Systemic means of dedifferentiation

Abstract

Means of rejuvenating a subject through exposure to soluble factors or cell-free means, wherein the soluble factors are elaborated by immature or tolerogenic cells, and wherein the soluble factors comprise peptides, proteins, nucleic acids, microvesicles, nanovesicles, apoptotic bodies, and exosomes. Administration of the soluble factors is regenerative and to treat or prevent diseases associated with aging. Ultrasound can be utilized to enhance uptake, wherein said ultrasound may be delivered through extracorporeal shock wave therapy or other means. Cell secretions comprising soluble factors are collected from a bioreactor in which immature cells are viable in a basal state or stimulated by inflammatory or age-associated stimuli.

Description

SYSTEMIC MEANS OF DEDIFFERENTIATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and benefit from U.S Provisional Application No. 63 / 548,725, titled SYSTEMIC MEANS OF DEDIFFERENTIATION and filed on February 1, 2024, the entire contents of which are hereby expressly incorporated by reference.FIELD OF INVENTION

[0002] The invention relates to the field of regenerative medicine, more specifically the invention pertains to utilization of cellular derivatives to stimulate regenerative / dedifferentiation processes without relying on administration of cells.BACKGROUND

[0003] The basic concept of cellular therapy has been discussed since dawn of medicine. Unfortunately, outside of hematology, the use of cell therapy has been relatively stagnant with regards to clinical implementation. Despite numerous successful Phase I and II clinical trials, stem cell mediated regeneration trials have yielded somewhat unpredictable results in the Phase III setting. One reason is because allogeneic stem cells are usually rejected by the host, thus not allowing for sufficient number of cells to be implanted for a long enough period of time. Moreover, autologous therapies do not provide enough stem cells due to limitations of starting materials.

[0004] The present invention provides methods for applying therapeutic cells and their soluble products for treating disorders and symptoms related to aging to overcome the limitations of previous approaches that have failed to provide efficacious cell sources.

[0005] The present invention provides therapeutic cells derived from pluripotent stem cells and soluble factors derived thereof, and methods of use thereof, for treating disorders and symptoms related to aging. Numerous types of stem cells mediate therapeutic activities through secretion of soluble factors such as cytokines, growth factors, exosomes, microvesicles, microRNAs (miRNAs), and various other proteins. Disclosed herein are compositions of therapeutic factors (or soluble factors) from regenerative cells and methods of use thereof. Also disclosed are methods for treating a subject with said therapeutic factors, which may be used to enhance cell therapies.SUMMARY

[0006] Teachings herein are directed to methods of rejuvenating an organism by exposure to compositions of soluble factors or cell-free means at concentrations and sufficient to induce one or more regenerative processes or functions in the body.

[0007] The invention discloses methods of treating a subject using paracrine factors (i.e., soluble factors or cell free means) that are produced by pluripotent stem cells and derivatives thereof, which can mimic specific therapeutic benefits of the regenerative cells. In one embodiment, the invention provides populations of therapeutic cells that produce soluble factors, wherein the soluble factors can be co-administered with the therapeutic cells to a subject for treating a disease, disorder, or symptom related to aging. In another embodiment, the invention provides soluble factors derived from regenerative cells that are administered to a subject to reduce some limitations and drawbacks regarding the clinical uses of regenerative cells themselves.

[0008] In certain embodiments, a soluble factor comprises one or a plurality of entities released by a cell, wherein these one or plurality of entities may be secreted by a cell or cleaved from the surface cell surface, detached as a cell fragment, or any cell product that is released into the extracellular environment. Embodiments of the invention provide methods for inducing or maintaining the release of one or a plurality of soluble factors from a cell into a fluid or a medium, for example, in a tissue culture plate / flask or in a bioreactor. In certain embodiments, a soluble factor comprises a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, an apoptotic body, a DNA molecule, or an RNA molecule such as a microRNA (RNA), messenger RNA (mRNA), and other non-coding RNAs. In certain embodiments, tissue culture medium or a conditioned medium is a source of one or a plurality of soluble factors, wherein the medium comprises distinct entities including but not limited to a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, an apoptotic body, a DNA molecule, or an RNA molecule. In certain embodiments, conditioned media may comprise a plurality of soluble factors released by a cell population. In certain embodiments, the term “soluble factor” may refer to a biological factor comprising an antigen; for example, a member of the “cluster of differentiation” family of cell surface markers. In certain embodiments, a soluble factor comprises a cytokine, a growth factor, an adhesion molecule, an addressin, a chemokine, or another type of molecule that exerts a function on a cell or tissue. In certain embodiments, asoluble factor comprises a molecule or antigen that is free-floating in a fluid such as culture medium. In other embodiments, a soluble factor comprises a molecule or antigen that is a component of an extracellular vesicle, an exosome, or an apoptotic body, wherein the molecule or antigen may be expressed on the surface or within the membrane of a vesicle, or both. In yet other embodiments, the term “soluble factor” is used to describe a vesicle such as an exosome that contains one or a plurality of antigens or molecules. In certain embodiments, a soluble factor is isolated or separated from a cellular component in fluid or culture medium, thereby providing a cell-free composition reflecting the secretome of the cell type(s) present in the fluid or culture.

[0009] In certain embodiments, cell-free treatments are provided that leverage the secretome of regenerative cells as a therapeutic modality for treating certain disorders, in particular, diseases and conditions that are associated with aging. In certain embodiments, the secretome comprises one or a plurality of molecules comprising a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, a DNA molecule, or an RNA molecule. The inventors have optimized methods for producing regenerative cell populations and derivatives thereof as regenerative agents or senolytic agents, wherein the regenerative cell populations and derivatives thereof produce a secretome that is useful for preventing disorders and symptoms that are related to aging of one or a plurality of organ systems in the body. Accordingly, cell populations that are the source of the cell-free treatments of the invention are disclosed. The invention provides methods of treating a subject with a disease, condition, or symptom related to aging using therapeutic soluble factors from regenerative cells or from derivatives thereof.

[0010] In one embodiment, a method for treating a subject with an age-related disease, condition, or symptom is provided, the method comprising: a) identifying a subject who is affected by an age-related disease, condition, or symptom; b) isolating a primary somatic cell from a biological fluid or a tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is exposed to culture conditions and treatments that induce dedifferentiation into an induced pluripotent stem cell; d) providing a second cell culture system, wherein the induced pluripotent stem cell is exposed to culture conditions to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative functions, immune modulatory functions, or both; e) harvesting or isolating the one or aplurality of soluble factors from the culture medium of the second culture system; and f) administering the one or plurality of soluble factors to the subject, wherein the one or plurality of soluble factors exert regenerative functions, immune modulatory functions, or both. In certain embodiments, immune modulatory functions refer to functions related to either stimulating the immune system or inhibiting the immune system. In certain embodiments, additional growth factors or immune modulatory proteins or therapeutics may be optionally administered to the subject, either systemically or locally to a tissue site, to enhance the effects of the administered soluble factors or to provide distinctive regenerative capabilities to combat aging or age-associated diseases. Other embodiments of the invention provide a method for preventing a disease, disorder, or symptom associated with aging using the compositions of cells of the invention, the compositions of soluble factors of the invention, or combinations thereof. In this embodiment, a subject at risk for an age-related disease, condition, or symptom is selected for treatment with a method disclosed herein, wherein risk may be assessed based on the subject’s age, environmental factors, genetics, symptoms displayed by the subject, or combinations thereof. In certain embodiments, a primary somatic cell is isolated from the subject who is afflicted or at risk for developing an age-related condition or disease, wherein the autologous cell is subjected to the methods of the invention. In other embodiments, a primary somatic cell is from an allogeneic donor who is not the recipient of the therapies disclosed herein.

[0011] In another embodiment, a method for treating or a preventing a disease, disorder, or symptom associated with aging in a subject is provided, the method comprising: a) identifying a subject who is affected by or at risk for developing an age- related disease, condition or symptom; b) obtaining a primary somatic cell from a biological fluid or from a tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce its dedifferentiation into a pluripotent stem cell; d) providing a second cell culture system, wherein the pluripotent stem cell is provided with culture conditions to induce its differentiation into a somatic cell; e) providing a third cell culture system, wherein the differentiated somatic cell is provided with culture conditions to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative functions, immune modulatory functions, or both ; f) harvesting or isolating the one or a plurality of autologous soluble factors from the culturemedium of the third culture system; and g) administering the one or plurality of autologous soluble factors to the subject, wherein the one or plurality of soluble factors exert regenerative functions, immune modulatory functions, or both. In certain embodiments, the soluble factors can be autologous or allogeneic. In certain embodiments, the differentiated somatic cell from the second culture system is selected from the group comprising a progenitor cell, a tissue-specific progenitor cell, a multipotent cell, a mesenchymal stem cell, a hematopoietic stem cell, a fibroblast, a granulocyte, a regulatory T cell, a B cell, a T cell, a neural -lineage cell, or another cell type. Certain embodiments of the invention also provide methods for generating a plurality of differentiated cell types from induced pluripotent stem cells. In this embodiment, the method provides a plurality of culture conditions in step d) to support differentiation of a plurality cell types from a population of induced pluripotent stem cells. In an exemplary embodiment, a population of induced pluripotent stem cells may be partitioned into a first culture system for generation of a first differentiated cell type (e.g., mesenchymal stem cells) and a second culture system is seeded with induced pluripotent stem cells for generation of a second differentiated cell type (e.g., regulatory T cells). Additionally, a plurality of distinct cell culture conditions may be provided in step e) to elicit soluble factors from each differentiated cell type generated in step d), wherein the soluble factors from each differentiated cell type are subsequently isolated for therapeutic administration to a subject. In yet another embodiment, soluble factors are harvested from cultures of both induced pluripotent stem cells and from their derivatives (i.e., differentiated cells) for administration to a subject in need thereof.

