ischemia, said ischemia is selected from the group consisting of myocardial ischemia, cerebral ischemia, renal ischemia, liver ischemia, peripheral muscle tissue ischemia, retinal ischemia and spinal cord ischemia. As described supra, ischemia may occur in any tissue and / or organ suffering from a lack of oxygen and / or metabolites for a prolonged time which results in organic defects. The term "organ defect" as used herein relates to dysfunctional myocardium, brain, kidney, liver, peripheral muscle, retina or spinal cord defects. Said organ defects are caused by myocardial ischemia, e.g., due to heart failure, hypertension, coronary artery disease (CAD), myocardial infarction, thrombo-embolic events, trauma and / or surgical procedures; cerebral ischemia, e.g., due to trauma, stroke, thromboembolic events, malformation of blood-supplying vessels, multi-infarct disease, cerebral hemorhage, surgical and / or interventional measures; renal ischemia, e.g., due to thromboembolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and / or surgical procedures; liver ischemia, e.g., due thrombo-embolic events, malformation of blood-supplying vessels, trauma and / or surgical procedures; peripheral muscle tissue ischemia, e.g., is caused by thrombo-embolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and / or surgical procedures; retinal ischemia, e.g., is caused by thrombo-embolic events, malformation of blood-supplying vessels, traumaand / or surgical procedures; and spinal cord ischemia, e.g., is caused by thrombo-embolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and / or surgical procedures. The myocardial ischemia which is treated with the present invention is caused by heart failure, hypertension, coronary artery disease (CAD), myocardial infarction, thrombo-embolic events, trauma and / or surgical procedures. The cerebral ischemia which is treated with the present invention is caused by trauma, stroke, thrombo-embolic events, malformation of blood-supplying vessels, multi -infarct disease, cerebral hemorrhage, surgical and / or interventional measures. The renal ischemia which is treated with the present invention is caused by thrombo-embolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and / or surgical procedures. The liver ischemia or retinal ischemia which is treated with the present invention is caused by thrombo-embolic events, malformation of blood-supplying vessels, trauma and / or surgical procedures. The peripheral muscle tissue ischemia or spinal cord ischemia which is treated with the present invention is caused by thrombo-embolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and / or surgical procedures.

[0172] In a one embodiment, the present invention provides method of treating a subject having an ischemic tissue or a tissue damaged by ischemia comprising: administering a therapeutically effective amount of a composition comprising bone marrow mononuclear cells coinjected with natural antibodies, wherein said natural antibodies is administered by time release means. Furthermore, in one embodiment, said bone marrow mononuclear cells may be treated ex vivo with a prostaglandin pathway agonist and optionally, a glucocorticoid, under conditions sufficient to increase CXCR4 gene expression at least two-fold in the treated stem or progenitor cells compared to nontreated bone marrow mononuclear cells. In certain embodiment, the present invention contemplates, in part, a method of ameliorating at least one symptom associated with an ischemic tissue or a tissue damaged by ischemia in a subject comprising: administering a therapeutically effective amount of a composition comprising bone marrow mononuclear cells treated ex vivo with a prostaglandin pathway agonist and optionally, a glucocorticoid, under conditions sufficient to increase CXCR4 gene expression at least two fold in the treated stem or progenitor cells compared to non-treated bone marrow mononuclear cells, wherein said cells are administered together with natural antibodies. In some embodiments, natural antibodies is utilized as a means of increasing expression of CXCR4. Furthermore, in one embodiment, the present invention contemplates, in part,a method of increasing bone marrow mononuclear cell homing to an ischemic tissue or a tissue damaged by ischemia, comprising treating stem or progenitor cells ex vivo with a prostaglandin pathway agonist and optionally, a glucocorticoid, under conditions sufficient to increase the percent (%) migration in an SDF-1 transwell migration assay at least two fold in the treated stem or progenitor cells compared to non-treated stem or progenitor cells; and administering a composition comprising said bone marrow mononuclear cells to a subject having an ischemic tissue or a tissue damaged by ischemia. In some embodiments of the invention natural antibodies is administered ex vivo to enhance migration towards SDF-1 alone, or through coadministration with said prostaglandin pathway inhibitor and / or glucocorticoids.