[0012] In another embodiment, a method for treating or a preventing a disease, disorder, or symptom associated with aging in a subject is provided, the method comprising: a) identifying a subject who is affected by or at risk for developing an age- related disease, condition or symptom; b) obtaining a somatic cell directly from a biological fluid or a tissue of a subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce its dedifferentiation into a pluripotent stem cell; d) providing a second cell culture system, wherein the pluripotent stem cell is provided with culture conditions to induce its differentiation into a somatic cell; e) providing a third cell culture system, wherein the differentiated somatic cell is provided with culture conditions to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possessregenerative functions, immune modulatory functions, or both ; f) harvesting or isolating the one or a plurality of autologous soluble factors from the culture medium of the third culture system; g) administering the one or plurality of soluble factors to the subject; and h) optionally, administering the differentiated somatic cell the subject. In certain embodiments, other agents may also be administered to the subject to enhance the regenerative capacity of the administered cells and / or soluble factors that are generated according to the methods of the invention. By way of example, an agent that promotes cell migration or homing (e.g., a chemotactic agent), an immune inhibitory agent, and / or an immune stimulatory agent may be administered to the subject prior to, subsequent to, or concurrent with administration of the therapies of the invention comprising soluble factors and / or cells. In other embodiments, a therapeutic cell population generated by other means may be co-administered to the subject in addition to the therapies disclosed herein.

[0013] In some embodiments, the invention teaches selecting a population of cells that produces one or a plurality of soluble factors, and subsequently selecting or harvesting the one or plurality of soluble factors that are clinically useful for treating or preventing a disease, condition, or symptom that is related to aging. In one embodiment, a disease of aging is selected from the group comprising autoimmunity, arthritis, cardiovascular disease, hypertension, stroke, heart failure, diabetes mellitus, hearing loss, osteoporosis, osteoarthritis, cataracts, pain, chronic obstructive pulmonary disease (COPD), osteoporosis, dementia, Alzheimer’s disease, Parkinson’s disease, depression, and cancer. In one embodiment, a disease of aging comprises a type of cancer selected from the list comprising melanoma (e.g., metastatic malignant melanoma), renal cancer, prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, and T-cell lymphoma.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure l is a bar graph showing the effects of pluripotent iPSC conditioned media extracted in serum free media and added at 5% v / v; 10% v / v; and 15% v / v to fibroblasts exposed to doxorubicin on pl6 expression

[0015] Figure 2 is a bar graph showing the effects of pluripotent MSC conditioned media extracted in serum free media and added at 5% v / v; 10% v / v; and 15% v / v to fibroblasts exposed to doxorubicin on pl6 expression.

[0016] Figure 3 is a bar graph showing the effects of various concentrations of pluripotent stem cell conditioned media on IL-11 expression in BALB / c mice.

[0017] Figure 4 is a bar graph showing of various concentrations of pluripotent stem cell derived MSC conditioned media on IL-11 expression in BALB / c mice.DETAILED DESCRIPTION

[0018] 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.Definitions

[0019] As used herein, “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0020] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0021] 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.

[0022] In those cases where a single numerical value is given for a characteristic or property, it is intended to be interpreted as at least covering deviations of that value within one significant digit of the numerical value given.

[0023] If a numerical value or range of numerical values is provided to define a characteristic or property of a thing or act described herein, whether the value or range is qualified with a term of degree, a specific method of measuring the characteristic or property may be defined herein as well. In the event no specific method of measuring the characteristic or property is defined herein, and there are different generally accepted methods of measurement for the characteristic or property, then the measurement method should be interpreted as the method of measurement that would most likely be adopted by one of ordinary skill in the art given the description and context of the characteristic or property.

[0024] As used herein, the terms “subject” or “patient” refers to a human or another mammal to whom a method or therapy of the invention is applied or administered.

[0025] As used herein, “committed” refers to cells which are considered to be permanently committed to a specific function. Committed cells are also referred to as “terminally differentiated cells.”

[0026] As used herein, “cytoplast extract modification” refers to the process wherein a cellular extract consisting of the cytoplasmic contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.

[0027] As used herein, the terms “dedifferentiation” or “retrodifferentiation” may be used to refer to the reversal of differentiation of a cell, whereby a cell loses its specialization in form or function and becomes a less committed cell.

[0028] As used herein, “differentiation” refers to the adaptation of cells for a particular form or function. In cells, differentiation leads to a more committed cell.

[0029] As used herein, “donor cell” refers to any diploid (2N) cell derived from a pre-embryonic, embryonic, fetal, or post-natal multi-cellular organism or a primordial sex cell which contributes its nuclear genetic material to the hybrid stem cell. The donor cell is not limited to those cells that are terminally differentiated or cells in the process of differentiation. For the purposes of this invention, donor cell refers to both the entire cell or the nucleus alone.

[0030] As used herein, “germ cell” refers to a reproductive cell such as a spermatocyte or an oocyte, or a cell that will develop into a reproductive cell.

[0031] As used herein, “host cell” refers to any multipotent stem cell derived from a pre-embryonic, embryonic, fetal, or post-natal multicellular organism that contributes the cytoplasm to a hybrid stem cell.

[0032] As used herein, “host cell preparation” refers to the process wherein the host cell is enucleated.

[0033] As used herein, “hybrid stem cell” refers to any cell that is multipotent and is derived from an enucleated host cell and a donor cell, or nucleus thereof, of a multicellular organism. Hybrid stem cells are further disclosed in co-pending U.S. patent application Ser. No. 10 / 864,788.

[0034] As used herein, “karyoplasf ’ refers to the nucleus of the cell that is surrounded by a thin layer of cytoplasm and a plasma membrane, or the nucleus that has been removed from a cell.

[0035] As used herein, “karyoplast extract modification” refers to the process wherein a cellular extract consisting of the nuclear contents of a cell, lacking the DNA, are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation or receptive to the host cell cytoplasm.

[0036] As used herein, “maturation” refers to a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation or de-differentiation. As used herein, maturation is synonymous with the terms develop or development when applied to the process described herein.

[0037] As used herein, a “modified germ cell” refers to a cell comprised of a host enucleated ovum and a donor nucleus from a spermatogonia, oogonia or a primordial sex cell. The host enucleated ovum and donor nucleus can be from the same or different species. A modified germ cell may also be referred to as a “hybrid germ cell.”

[0038] As used herein, “multipotent” refers to the capability of a cell to give rise to several other cell types, wherein the other cell types are limited in number. By way of example, a multipotent cell may be a hematopoietic cell, i.e., a blood stem cell that can develop into several types of blood cells but cannot develop into brain cells.

[0039] As used herein, a “multipotent adult progenitor cell” refers to a multipotent cell isolated from the bone marrow which has the potential to differentiate into a mesenchymal, endothelial, and endodermal lineage cell.

[0040] As used herein, a “pre-embryo” refers to a fertilized egg in the early stage of development prior to cell division. During the pre-embryonic stage, the initial stages of cleavage are occurring.

[0041] As used herein, a “post-natal stem cell” refers to a cell that is multipotent and derived from a multi-cellular organism after birth.

[0042] As used herein, “pluripotent” refers to the state of a cell that enables the cell to rise to any cell type except the cells of the placenta or other supporting cells of the uterus.

[0043] As used herein, a “primordial sex cell” refers to a diploid cell that is derived from the male or female mature or developing gonad, is able to generate cells that propagate a species and contains a diploid genomic state. A primordial sex cell can be quiescent or actively dividing. These cells include male gonocytes, female gonocytes, spermatogonial stem cells, ovarian stem cells, oogonia, type-A spermatogonia, Type-B spermatogonia. Also known as germ-line stem cells.

[0044] As used herein, a “primordial germ cell” refers to a cell present in early embryogenesis that are destined to become germ cells.

[0045] As used herein, “somatic stem cells” refers to diploid multipotent or pluripotent stem cells. Somatic stem cells are not totipotent stem cells. Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the “master” cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to fourdivisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus. At the next stage, cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number. At the end of the long chain of cell divisions that make up the embryo are “terminally differentiated” cells — cells that are considered to be permanently committed to a specific function.