[0173] In one embodiment of the invention, bone marrow mononuclear cells are genetically modified to enhance desirable properties of the cells. For modification, in some embodiments, specific types of cells with regenerative activity may be isolated from said bone marrow mononuclear cells before transfection. In one embodiment, cells are transfected with anti-apoptotic proteins to enhance in vivo longevity. The present invention includes a method of using cells that have been cultured under conditions to express increased amounts of at least one anti-apoptotic protein as a therapy to inhibit or prevent apoptosis. In one embodiment, the cells which are used as a therapy to inhibit or prevent apoptosis have been contacted with an apoptotic cell. The invention is based on the discovery that bone marrow that have been contacted with natural antibodies express high levels of anti-apoptotic molecules. In some instances, the bone marrow cells that have been contacted with an apoptotic cell secrete high levels of at least one anti- apoptotic protein, including but not limited to, STC-1, BCL-2, XIAP, Survivin, and Bcl- 2XL. Methods of transfecting antiapoptotic genes into cells have been previously described which can be applied to the current invention, said antiapoptotic genes that can be utilized for practice of the invention, in a nonlimiting way, include GATA-4

[0021] , FGF-2

[0022] , bcl-2 [23, 24], and HO-1

[0025] ,

[0174] The autologous bone-marrow mononuclear cells are transplanted by injection into the center, bordering zone, or neighboring areas of the ischemic tissue. In additional embodiments of the present invention, the autologous bone-marrow mononuclear cells may be transplanted into or near any site of any tissue in which angiogenesis or repair is required. Such tissue includes but is not limited to underperfused tissue of any end-organ, e.g. tissues with chronic ischemia. Such underperfused tissue includes but is not limited to the heart, brain, skeletal muscle, kidney, liver, organs of the gastrointestinal tract andother organs and tissues requiring repair. The transplanted autologous bone-marrow mononuclear cells are delivered to the desired tissue site(s) in an effective amount of approximately 105cells to about IO10cells, preferably about 107cells to about 108cells per injection site, preferably by needle injection. Preferably, a tissue receives at least 5 injections, at least 10 injections, at least 15 injections, or at least 20 injections over the course of a treatment or multiple treatments. Preferably, a tissue receives a total of about fifty injections, e.g. for a leg or arm, and about ten injections into heart muscle. Alternatively, the autologous bone-marrow mononuclear cells are delivered by intravascular injection or infusion into arteries or veins, endoluminal injection directly into an occlusion, retrograde perfusion, pericardial delivery, implants (biodegradable or biostable), e.g. local implant scaffold, patch, needle-free injection using propulsion by gas such as CO2, acceleration or transfer into tissue by other means such as iontophoresis or electroporation, pressure or application to a tissue or organ surface. In general, delivery may be accomplished with the use of any medical device for delivery of transplanted cells. In preferred embodiments of any of the methods described herein, the tissue into which autologous bone-marrow mononuclear cells are transplanted includes any diseased or damaged tissue and any tissue in need of repair or regeneration, including but not limited to underperfused tissue such as tissue found in chronic ischemia. Preferably, the tissue includes but is not limited to ischemic tissue. More preferably the tissue includes such tissue as cardiac muscle tissue, skeletal muscle tissue, brain tissue e.g., affected by stroke or AV malformations, coronary vessels, kidney, liver, organs of the gastrointestinal tract, muscle tissue afflicted by atrophy, including neurologically based muscle atrophy. In further embodiments the subject is preferably a mammal. Most preferably, the mammal is a human.

[0175] In the present invention, autologous bone marrow mononuclear cells locally transplanted into ischemic tissues. There are several advantages of local transplantation rather than intravenous transfusion of bone marrow mononuclear cells for therapeutic neovascularization. First, through local transplantation, one can increase the density of endothelial progenitor cells at the target tissue compared with intravenous infusion. In the present invention, approximately 105cells to about IO10cells, preferably about 107cells to about 108cells per injection site are delivered, preferably by needle injection within or near the diseased or damaged tissue or any tissue in need of repair or tissue regeneration, e.g. ischemic tissues. This may be an advantage for cell survival in the tissues, because it is believed that cells must form clusters to survive in tissues. In cancer cells, for example,there must be a clump of tumor cells to form a new metastasis colony in remote tissues. Second, local transplantation may reduce the systemic side effects of transplanted bone marrow mononuclear cells compared with systemic infusion. Other preferred means of delivery of autologous bone marrow mononuclear cells to the tissue include but are not limited to delivery by intravascular injection or infusion into arteries or veins, endoluminal injection directly into an occlusion, retrograde perfusion, pericardial delivery, implants (biodegradable or biostable), e.g. local implant scaffold, patch, needle- free injection using propulsion by gas such as CO2, acceleration or transfer into tissue by other means such as iontophoresis or electroporation, pressure or application to a tissue or organ surface. In general, delivery may be accomplished with the use of any medical device for delivery of transplanted cells. Preferably, each tissue receives a total of about ten to fifty injections.

[0176] For the practice of the invention, autologous bone marrow mononuclear cells are transplanted to an ischemic tissue where they become incorporated into or participate in the formation of new blood vessels and / or capillaries. Alternatively, said bone marrow mononuclear cells may provide trophic support for augmentation of activity of residing progenitor cells.

[0177] The choice of formulation for administering bone marrow derived mononuclear cells a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the disorder, dysfunction, or disease being treated and its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration of the bone marrow derived mononuclear cells, survivability of bone marrow derived mononuclear cells via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art. In particular, for instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, for example, liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0178] For example, cell survival can be an important determinant of the efficacy of cell-based therapies. This is true for both primary and adjunctive therapies. Another concern arises when target sites are inhospitable to cell seeding and cell growth. This may impede access to the site and / or engraftment there of therapeutic bone marrow derivedmononuclear cells. Various embodiments of the invention comprise measures to increase cell survival and / or to overcome problems posed by barriers to seeding and / or growth.

[0179] Examples of compositions comprising bone marrow derived mononuclear cells include liquid preparations, including suspensions and preparations for intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may comprise an admixture of bone marrow derived mononuclear cells with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.

[0180] Compositions of the invention often are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Various additives often will be included to enhance the stability, sterility, and isotonicity of the compositions, such as antimicrobial preservatives, antioxidants, chelating agents, and buffers, among others. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents that delay absorption, for example, aluminum monostearate, and gelatin.According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the cells. Bone marrow derived mononuclear cells solutions, suspensions, and gels normally contain a major amount of water (preferably purified, sterilized water) in addition to the cells. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents and jelling agents (e.g., methylcellulose) may also bepresent. Typically, the compositions will be isotonic, i.e., they will have the same osmotic pressure as blood and lacrimal fluid when properly prepared for administration.

[0181] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions. Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount, which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0182] A pharmaceutically acceptable preservative or cell stabilizer can be employed to increase the life of bone marrow derived mononuclear cells compositions. If such preservatives are included, it is well within the purview of the skilled artisan to select compositions that will not affect the viability or efficacy of the bone marrow derived mononuclear cells.

[0183] Those skilled in the art will recognize that the components of the compositions should be chemically inert. This will present no problem to those skilled in chemical and pharmaceutical principles. Problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation) using information provided by the disclosure, the documents cited herein, and generally available in the art.

[0184] Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.

[0185] In some embodiments, bone marrow derived mononuclear cells are formulated in a unit dosage injectable form, such as a solution, suspension, or emulsion. Pharmaceutical formulations suitable for injection of bone marrow derived mononuclear cells typically are sterile aqueous solutions and dispersions. Carriers for injectable formulations can be a solvent or dispersing medium containing, for example, water,saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0186] The skilled artisan can readily determine the numbers or concentrations of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the invention. Typically, any additives (in addition to the cells) are present in an amount of 0.001 to 50 wt % in solution, such as in phosphate buffered saline. The active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.

[0187] For any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodent such as mouse or rat; and, the dosage of the composition(s), concentration of components therein, and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure, and the documents cited herein. The time for sequential administrations can be ascertained without undue experimentation.