[0046] As used herein, “reprogramming” when used in the context of a cell refers to a process that uses reprogramming factors such as transcription factors to convert one cell type into another cell type. For example, “reprogramming” may comprise conversion of a somatic cell into an induced pluripotent stem cell. Reprogramming of a cell may involve introducing reprogramming factors into a cell type through genetic manipulations and / or using chemical compounds. In some contexts, reprogrammed cells are useful for implantation into a host to replace or repair diseased, damaged, defective or genetically impaired tissue.

[0047] As used herein, “totipotent” is used to describe cells that contain all the genetic information needed to create all the cells of the body plus the placenta. Human cells have the capacity to be totipotent only during the first few divisions of a fertilized egg-10048] As used herein, “whole cell extract modification” refers to the process wherein a cellular extract consisting of the cytoplasmic and nuclear contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.

[0049] As used herein, “induced pluripotent stem cell” (“iPSC”), also referred to herein as a “personalized regenerative cell” refers to a pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., a primary cell isolated directly from skin, from peripheral blood cells, from bone marrow, or from another tissue source). For example, an induced pluripotent stem cell may be reprogrammed by the introduction of specific genes encoding transcription factors Oct3 / 4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under embryonic stem (ES) cell culture conditions. A personalized regenerative cell may refer to a pluripotent stem cell that is amenable to modification or differentiation to provide an autologous therapeutic cell for treating the same subject. Forexample, in certain embodiments, a personalized progenitor cell is used to generate an autologous cell that is amenable to gene modification using the methods of the invention, wherein the autologous cell may comprise a progenitor cell, a tissue-specific progenitor cell, a multipotent cell, a mesenchymal cell, a hematopoietic cell, a fibroblast, a lymphocyte, or another cell type.

[0050] As used herein, “mesenchymal stem cell” or “MSC” refers to cells that are adherent to plastic, express CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and also possess ability to differentiate to osteogenic, chondrogenic and adipogenic lineage. As used herein, “mesenchymal stromal cell” or “MSC” can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and tooth. As used herein, “mesenchymal stromal cell” or “MSC” includes cells that are CD34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally. As used herein, “MSC” includes cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof and satisfy the ISCT criteria either before or after expansion. As used herein, mesenchymal stem cells may comprise cells described in the literature as bone marrow stromal stem cells (BMSSC), marrow-isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MultiStem®, Prochymal®, remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX-PAD, AlloStem®, Astrostem®, Ixmyelocel-T, MSC-NTF, NurOwn™, Stemedyne™-MSC, Stempeucel®, StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, Revascor®, Cardiorel®, Cartistem®, Pneumostem®, Promostem®, Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs).Methods and Compositions of the Invention

[0051] The invention discloses methods of treating a subject using paracrine factors(i.e., soluble factors or cell free means) that are produced by pluripotent stem cells and derivatives thereof, which can mimic specific therapeutic benefits of the regenerative cells. In one embodiment, the invention provides populations of therapeutic cells thatproduce soluble factors, wherein the soluble factors can be co-administered with the therapeutic cells to a subject for treating a disease, disorder, or symptom related to aging. In another embodiment, the invention provides soluble factors derived from regenerative cells that are administered to a subject to reduce some limitations and drawbacks regarding the clinical uses of regenerative cells themselves. The methods of the invention are provided to promote tissue regeneration and cellular rejuvenation in one or a plurality of organ systems may be affected by aging in a subject, to treat diseases that are associated with biological aging, and to correct or prevent cellular and molecular changes in the body that precipitate age-associated diseases.

[0052] In certain embodiments, a soluble factor comprises one or a plurality of entities released by a cell, wherein these one or plurality of entities are secreted by a cell or cleaved or pinched off from the cell surface, detached as a cell fragment, or comprise any cell-derived product that is released into the extracellular environment such as through cellular apoptosis or necrosis. Culture systems and methods for producing and maintaining conditioned media comprising soluble factors derived from regenerative cell populations are disclosed herein. In certain embodiments, methods are provided for inducing or maintaining the release of one or a plurality of soluble factors from a cell into a fluid or a medium, for example, in a tissue culture plate / flask or in a bioreactor. In certain embodiments, a soluble factor comprises a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, an apoptotic body, a DNA molecule, or an RNA molecule such as a microRNA (RNA), messenger RNA (mRNA), and other non-coding RNAs. In certain embodiments, tissue culture medium or a conditioned medium is a source of one or a plurality of soluble factors, wherein the medium comprises distinct entities including but not limited to a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, an apoptotic body, a DNA molecule, or an RNA molecule. In certain embodiments, conditioned media may comprise a plurality of soluble factors released by a cell population. In certain embodiments, the term “soluble factor” may refer to a biological factor comprising an antigen; for example, a member of the “cluster of differentiation” family of cell surface markers. In certain embodiments, a soluble factor comprises a cytokine, a growth factor, an adhesion molecule, an addressin, a chemokine, or another type of molecule that exerts a function on a cell or tissue. In certain embodiments, a soluble factor comprises a molecule or antigen that is free- floating in a fluid such as a culture medium. In other embodiments, a soluble factorcomprises a molecule or antigen that is a component of an extracellular vesicle, an exosome, or an apoptotic body, wherein the molecule or antigen may be expressed on the surface or within the membrane of the vesicle or body, or both. In yet other embodiments, the term “soluble factor” is used to describe a vesicle such as an exosome that contains one or a plurality of antigens or molecules. In certain embodiments, a soluble factor is isolated or separated from a cellular component in fluid or culture medium, thereby providing a cell-free composition reflecting the secretome of the cell type(s) present in the fluid or culture.

[0053] In certain embodiments, cell-free treatments are provided that leverage the secretome of regenerative cells as a therapeutic modality for treating certain disorders, in particular, diseases and conditions that are associated with aging. In certain embodiments, the secretome comprises one or a plurality of molecules comprising a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, a DNA molecule, or an RNA molecule. The inventors have optimized methods for producing regenerative cell populations and derivatives thereof as regenerative agents or senolytic agents, wherein the regenerative cell populations and derivatives thereof produce a secretome that is useful for preventing disorders and symptoms that are related to aging of one or a plurality of organ systems in the body. Accordingly, cell populations that are the source of the cell-free treatments of the invention are disclosed. The invention provides methods of treating a subject with a disease, condition, or symptom related to aging using therapeutic soluble factors from stem cell populations or from derivatives thereof.

[0054] In one embodiment, a therapeutic soluble factor is derived from an autologous regenerative cell comprising an induced pluripotent stem cell or a derivative thereof (i.e., a differentiated cell product thereof), wherein the soluble factor is secreted or otherwise released by the cell into the extracellular environment. In another embodiment, a soluble factor that is applied for treating a subject is derived from an allogeneic regenerative cell or a derivative thereof. In one embodiment, a soluble factor is derived from a cell derivative of an induced pluripotent stem cell, i.e., a cell that is generated or differentiated from an induced pluripotent stem cell. In one embodiment, a therapeutic soluble factor is produced by a cell comprising a progenitor cell, a tissue-specific progenitor cell, a multipotent cell, a mesenchymal stem cell, a hematopoietic cell, a fibroblast, a lymphocyte, a granulocyte, or another cell type. In certain embodiments, an autologous regenerative cell comprising a pluripotent stem cell is produced from asomatic cell that is isolated directly from a tissue source or a biological fluid of a subject, e.g., from bone marrow, blood, or adipose tissue. In some embodiments, a somatic cell is isolated directly from a tissue source or a biological fluid of a subject and is cultured in vitro under specific conditions to produce an autologous regenerative cell. In certain embodiments, a regenerative cell comprising a stem cell is further cultured in vitro under conditions to induce differentiation or reprogramming of the cell. In one specific embodiment, a regenerative cell comprises an induced pluripotent stem cell, wherein the induced pluripotent stem cell is cultured in vitro to induce reprogramming, and wherein reprogramming is achieved by introduction of specific genes encoding transcription factors. In an exemplary embodiment, an induced pluripotent stem cell is reprogrammed by introducing specific genes encoding one or a plurality of transcription factors comprising Oct3 / 4, c-Myc, and Klf4 into a primary cell under embryonic stem cell culture conditions. In certain embodiments, a regenerative cell is amenable to gene modification or to treatment with one or a plurality of soluble factors. In certain embodiments, a regenerative cell is amenable to differentiation into one or a plurality of cell types in vitro, wherein the differentiated cell types may comprise cells at various stages of terminal differentiation; for example, stem cells, progenitor cells, tissue-specific progenitor cells, multipotent cells, terminally differentiated cells. In a specific embodiment, an autologous regenerative cell comprises a pluripotent stem cell that is amenable to differentiation into a cell type comprising a mature dendritic cell, an immature dendritic cell, a lymphoid dendritic cell, a myeloid dendritic cell, a monocyte, a macrophage, an alternatively activated macrophage, a mesenchymal stem cell, a T cell, a B cell, a CD5-positive B cell, a T regulatory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloblast, a common myeloid progenitor, a common lymphoid progenitor, a hematopoietic stem cell, a natural killer (NK) cell, an NKT cell, a fibroblast, a synovial cell, an epithelial cell, an epithelial progenitor cell, an endothelial cell, an endothelial progenitor cell, a hepatocyte-like cell, a hepatic stellate cell, a cardiomyocyte, a keratinocyte, a renal progenitor cell, a neural stem cell, a neural progenitor cell, a neuron, or any tissue-specific stem or progenitor cell.