[0188] In some embodiments bone marrow derived mononuclear cells are encapsulated for administration, particularly where encapsulation enhances the effectiveness of the therapy, or provides advantages in handling and / or shelf life. Encapsulation in some embodiments where it increases the efficacy of bone marrow derived mononuclear cells mediated immunosuppression may, as a result, also reduce the need for immunosuppressive drug therapy.

[0189] Also, encapsulation in some embodiments provides a barrier to a subject's immune system that may further reduce a subject's immune response to the bone marrow derived mononuclear cells (which generally are not immunogenic or are only weakly immunogenic in allogeneic transplants), thereby reducing any graft rejection or inflammation that might occur upon administration of the cells.

[0190] In a variety of embodiments where bone marrow derived mononuclear cells are administered in admixture with cells of another type, which are more typically immunogenic in an allogeneic or xenogeneic setting, encapsulation may reduce or eliminate adverse host immune responses to the non-enhanced MSC cells and / or graft- versus-host disease (GVHD) that might occur in an immunocompromised host if theadmixed cells are immunocompetent and recognize the host as non-self. In some embodiments bone marrow derived mononuclear 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 Enhanced bone marrow derived mononuclear 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 poly ethersulfone (PES) hollow fibers. Techniques for microencapsulation of cells that may be used for administration of enhanced MSC are known to those of skill in the art. For example, a biocompatible capsule for long-term maintenance of cells that stably express biologically active molecules may be selected for encapsulation of bone marrow derived mononuclear cells.

[0191] Certain embodiments incorporate bone marrow derived mononuclear cells into a polymer, such as a biopolymer or synthetic polymer. Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. Other factors, such as the cytokines discussed above, can also be incorporated into the polymer. In other embodiments, bone marrow derived mononuclear cells may be incorporated in the interstices of a three-dimensional gel. A large polymer or gel, typically, will be surgically implanted. A polymer or gel that can be formulated in small enough particles or fibers can be administered by other common, more convenient, non-surgical routes.

[0192] In one embodiment, a method for treating a subject with an age-related disease or condition is provided, the method comprising: a) identifying a subject with an age-related disease or condition; b) administering a bone marrow-derived mononuclear cell population to the subject; wherein the bone marrow-derived mononuclear cells have at least one regenerative effect in the subject; and c) administering natural antibodies to the subject, wherein the natural antibodies reduce the quantities of senescent cells in the subject, and wherein the natural antibodies enhance the regenerative effect of the bone marrow-derived mononuclear cells. In certain embodiments, the regenerative effect ofbone marrow mononuclear cells comprises immune modulation. In certain embodiments, immune modulation by bone marrow-derived mononuclear cells is related to expression of one or a plurality of molecules selected from the group comprising IL-10, TGF-P, IL- IRa, hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), HLA-G, and indoleamine 2,3 dioxygenase (IDO). In one embodiment, the bone marrow-derived mononuclear cells are selected from the group comprising hematopoietic stem cells, mesenchymal stem / stromal cells, monocytes, macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells, endothelial progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, and plasma cells. In a specific embodiment, the bone marrow-derived mononuclear cells comprise tissue-specific progenitor cells, wherein the tissue-specific progenitor cells can be induced to differentiate into at least one tissue selected from a 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. In one embodiment, the natural antibodies comprise polyclonal IgM antibodies. In one embodiment, the natural antibodies comprise intravenous immunoglobulin, wherein the intravenous immunoglobulin comprises polyclonal IgG. In one embodiment, the natural antibodies are produced by B-l cells. In other embodiments, the natural antibodies are administered to a subject in the form of antibody-producing cells. Embodiments of the invention provide natural antibodies that target senescent cells, wherein the senescent cells can be identified based on expression of antigens such as pl 6, p21, or both. In one embodiment, the natural antibodies elicit antibody-dependent cell cytotoxicity against senescent cells. In one embodiment, the natural antibodies elicit complement activation against senescent cells.

[0193] In one embodiment, the methods of the invention further comprise administering a senolytic agent to the subject in addition to a regenerative cell population and natural antibodies. In one embodiment, a senolytic agent is selected from the group comprising dasatinib, quercetin, fisetin, navitoclax (ABT-263), UBX1325, UBX0101, FOXO4-DRI, AZD8055, piperlongumine, curcumin analogs, resveratrol, epigallocatechingallate (EGCG), proxofim, galacto-conjugated prodrugs, senolytic CAR T cells, and dendritic cells expressing one or a plurality of antigens derived from senescent cells.

[0194] In certain embodiments, the methods of the invention are applied for treatment or prevention of a age-related disease or condition selected from the group comprising Alzheimer’s disease, Parkinson’s disease, osteoarthritis, osteoporosis, age- related macular degeneration, cataracts, presbycusis (age-related hearing loss), presbyopia, sarcopenia, atherosclerosis, hypertension, cardiovascular disease, type 2 diabetes, chronic kidney disease, heart failure, stroke, atrial fibrillation, chronic obstructive pulmonary disease, benign prostatic hyperplasia, urinary incontinence, frailty syndrome, immune senescence, anemia of aging, depression, insomnia, glaucoma, degenerative disc disease, and cancer. In one embodiment, the age-related disease or condition involves ischemia in an organ or tissue. In one embodiment, the age-related disease or condition involving ischemia is selected from the group comprising peripheral artery disease, critical limb ischemia, diabetic foot ulcer, and chronic limb-threatening ischemia, atherosclerosis-related limb ischemia, Buerger's disease, post-surgical limb ischemia, traumatic limb ischemia, and ischemia-reperfusion injury. In certain embodiments, the disease or condition involving ischemia is reduced or ameliorated by clearance of senescent cells causing occlusion of blood vessels.Examples

[0195] The following examples are not intended as limitations. Rather they demonstrate illustrative embodiments of the present invention.

[0196] Example 1 : Augmentation of Bone Marrow Angiogenesis by Senolytic Cell Removal using Intravenous Immunoglobulin (IVIG)

[0197] These experiments utilized a mouse model of critical limb ischemia to assess the efficacy of natural antibodies as senolytic agents. Specifically, femoral artery ligation in mice was used to induce ischemia. The therapeutic interventions tested comprised bone marrow mononuclear cells as regenerative cells and natural antibodies used alone or in combination. As the endpoint, blood flow recovery, or perfusion, was measured in the affected limb.

[0198] Ten female BALB / c mice (age 40-50 weeks) per group were subjected to unilateral femoral artery ligation on day zero. Groups consisted of a) Phosphate Buffered Saline (PBS) control injection intramuscularly downstream of ligation (500 microliters); b) 500,000 syngeneic bone marrow mononuclear cell in 500 microliters of PBS); c) 100 micrograms of IVIG; and d) the combination. Cells were injected on day 1 of theexperiment, and IVIG was provided every second day. IVIG was administered via the intraperitoneal route. Assessment of perfusion was performed in the ligated limb and reported as a percentage from the contralateral non -ligated control limb of the animal. Results are shown in Tables 1 and 2 provided below, corresponding to Day 14 and Day 21 following administration of IVIG, respectively.

[0199] The results show that bone marrow mononuclear cells and IVIG alone afforded increased limb perfusion, however the combination of these two interventions afforded the most significant recovery at both 14 days (Table 1) and 21 days (Table 2).

[0200] Table 1 : Day 14 Perfusion in the Femoral Artery Ligation Model in Mice treated with Bone Marrow Mononuclear Cells, IVIG (natural antibodies), and the CombinationDay 14

[0201] Table 2: Day 21 Perfusion in the Femoral Artery Ligation Model in Mice treated with Bone Marrow Mononuclear Cells, IVIG (natural antibodies), and the CombinationDay 21Control BM Natural AB BM + Natural1.0 21.0 32.0 41.0 76.0 2.0 22.0 33.0 42.0 77.0 3.0 23.0 33.0 41.0 75.0 4.0 21.0 34.0 44.0 76.0 5.0 25.0 33.0 41.0 73.0 6.0 22.0 32.0 42.0 75.0 7.0 23.0 35.0 42.0 75.0 8.0 21.0 32.0 43.0 77.0 9.0 25.0 35.0 45.0 74.0 10.0 21.0 32.0 43.0 75.0Average 22.4 33.1 42.4 75.3 STDEV 1.6 1.2 1.3 1.3