[0055] Embodiments of the invention provide methods for generating one or a plurality of therapeutic factors from an autologous regenerative cell or a derivative thereof, and methods of use of the one or plurality of regenerative factors for treating a subject. In certain embodiments, a conditioned medium comprising one or a plurality of soluble factors is produced by culture the autologous regenerative cell or a derivativethereof, wherein the conditioned medium may serve as a cell-free therapeutic composition. In certain embodiments, one or a plurality of soluble factors such as proteins, glycoproteins, vesicles, or other molecules are isolated from the conditioned medium to provide a therapeutic composition for treating a subject.

[0056] In one embodiment, a method for treating a subject with a disease, condition, or symptom of biological aging is provided, the method comprising: a) identifying a subject who is affected by an age-related disease, condition, or symptom; b) isolating a primary somatic cell from a biological fluid or a tissue of the subject; c) providing a first culture system, wherein the primary somatic cell is exposed to culture conditions and treatments that induce dedifferentiation into an induced pluripotent stem cell; d) providing a second culture system, wherein the induced pluripotent stem cell is exposed to culture conditions and treatments to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative functions, immune modulatory functions, or both; e) harvesting or isolating the one or a plurality of soluble factors from the culture medium of the second culture system; and f) administering the one or plurality of soluble factors to the subject, wherein the one or plurality of soluble factors exert regenerative functions, immune modulatory functions, or both. Optionally, additional growth factors or immune modulatory proteins or therapeutics may be administered to the subject, either systemically or locally to a tissue site, to enhance the effects of the administered soluble factors or to provide distinctive regenerative capabilities to combat aging. Other embodiments of the invention provide a method for preventing a disease, disorder, or symptom associated with aging using the compositions of cells of the invention, the compositions of soluble factors of the invention, or combinations thereof. In this embodiment, a subject at risk for an age- related disease, condition, or symptom is selected for treatment with a method disclosed herein, wherein risk may be assessed based on the subject’s age, environmental factors, genetics, symptoms displayed by the subject, or combinations thereof. In certain embodiments, a primary somatic cell is isolated from the subject who is afflicted or at risk for developing an age-related condition or disease, wherein the autologous cell is subjected to the methods of the invention. In other embodiments, a primary somatic cell is from an allogeneic source, i.e., from a subject who is not the recipient of the therapies disclosed herein.

[0057] In another embodiment, a method for treating or a preventing a disease, disorder, or symptom associated with biological aging in a subject is provided, the method comprising: a) identifying a subject who is affected by or is at risk for developing an age-related disease, condition or symptom; b) obtaining a primary somatic cell from a biological fluid or from a tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is exposed to culture conditions and treatments to induce its dedifferentiation into an induced pluripotent stem cell; d) providing a second culture system, wherein the induced pluripotent stem cell is exposed to culture conditions and treatments to induce its differentiation into a somatic cell; e) providing a third culture system, wherein the differentiated somatic cell is exposed to culture conditions and treatments to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative functions, immune modulatory functions, or both; f) harvesting or isolating the one or a plurality of soluble factors from the culture medium of the third culture system; and g) administering the one or plurality of soluble factors to the subject, wherein the one or plurality of soluble factors exert regenerative functions, immune modulatory functions, or both. In certain embodiments, the soluble factors are either autologous or allogeneic. In one embodiment, the distinctive culture systems disclosed herein comprise different cell culture media or different additives, wherein the additives may be selected from the group comprising carbon sources, nitrogen sources, phosphate sources, essential minerals, amino acids vitamins, buffering agents, pH indicators, antibiotics, and specific growth factors, cytokines, hormones, as well as other molecules. In other embodiments, there is overlap in the composition of the culture systems for each distinctive step of the methods of the invention. In certain embodiments, the differentiated somatic cell from the second culture system is selected from the group comprising a stem cell, a progenitor cell, a mature dendritic cell, an immature dendritic cell, a lymphoid dendritic cell, a myeloid dendritic cell, a monocyte, a macrophage, an alternatively activated macrophage, a mesenchymal stem cell, a T cell, a B cell, a CD5-positive B cell, a T regulatory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloblast, a common myeloid progenitor, a common lymphoid progenitor, a hematopoietic stem cell, a natural killer (NK) cell, an NKT cell, a fibroblast, an epithelial cell, an epithelial progenitor cell, an endothelial cell, an endothelial progenitor cell, a hepatocyte-like cell, a hepatic stellate cell, acardiomyocyte, a keratinocyte, a renal progenitor cell, a neural stem cell, a neural progenitor cell, a neuron, or a tissue-specific stem or progenitor cell.

[0058] Certain embodiments of the invention also provide methods for generating a plurality of differentiated cell types from induced pluripotent stem cells. In this embodiment, the method provides a plurality of culture conditions in step d) to support differentiation of a plurality cell types from a population of induced pluripotent stem cells. In an exemplary embodiment, a population of induced pluripotent stem cells may be partitioned into a first culture system for generation of a first differentiated cell type (e.g., mesenchymal stem cells) and a second culture system is seeded with induced pluripotent stem cells for generation of a second differentiated cell type (e.g., regulatory T cells). Additionally, a plurality of distinct cell culture conditions may be provided in step e) to elicit soluble factors from each differentiated cell type generated in step d), wherein the soluble factors from each differentiated cell type are subsequently isolated for therapeutic administration to a subject. In yet another embodiment, soluble factors are harvested from cultures of both induced pluripotent stem cells and from their derivatives (i.e., differentiated cells) for administration to a subject in need thereof.

[0059] In another embodiment, a method for treating or a preventing a disease, disorder, or symptom associated with aging in a subject is provided, the method comprising: a) identifying a subject who is affected by or at risk for developing an age- related disease, condition or symptom; b) obtaining a somatic cell directly from a biological fluid or a tissue of a subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions to induce its dedifferentiation into an induced pluripotent stem cell; d) providing a second culture system, wherein the induced pluripotent stem cell is provided with culture conditions to induce its differentiation into a somatic cell; e) providing a third culture system, wherein the differentiated somatic cell is provided with culture conditions to elicit or maintain production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative or immune modulatory functions ; f) harvesting or isolating the one or a plurality of autologous soluble factors from the culture medium of the third culture system; g) administering the one or plurality of autologous soluble factors to the subject; and h) optionally, administering the differentiated somatic cell the subject. In certain embodiments, other agents may also be administered to the subject to enhance the regenerative capacity of the administered cells and / or soluble factors that are generatedaccording to the methods of the invention. By way of example, an agent that promotes cell migration or homing (e.g., a chemotactic agent), an immune modulatory agent, and / or an immune stimulatory agent may be administered to the subject prior to, subsequent to, or concurrent with administration of the therapies of the invention comprising soluble factors and / or cells. In other embodiments, a therapeutic cell population generated by other means may be co-administered to the subject in addition to the therapies disclosed herein.

[0060] In some embodiments, the invention teaches selecting a population of cells producing one or a plurality of soluble factors and selecting or harvesting the one or plurality of soluble factors that are clinically useful for treating or preventing a disease, condition, or symptom that is related to aging or that is exacerbated by the mechanisms underlying biological aging. In one embodiment, a disease of aging is selected from the group comprising autoimmunity, arthritis, cardiovascular disease, hypertension, stroke, heart failure, diabetes mellitus, hearing loss, osteoporosis, osteoarthritis, cataracts, pain, chronic obstructive pulmonary disease (COPD), osteoporosis, dementia, Alzheimer’s disease, Parkinson’s disease, depression, and cancer. In one embodiment, a disease of aging comprises a type of cancer selected from the list comprising melanoma (e.g., metastatic malignant melanoma), renal cancer, prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, and T-cell lymphoma. Certain embodiments of the invention provide methods for addressing the molecular changes of aging that occur at the cellularor tissue level. In certain embodiments, a method is provided for reducing, ameliorating, or preventing fibrosis that is associated with biological aging. In one embodiment, a method is provided for preventing or treating fibrotic diseases. In a specific embodiment, a fibrotic disease comprises one or a plurality of fibrotic diseases selected from the group consisting of idiopathic pulmonary fibrosis, cardiac fibrosis, liver fibrosis, chronic kidney disease, or other tissue-specific disorders. In another embodiment, a method is provided for reducing one or a plurality of symptoms associated with a fibrotic disease in a subject, wherein the one or plurality of symptoms comprise shortness of breath, fatigue, cough, muscular weakness, muscular stiffness, impaired healing from injuries, and unintended weight loss. Certain embodiments also provide a method for reducing the accumulation of senescent cells in an organ or in a tissue such as connective tissue or blood. Yet other embodiments provide a method for improving immune function in a subject. Biological aging can have a progressive, negative impact on immune function, wherein one or a plurality of the following manifestations are occurring in a subject: impaired immunity against certain infectious pathogens, dysregulated or overactive immune responses against certain infectious pathogens or against self-tissue (i.e., autoimmunity), increased susceptibility to cancer, impaired wound and tissue healing in response to injury, and susceptibility to various chronic inflammatory diseases