Claims

WHAT IS CLAIMED IS:

1. A method for treating a subject with an age-related disease or condition, the method comprising: a) identifying a subject with an age-related disease or condition. b) administering a bone marrow-derived mononuclear cell population to the subject; wherein the bone marrow-derived mononuclear cells have at least one regenerative effect in the subject; c) administering natural antibodies to the subject, wherein the natural antibodies reduce the quantities of senescent cells in the subject, and wherein the natural antibodies enhance the regenerative effect of the bone marrow-derived mononuclear cells.

2. The method of Claim 1, wherein the regenerative effect of bone marrow mononuclear cells comprises immune modulation.

3. The method of Claim 2, wherein immune modulation is mediated by expression of one or a plurality of molecules selected from the group comprising IL- 10, TGF-P, IL-IRa, hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), HLA-G, and indoleamine 2,3 dioxygenase (IDO).

4. The method of Claim 1, wherein the bone marrow-derived mononuclear cells are selected from the group comprising hematopoietic stem cells, mesenchymal stem / stromal cells, monocytes, macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells, endothelial progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, and plasma cells.

5. The method of Claim 1, wherein the bone marrow-derived mononuclear cells comprise tissue-specific progenitor cells.

6. The method of Claim 5, wherein the tissue-specific progenitor cell can be induced to differentiate into at least one tissue selected from a 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, testiculartissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.

7. The method of Claim 1, wherein the natural antibodies comprise polyclonal IgM antibodies8. The method of Claim 1, wherein the natural antibodies comprise intravenous immunoglobulin.

9. The method of Claim 8, wherein the intravenous immunoglobulin comprises polyclonal IgG antibodies.

10. The method of Claim 1, wherein the natural antibodies are produced by B- 1 cells.

11. The method of Claim 1, wherein the natural antibodies are administered in the form of an antibody-producing cell.

12. The method of Claim 1, wherein the senescent cells express pl6, p21, or both.

13. The method of Claim 1, wherein the natural antibodies elicit antibodydependent cell cytotoxicity against the senescent cells.

14. The method of Claim 1, wherein the natural antibodies elicit complement activation against the senescent cells.

15. The method of Claim 1, wherein the method further comprises administering a senolytic agent to the subject.

16. The method of Claim 15, wherein the senolytic agent is selected from the group comprising dasatinib, quercetin, fisetin, navitoclax (ABT-263), UBX1325, UBX0101, FOXO4-DRI, AZD8055, piperlongumine, curcumin analogs, resveratrol, epigallocatechin gallate (EGCG), proxofim, galacto-conjugated prodrugs, senolytic CAR T cells, and dendritic cells expressing one or a plurality of antigens derived from senescent cells.

17. The method of Claim 1, wherein the age-related disease or condition is selected from the group comprising Alzheimer’s disease, Parkinson’s disease, osteoarthritis, osteoporosis, age-related macular degeneration, cataracts, presbycusis (age- related hearing loss), presbyopia, sarcopenia, atherosclerosis, hypertension, cardiovascular disease, type 2 diabetes, chronic kidney disease, heart failure, stroke, atrial fibrillation, chronic obstructive pulmonary disease, benign prostatic hyperplasia, urinary incontinence, frailty syndrome, immune senescence, anemia of aging, depression, insomnia, glaucoma, degenerative disc disease, or cancer.

18. The method of Claim 1, wherein the age-related disease or condition involves ischemia in an organ or tissue.

19. The method of Claim 18, wherein the age-related disease or condition involving ischemia is selected from the group comprising peripheral artery disease, critical limb ischemia, diabetic foot ulcer, chronic limb -threatening ischemia, atherosclerosis-related limb ischemia, Buerger's disease, post-surgical limb ischemia, traumatic limb ischemia, and ischemia-reperfusion injury.

20. The method of Claim 18, wherein the age-related disease or condition involving ischemia is reduced or ameliorated by clearance of senescent cells causing occlusion of blood vessels.

Images