[0061] Various aspects of the invention are enumerated in the following paragraphs:

[0062] Aspect 1 : A method of inducing one or a plurality of regenerative processes or functions in a subject with a disease, disorder, or symptom related to biological aging, the method comprising: a) identifying a subject with an age-related disease, disorder, or symptom; b) harvesting primary somatic cells from a tissue or from blood of the subject, or alternatively from an allogeneic source; c) performing a first cell culture method or step, wherein the primary somatic cells are cultured in medium and conditions to induce dedifferentiation into pluripotent stem cells; d) performing a second culture method or step, wherein the induced pluripotent stem cells are cultured in medium and conditions for generating a differentiated somatic cell type; e) optionally, performing an additional culture method or step for generating one of a plurality of additional differentiated somatic cell types from the induced pluripotent stem cells; f) performing a third cell culture method or step, wherein the induced pluripotent stem cells from the first culture method and / or the differentiated somatic cells from the subsequent culture methods are provided with culture media and conditions that induce or maintain the release of one or a plurality of soluble factors into the culture media; g) harvesting or isolating the one orplurality of soluble factors derived from the third cell culture method; h) administering the one or plurality of soluble factors from the third cell culture method to the subject at a concentration and for a duration of time sufficient to induce one or a plurality of regenerative processes or functions in the subject; and i) optionally, also administering the one or plurality of differentiated somatic cell types generated from the second or additional culture methods to the subject.

[0063] Aspect 2: The method of Aspect 1, wherein the harvested one or plurality of soluble factors comprise a cell-free composition for administration to the subject.

[0064] Aspect 3 : The method of Aspect 2, wherein the cell-free composition comprises conditioned media, protein, nucleic acids, microRNA, apoptotic bodies, extracellular vesicles, or exosomes.

[0065] Aspect 4: The method of Aspect 1, wherein the disease, disorder, or symptom of biological aging is associated with decreased endothelial function in an organ or tissue.

[0066] Aspect 5: The method of Aspect 4, wherein endothelial function is assessed based on flow-mediated vasodilation, nitric oxide production, maintenance of an anti coagulated surface, vasodilation in response to inflammation, upregulation of integrins in response to inflammation, upregulation of addressins in response to inflammation, and upregulation of adhesion molecules in response to inflammation.

[0067] Aspect 6: The method of Aspect 5, wherein vasodilation in response to inflammation is elicited by one or a plurality of the following factors: prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), interleukin- 1 beta (IL-lbeta), interferon gamma (IFN-g), IL-6, IL-8, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IL-33, TRANCE, TRAIL, TWEAK, LIGHT, PGE-1, lymphotoxin, neutrophil extracellular traps, free DNA, double stranded RNA, circular DNA, HMGB1, denatured proteins, endoplasmic reticulum stress, apoptotic bodies, necrotic bodies, free mitochondria, tissue injury, tissue factor, ferroptotic bodies.

[0068] Aspect 7: The method of Aspect 5, wherein adhesion molecules upregulated in response to inflammation comprise one or a plurality of LFA-1, ICAM-1, HLA-1, HLA-II, HLA-G, CD37, ICOS, CD40, CD80, CD86, programmed cell death protein 1 (PD-1), PD-2, programmed death-ligand 1 (PD-L1), PD-L2, B and T lymphocyte attenuator (BTLA-4), T-cell immunoglobulin and mucin protein-1 (TIM-1(, TIM-3, galectin-1, galectin-3, galectin-5, galectin-9, LPR-1, neuropilin (NRP), CD40 ligand, and Receptor for Advanced Glycation Endproducts (RAGE).

[0069] Aspect 8: The method of Aspect 4, wherein decreased endothelial function is determined based on a reduced ability of endothelium to allow a cell to exit circulation and enter tissue.

[0070] Aspect 9: The method of Aspect 8, wherein the cell’s exit from circulation and entry into tissue occurs through extravasation through high endothelial venules.

[0071] Aspect 10: The method of Aspect 8, wherein the cell is a stem cell.

[0072] Aspect 11 : The method of Aspect 10, wherein the stem cell is a hematopoietic stem cell.

[0073] Aspect 12: The method of Aspect 11, wherein the hematopoietic stem cell expresses one or a plurality of molecules comprising CD34, CD38, CXCR4, IL-1 receptor antagonist, c-kit, VEGF receptor, CD 127, PD-L1, ILT3, ILT4, fas ligand, membrane-bound TNF, membrane-bound TWEAK, membrane-bound TRANCE, membrane-bound TRAIL, membrane-bound leukemia inhibitory factor receptor, IL-6 receptor, IL-8 receptor, IL- 11 receptor, IL- 15 receptor, IL- 17 receptor, IL-22 receptor, IL- 37 receptor, IL-38 receptor, G-CSF receptor M-CSF receptor, angiopoietin receptor, FGF-1 receptor, FGF-2 receptor, FGF-5 receptor, MMP9, thrombospondin, SMAD2, and TGF-beta.

[0074] Aspect 13: The method of Aspect 11, wherein the hematopoietic stem cell is capable of inducing multilineage engraftment in an immunodeficient host.

[0075] Aspect 14: The method of Aspect 10, wherein the stem cell is a mesenchymal stem cell.

[0076] Aspect 15: The method of Aspect 14, wherein the mesenchymal stem cell has a fibroblast-like morphology.

[0077] Aspect 16: The method of Aspect 14, wherein the mesenchymal stem cell migrates toward an SDF-1 gradient.

[0078] Aspect 17: The method of Aspect 14, wherein the mesenchymal stem cell expresses one or a plurality of molecules comprising CD37, CD133, CD74, CD90, CD 105, c-kit, and PD-L1.

[0079] Aspect 18: The method of Aspect 14, wherein the mesenchymal stem cell lacks expression of one or a plurality of molecules comprising CD14, CD16, CD19, CD45, CD34, and HLA-G.

[0080] Aspect 19: The method of Aspect 14, wherein the mesenchymal stem cell is adherent to plastic.

[0081] Aspect 20: The method of Aspect 14, wherein the mesenchymal stem cell is capable of suppressing T cell proliferation

[0082] Aspect 21 : The method of Aspect 20, wherein the T cell proliferation is induced by stimulating the T cells with one or more mitogens.

[0083] Aspect 22: The method of Aspect 21, wherein the mitogen comprises one or more of a lectin, phytohemagglutinin, and concanavalin A.

[0084] Aspect 23 : The method of Aspect 21, wherein the mitogen comprises one or a plurality of antibodies capable of cross-linking the T cell receptor.

[0085] Aspect 24: The method of Aspect 21, wherein the mitogen comprises one or a plurality of antibodies capable of cross-linking the T cell receptor and one or more antibodies crosslinking a costimulatory receptor.

[0086] Aspect 25: The method of Aspect 24, wherein the antibody capable of crosslinking the T cell receptor comprises one or a plurality of an anti-CD3 antibody, an anti-T cell receptor (TCR) antibody, and an anti-CD52 antibody.

[0087] Aspect 27: The method of Aspect 24, wherein the antibody capable of crosslinking a costimulatory receptor comprises one or a plurality of an anti-CD40 antibody, an anti-CD5 antibody, an anti-CD25 antibody, and an anti-ICAM antibody.

[0088] Aspect 28: The method of Aspect 1, wherein the pluripotent stem cell is an induced pluripotent stem cell.

[0089] Aspect 29: The method of Aspect 1, wherein the one or plurality of soluble factors are harvested from an induced pluripotent stem cell.

[0090] Aspect 30: The method of Aspect 28, wherein the induced pluripotent stem cell comprises a dedifferentiated monocyte.

[0091] Aspect 31 : The method of Aspect 30, wherein the dedifferentiated monocyte is generated by exposure to conditioned media from induced pluripotent stem cells.

[0092] Aspect 32: The method of Aspect 31, wherein the induced pluripotent stem cells are cultured in the form of embryoid bodies in a liquid medium.

[0093] Aspect 33: The method of Aspect 32, wherein the liquid medium comprises Iscove’s medium, DMEM medium, OptiMEM medium, EMEM medium, RPMI-1640 medium, or AIM-V medium.

[0094] Aspect 34: The method of Aspect 32, wherein the embryoid body is disassembled or disassociated once every 2 days, once every 5 days, or once every 10 days.

[0095] Aspect 35: The method of Aspect 32, wherein the embryoid body is comprised of induced pluripotent stem cells and monocytes.

[0096] Aspect 36: The method of Aspect 35, wherein the monocytes are exposed to a stimulatory agent or a stressor prior to admixing with induced pluripotent stem cells for generation of conditioned medium.

[0097] Aspect 37: The method of Aspect 36, wherein the stressor comprises one or a plurality of hypoxia, hypertonicity, hypotonicity, hyperthermia, serum starvation, mTOR inhibition, AMPK activation, or activation of inflammatory pathways.

[0098] Aspect 38: The method of Aspect 37, wherein the inflammatory pathway comprises one or more of the following pathways: MAP kinase, Janus activated kinase, Signal Transducer and Activator of Transcription-3 (STAT3), STAT5, STAT6, TLR2, TLR3, and TLR4.

[0099] Aspect 39: The method of Aspect 38, wherein the TLR2 pathway is activated using peptidoglycan.

[0100] Aspect 40: The method of Aspect 38, wherein the TLR3 is activated using one or a plurality of the following: double stranded RNA, polyinosinic:polycytidylic acid (poly(I:C), polyinosinic-poly cytidylic acid-poly-L-lysine-carboxymethylcellulose (poly- ICLC), and inactivated influenza virus.

[0101] Aspect 41 : The method of Aspect 38, wherein the TLR4 pathway is activated by one or a plurality of the following: neutrophil extracellular traps, beta glucan, HMGB1, lipopolysaccharide, yeast cell wall extract, or anti-TLR4 antibody.

[0102] Aspect 42: The method of Aspect 31, wherein the induced pluripotent stem cells comprise genetically engineered cells expressing an inhibitor of TGF-beta.

[0103] Aspect 43 : The method of Aspect 42, wherein the inhibitor of TGF-beta comprises an antibody molecule.

[0104] Aspect 44: The method of Aspect 43, wherein the antibody comprises a cameloid antibody, a microbody, or an aptamer.

[0105] Aspect 45: The method of Aspect 42, wherein the inhibitor of TGF-beta comprises a molecule that induces RNA interference.

[0106] Aspect 46: The method of Aspect 45, wherein the molecule that induces RNA interference comprises a short interfering RNA, a short hairpin RNA, a microRNA, or a long noncoding RNA.

[0107] Aspect 47: The method of Aspect 1, wherein the first culture method provides an agent that induces dedifferentiation or reprogramming of the primary somatic cell and / or promotes pluripotency of a stem cell.

[0108] Aspect 48: The method of Aspect 47, wherein the agent comprises one or a plurality of the following GDF-11, GDF-15, amniotic fluid, umbilical cord blood plasma, and BMP2.

[0109] Aspect 49: The method of Aspect 47, wherein the agent suppresses activation of nuclear factor-kappa B.

[0110] Aspect 50: The method of Aspect 47, wherein the agent increases nuclear factor erythroid 2-related factor 2 (NRF2) activation.

[0111] Aspect 51 : The method of Aspect 47, wherein the agent increases heme- oxygenase-1 activation.

[0112] Aspect 52: The method of Aspect 47, wherein the agent increases activation of bcl-2.

[0113] Aspect 53 : The method of Aspect 47, wherein the agent increases activation ofbcl-2XL.

[0114] Aspect 54: The method of Aspect 47, wherein the agent increases activation of survivin.

[0115] Aspect 55: The method of Aspect 47, wherein the agent increases activation of livin.

[0116] Aspect 56: The method of Aspect 47, wherein the agent is an HD AC inhibitor.

[0117] Aspect 57: The method of Aspect 56, wherein the HD AC inhibitor comprises one or a plurality of the following: valproic acid, trichostatin A, sodium phenylbutyrate, and butyrate.

[0118] Aspect 58: The method of Aspect 47, wherein the agent comprises a GSK-3 inhibitor.

[0119] Aspect 59: The method of Aspect 58, wherein the GSK inhibitor comprises lithium.

[0120] Aspect 60: The method of Aspect 47, wherein the agent comprises PIM1.

[0121] Aspect 61 : The method of Aspect 47, wherein the agent comprises an immortalizing oncogene.

[0122] Aspect 62: The method of Aspect 61, wherein the oncogene comprises one or a plurality of the following: SV40 large T antigen, abll, AFF4, AKT2, AML1, MTG8,BCL6, MCF6, DCF3, EGFR, MLLT11, ERBB2, CSF1R, FOS, FES, GNAS, HER2, FGF3, FGF4, JUN, c-kit, K-SAM, AKAP13, LCK, LM01, LYL1, MASI, MDM2, MOS, MYH1 1, MYB, MYCN, PAX5, RAF, RAS, REL, ROS1, SKI (PDGF-BB), SET, SRC, TALI, TAN1, TIAN, TSC2, and TRK.

[0123] Aspect 63 : The method of Aspect 47, wherein the agent causes a change in the genotype of the pluripotent stem cell.

[0124] Aspect 64: The method of Aspect 47, wherein the agent can be conditionally inactivated.

[0125] Aspect 65: The method of Aspect 1, wherein the disease, disorder, or symptom related to biological aging is associated with a decrease in immune function.

[0126] Aspect 66: The method of Aspect 65, wherein the decrease in immune function is associated with a loss in thymic volume.

[0127] Aspect 67: The method of Aspect 65, wherein the decrease in immune function is associated with a loss of T cell activity.

[0128] Aspect 68: The method of Aspect 1, wherein reprogramming the first somatic cell is performed using one or a plurality of retroviral expression vectors designed to deliver one or a plurality of specific genes to dedifferentiate the first somatic cell into a pluripotent stem cell.

[0129] Aspect 69: The method of Aspect 68, wherein the one or plurality of specific genes encode one or a plurality of transcription factors.

[0130] Aspect 70: The method of Aspect 68, wherein the retroviral expression vector comprises one or a plurality of the following vectors: a pMX vector, a lentiviral vector, an adenoviral vector, a pMX adeno-associated vector, and a herpesvirus vector.

[0131] Aspect 71 : The method of Aspect 68, wherein the retroviral expression vector comprises one or a plurality of the following types of vectors: a plasmid-based vector, an RNA-based vector, an mRNA-based vector, and a microRNA-based vector.

[0132] Aspect 72: The method of Aspect 47, wherein the first cell culture system also comprises one or a plurality of the following molecules or compounds: ascorbic acid, a stimulator of the RAS pathway, a stimulator of the myc pathway, a stimulator of the PIM1 pathway, and a stimulator of the j anus activated kinase pathway.

[0133] Aspect 73: The method of Aspect 1, wherein a disease, disorder, or symptom related to biological aging is associated with fibrosis in an organ or tissue.

[0134] Aspect 74: A method for treating or preventing age-related fibrosis, the method comprising: a) identifying a subject presenting with evidence or at-risk for age-related fibrosis in an organ or tissue; b) harvesting a first somatic cell from a tissue or from blood of the subject; c) providing a first cell culture system for reprogramming the first somatic cell into a pluripotent stem cell; d) providing a second cell culture system for inducing differentiation of the pluripotent stem cell into a regulatory T cell; e) providing a third cell culture system for inducing or maintaining the production of one or a plurality of soluble factors from the regulatory T cell produced in the second cell culture system; f) harvesting the one or plurality of soluble factors from the third cell culture system; and g) administering the one or plurality of soluble factors from step f) to the subject at a concentration and duration of time sufficient to induce one or a plurality of regenerative processes or functions in the subject.

[0135] Aspect 75: A method for treating age-related fibrosis, the method comprising: a) identifying a subject presenting with evidence of age-related fibrosis in an organ or tissue; b) harvesting a first somatic cell from a tissue or from blood of the subject; c) providing a first cell culture system for reprogramming the first somatic cell into a pluripotent stem cell; d) providing a second cell culture system for inducing differentiation of the pluripotent stem cell into a mesenchymal stem cell; e) providing a third cell culture system for inducing or maintaining the production of one or a plurality of soluble factors from the mesenchymal stem cell produced in the second cell culture system; f) harvesting the one or plurality of soluble factors from the third cell culture system; and g) administering the one or plurality of soluble factors from step f) to the subject at a concentration and duration of time sufficient to induce one or a plurality of regenerative processes in the subject.

[0136] Aspect 76: The method of Aspect 1, wherein the second culture method generates a differentiated somatic cell type comprising a regulatory T cell.

[0137] Aspect 77: The method of Aspect 76, wherein the regulatory T cell produces the one or plurality of soluble factors.

[0138] Aspect 78: The method of Aspect 77, wherein the one or plurality of soluble factors induce the one or a plurality of regenerative processes upon administration to the subject.

[0139] Aspect 79: The method of Aspect 78, wherein the regenerative process comprises reducing or ameliorating fibrosis in an organ or tissue.

[0140] Aspect 80: The method of Aspect 1, wherein the second culture method generates a differentiated somatic cell type comprising a mesenchymal stem cell.

[0141] Aspect 81 : The method of Aspect 80, wherein the mesenchymal stem cell produces the one or plurality of soluble factors.

[0142] Aspect 82: The method of Aspect 81, wherein the one or plurality of soluble factors induces one or a plurality of regenerative processes upon administration to the subject.

[0143] Aspect 83: The method of Aspect 82, wherein the regenerative processes comprise reducing or ameliorating fibrosis in an organ or tissue.

[0144] Aspect 84: The method of Aspect 1, wherein the second culture method generates a plurality of differentiated somatic cell types, wherein the differentiated somatic cell types comprise regulatory T cells and mesenchymal stem cells.

[0145] Aspect 85: The method of Aspect 84, wherein the second culture method provides a first culture media or condition for the differentiation of regulatory T cells.

[0146] Aspect 86: The method of Aspect 85, wherein the first culture media or condition comprises one or a plurality of cytokines, chemokines, and growth factors.

[0147] Aspect 87: The method of Aspect 84, wherein the second culture method provides a second culture media or condition for the differentiation of mesenchymal stem cells.

[0148] Aspect 88: The method of Aspect 87, wherein the second culture media or condition comprises one or a plurality of cytokines, chemokines, and growth factors.

[0149] Aspect 89: The method of Aspect 84, wherein the regulatory T cells are capable of producing one or a plurality of soluble factors.

[0150] Aspect 90: The method of Aspect 84, wherein the mesenchymal stem cells are capable of producing one or a plurality of soluble factors.

[0151] Aspect 91 : The method of Aspect 1, wherein the one or plurality of differentiated somatic cell types that are administered to the subject comprise progenitor cells, endothelial progenitor cells, T cells, dendritic cells, myeloid dendritic cells, lymphoid dendritic cells, mature dendritic cells, immature dendritic cells, fibroblasts, B cells, neutrophils, monocytes, eosinophils, regulatory T cells, regulatory B cells, hematopoietic stem cells, or mesenchymal stem cells.

[0152] Aspect 92: The method of Aspect 91, wherein a first differentiated somatic cell type administered to a subject comprises mesenchymal stem cells and a second differentiated somatic cell type administered to a subject comprises regulatory T cells.

[0153] Aspect 93: The method of Aspect 92, wherein a mesenchymal stem cell is administered to the subject at least 1 day, at least 3 days, at least 5 days, at least 10 days, or at least 30 days prior to administration of regulatory T cells to the subject.

[0154] Aspect 94: The method of Aspect 89, wherein the one or plurality of soluble factors are administered to a subject, and wherein the one or plurality of soluble factors induce a regenerative process in the subject.

[0155] Aspect 95: The method of Aspect 90, wherein the one or plurality of soluble factors are administered to the subject, and wherein the one or plurality of soluble factors induce a regenerative process in the subject.

[0156] Aspect 96: The method of Aspect 80, wherein the mesenchymal stem cell produces a soluble factor comprising brain-derived neurotrophic factor.

[0157] Aspect 97: The method of Aspect 96, wherein the mesenchymal stem cell is selected for expression of CD56.

[0158] Aspect 98: The method of Aspect 96, wherein brain-derived neurotrophic factor is produced in response to molecular signals associated with tissue injury.

[0159] Aspect 99: The method of Aspect 98, wherein at least 10 pg, at least 25 pg, or at least 100 pg of brain-derived neurotrophic factor is produced by the mesenchymal stem cell subsequent to stimulation with poly(I:C).

[0160] Aspect 100: The method of Aspect 80, wherein the mesenchymal stem cell produces a soluble factor comprising ciliary neurotrophic growth factor.

[0161] Aspect 101 : The method of Aspect 100, wherein ciliary neurotrophic growth factor is produced in response to molecular signals associated with tissue injury.

[0162] Aspect 102: The method of Aspect 80, wherein the mesenchymal stem cell is selected for expression of CD73.

[0163] Aspect 103: The method of Aspect 101, wherein at least 4 pg, at least 8 pg, or at least 12 pg of ciliary neurotrophic factor is produced by the mesenchymal stem cell subsequent to stimulation with poly(I:C).

[0164] Aspect 104: The method of Aspect 80, wherein the mesenchymal stem cell produces a soluble factor comprising basic fibroblast growth factor.

[0165] Aspect 105: The method of Aspect 104, wherein basic fibroblast growth factor is produced in response to molecular signals associated with tissue injury.

[0166] Aspect 106: The method of Aspect 80, wherein the mesenchymal stem cell is selected for expression of CD 105.

[0167] Aspect 107: The method of Aspect 105, wherein at least 50 pg, at least 100 pg, or at least 200 pg of basic fibroblast growth factor is produced by the mesenchymal stem cell subsequent to stimulation with poly(I:C).

[0168] Aspect 108: The method of Aspect 1, wherein the second culture method generates a differentiated somatic cell type comprising a regulatory T cell, and wherein the regulatory T cell produces one or a plurality of soluble factors.

[0169] Aspect 109: The method of Aspect 1, wherein the one or plurality of soluble factors are administered prior to or subsequent to administration of a fibrinolytic enzyme to the subject.

[0170] Aspect 110: The method of Aspect 109, wherein the fibrinolytic enzyme comprises a matrix metalloproteinase.

[0171] Aspect 111 : The method of Aspect 110, wherein the matrix metalloproteinase comprises MMP-3, MMP-5, MMP-6, MMP-9, or MMP-12.

[0172] Aspect 112: The method of Aspect 1, wherein the one or plurality of soluble factors are administered prior to or subsequent to administration of an inhibitor of NOGO to the subject.

[0173] Aspect 113: The method of Aspect 112, wherein the inhibitor of NOGO comprises one or a plurality of the following molecules or compounds: an antisense oligonucleotide, a short interfering RNA, a short hairpin RNA, a small molecule inhibitor, an antibody, an aptamer, a SOMAmer®, a bispecific antibody, a ribozyme, an antibody, a microantibody, a hammerhead ribozyme, and a soluble receptor.

[0174] Aspect 114: The method Aspect 1, wherein a differentiated somatic cell type from the second culture method that is administered to the subject comprises myeloid- lineage cells.

[0175] Aspect 115: The method of Aspect 114, wherein the myeloid lineage cells comprise myeloid-derived dendritic cells.

[0176] Aspect 116: The method of Aspect 115, wherein the myeloid-derived dendritic cells are immature.

[0177] Aspect 117: The method of Aspect 115, wherein the myeloid-derived dendritic cells are generated from monocytes by specific culture conditions in vitro.

[0178] Aspect 118: The method of Aspect 117, wherein the monocytes are isolated from peripheral blood.

[0179] Aspect 119: The method of Aspect 118, wherein the monocytes in peripheral blood are first increased in numbers using a mobilization agent, and wherein themobilization agent is selected from the group comprising G-CSF, GM-CSF, M-CSF, IL- 3, IL- 10, beta glucan, and interferon gamma.

[0180] Aspect 120: The method of Aspect 117, wherein the monocytes are isolated from a source comprising menstrual blood, umbilical cord blood, Wharton’s jelly, bone marrow, adipose tissue, and omental tissue.

[0181] Aspect 121 : The method of Aspect 117, wherein the monocytes are cultured with one or a plurality of agents to induce a tolerogenic phenotype prior to differentiation into myeloid dendritic cells in vitro.

[0182] Aspect 122: The method of Aspect 117, wherein exosomes are isolated or harvested from the myeloid-derived dendritic cells generated in vitro.

[0183] Aspect 123: The method of Aspect 121, wherein the one or plurality of agents in culture comprise prostaglandin E2, genistein, quercetin, hypertonic saline, vascular endothelial growth factor (VEGF), platelet derived growth factor BB (PDGF- BB), insulin-like growth factor (IGF-1), salinomycin, erythropoietin, NF-kappa B decoy oligonucleotide, inhibitor of nuclear factor kappa-B kinase subunit beta (Ikk-beta) decoy oligonucleotides, IL-35, TGF-beta, ascorbic acid, N-acetylcysteine, alpha lipoic acid, methylene blue, and valproic acid.

[0184] Aspect 124: The method of Aspect 121, wherein the one or plurality of agents in culture induces production of IL- 10 by the monocytes.

[0185] Aspect 125: The method of Aspect 124, wherein at least 10 pg / mL, at least 20 pg / mL, or at least 40 pg / mL of IL- 10 are produced from monocytes in culture, and wherein the monocytes in the culture comprise at least 1 million cells, at least 2 million cells, or at least 5 million cells.

[0186] Aspect 126: The method of Aspect 116, wherein the immature dendritic cells express or produce one or a plurality of molecules comprising IL-10, IL-4, IL-13, IL-20, IL-22, IL-35, IL-37, IL-38, TFG-beta, IL-20, IL-22, IL-35, IL-37, IL-38, endoglin, HLA- G, and IL- 12 p40 homodimer.

[0187] Aspect 127: The method of Aspect 116, wherein the immature dendritic cells possess increased phagocytic activity as compared to mature dendritic cells.

[0188] Aspect 128: The method of Aspect 116, wherein the immature dendritic cells possess increased migratory activity towards a chemotactic gradient in vitro as compared to mature dendritic cells.

[0189] Aspect 129: The method of Aspect 128, wherein the chemotactic gradient is induced one or a plurality of chemotactic molecules comprising IL-8, MIP-1 alpha, MIP-1 beta, CXCL12, MCP-1, TNF-alpha, lymphotoxin, hyaluronic acid degradation products, and HMGB 1.

[0190] Aspect 130: The method of Aspect 116, wherein the immature dendritic cells possess decreased expression of one or a plurality of molecules or antigens comprising HLA I, HL A II, CD la, CD40, CD80, CD86, IL- 15 receptor, interferon gamma, IL- 18 receptor, progesterone receptor, and c-kit as compared to mature dendritic cells.

[0191] Aspect 131 : The method of Aspect 116, wherein the immature dendritic cells induce the generation of regulatory T cells upon culture with allogeneic naive T cells.

[0192] Aspect 132: The method of Aspect 116, wherein the immature dendritic cells upregulate expression of autoimmune regulator (AIRE) upon contact with allogeneic naive T cells.

[0193] Aspect 133: The method of Aspect 132, wherein the AIRE upregulation is dependent on production of one or a plurality of molecules comprising IL-10, TGF-beta, HLA-G, endoglin, FGF-1, FGF-2, FGF-5, and NOTCH.

[0194] Aspect 134: The method of Aspect 114, wherein a myeloid lineage cell comprises an immature neutrophil.

[0195] Aspect 135: The method of Aspect 134, wherein an immature neutrophil is a neutrophil progenitor.

[0196] Aspect 136: The method of Aspect 134, wherein an immature neutrophil expresses one or a plurality of molecules comprising PUL I, G-CSF receptor, stem cell factor receptor, c-met, M-CSF receptor, and GM-CSF receptor.

[0197] Aspect 137: The method of Aspect 134, wherein an immature neutrophil produces IL- 10 upon stimulation with a TLR-4 agonist.

[0198] Aspect 138: The method of Aspect 137, wherein a TLR-4 agonist is selected from the group comprising beta glucan, hyaluronic acid degradation products, HMGB1, and histones.

[0199] Aspect 139: The method of Aspect 114, wherein a myeloid lineage cell comprises a myeloid suppressor cell.

[0200] Aspect 140: The method of Aspect 139, wherein the myeloid suppressor cell is capable of differentiating into a monocyte when treated with a molecule selected from the group comprising All-Trans Retinoic Acid, vitamin D3, M-CSF, and GM-CSF.

[0201] Aspect 141 : The method of Aspect 139, wherein the myeloid suppressor cell is capable of inhibiting T cell proliferation by production of a molecule selected from the group comprising nitric oxide, reactive oxygen species, superoxide, hydrogen peroxide,arginase, IL- 10, soluble PD-L1, soluble VISTA, LAG-3, TIM3, PGE2, secreted vimentin, and secreted calreticulin.

[0202] Aspect 142: The method of Aspect 1, wherein a first differentiated somatic cell type administered to the subject comprises oligodendrocyte-lineage cells and a second differentiated somatic cell type administered to the subject comprises regulatory T cells.

[0203] Aspect 143: The method of Aspect 142, wherein oligodendrocyte-lineage cells are derived from induced pluripotent stem cells.

[0204] Aspect 144: The method of Aspect 1, wherein the pluripotent stem cell is generated by transfecting a somatic cell with cytoplasm from an oocyte.

[0205] Aspect 145: The method of Aspect 144, wherein the cytoplasm is transfected using one or a plurality of methods comprising electroporation, cell fusion, streptolysin O to generate transient holes in the cytoplasm of the recipient cell, cell penetrating peptides to generate transient holes in the cytoplasm of the recipient cell.

[0206] Aspect 146: The method of Aspect 1, wherein the pluripotent stem cell is an induced pluripotent stem cell, wherein the induced pluripotent stem cell is created by transfection of a somatic cell with pluripotency inducing factors to induce stem cell reprogramming, and wherein the pluripotency inducing factors comprise one or a plurality of OCT4, c-Myc, PIM-1, NANOG, c-met, hTERT, KLF4, RAS, NOTCH, BMP2, BMP4, and AIRE.

[0207] Aspect 147: The method of Aspect 76, wherein the regulatory T cells are treated with one or a plurality of histone deacetylase inhibitors.

[0208] Aspect 148: The method of Aspect 1, wherein the soluble factors comprise one or a plurality of entities or molecules comprising exosomes, and wherein the exosomes express one or a plurality of antigens or molecules.

[0209] Aspect 149: The method of Aspect 148, wherein the one or plurality of antigens or molecules are selected from the group comprising: CD4, CD8, CD63, CD9, CD81, TsglOl, Alix, flotillin, QSOX1, IFN-gamma, TGF-beta, IL-6, IL-8, MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2, MCP-1, MIP-1, MIP-2, VEGF, growth-regulated protein alpha (GRO-alpha), collagen alpha 1 (COL1 Al), CD34, CD44, CD73, FoxP3, miR-126, miR-296, miR-129-5p, miR-133b, miR-140-5p, miR-21-5p, miR-100-5p, and miR-143-3p.

[0210] Aspect 150: The method of Aspect 1, wherein the soluble factors comprise exosomes, and wherein the exosomes express phosphatidylserine on their outer membrane.

[0211] Aspect 151 : The method of Aspect 1, wherein the soluble factors are selected from the group comprising exosomes, apoptotic bodies, and extracellular vesicles, and wherein the soluble factors are present in conditioned medium.

[0212] Aspect 152: The method of Aspect 1, wherein the third culture step comprises culture media and conditions in which the cells are exposed to one or a plurality of agents that comprise cell stressors.

[0213] Aspect 153: The method of Aspect 152, wherein the one or a plurality of agents that comprise cell stressors induce the production of the one or plurality of soluble factors in the third cell culture step.

[0214] Aspect 154: The method of Aspect 153, wherein the one or plurality of cell stressors comprise molecules or agents that induce inflammation.

[0215] Aspect 155: The method of Aspect 154, wherein the molecules that induce inflammation activate one or a plurality of danger-associated receptors in cultured cells.

[0216] Aspect 156: The method of Aspect 155, wherein the one or plurality of danger-associated receptors comprise toll-like receptors.

[0217] Aspect 157: The method of Aspect 156, wherein the toll-like receptors are activated by one or a plurality of the following compounds or methods comprising beta glucan, poly(I:C), CpG DNA, BCG, flagellin, LPS, low molecular weight hyaluronic acid, free histone, isoxazolo[5,4 -d]pyrimidine, imiquimod, poly(A:U), MPL, poly G3, poly G10, or by contact with hyperthermia exposed cells.

[0218] Aspect 158: The method of Aspect 157, wherein the hyperthermia exposed cells express hsp90.

[0219] Aspect 159: The method of Aspect 158, wherein the expression of hsp90 is at least 50% higher than the expression level of hsp90 on cell-type matched untreated cells not been exposed to hyperthermia.

[0220] Aspect 160: The method of Aspect 1, wherein one or a plurality of soluble factors administered to the subject are derived from a regulatory T cell, and wherein a histone deacetylase inhibitor is also administered to the subject prior to or following the administration of the one or plurality of soluble factors, or both.

[0221] Aspect 161 : The method of Aspect 160, wherein the histone deactylase inhibitor is administered systemically to the subject.

[0222] Aspect 162: The method of Aspect 160, wherein the histone deactylase inhibitor is administered directly into an organ or tissue of the subject.

[0223] Aspect 163: The method of Aspect 162, wherein the histone deacetylase inhibitor induces one or a plurality of regenerative processes in the subject.

[0224] Aspect 164: The method of Aspect 1, wherein the regenerative processes of functions comprise one or a plurality of the following effects on cells, organs and tissues in the subject: reversing or preventing epigenetic alterations, halting or reducing telomere attrition, inducing telomere lengthening, improving genomic stability by promoting detoxification of DNA damaging agents, reducing or ameliorating proteostasis, modulating nutrient signaling pathways, preventing or reversing mitochondrial dysfunction and damage, preventing the cellular senescent state, combating stem cell exhaustion and loss of function, and exerting anti-inflammatory effects.

[0225] Aspect 165: The method of Aspect 160, wherein the regulatory T cell expresses one or a plurality of the following molecules or antigens comprising CD4, CD8, CD25, FoxP3, and CD103.

[0226] Aspect 166: The method of Aspect 160, wherein the one or plurality of soluble factors derived from a regulatory T cell are administered directly into an organ or tissue of the subject.

[0227] Aspect 167: The method of Aspect 160, wherein the one or plurality of soluble factors derived from a regulatory T cell are administered systemically.

[0228] Aspect 168: The method of Aspect 167, wherein systemic administration involves administration by the intravenous route.

[0229] Aspect 169: The method of Aspect 160, wherein the histone deacetylase inhibitor comprises one or a plurality of the following agents or compounds: CXD101, HDAClO-IN-1, tubastatin A TFA, ACY-775, Panobinostat, trichostatin A, vorinostat, entinostat, BML-210, abexinostat, dacinostat, quisinostat, mocetinostat, valproic acid, CUDC-101, GSK3117391, droxinostat, MC1568, pracinostat, divalproex sodium, diferuloylmethane, sodium butyrate, PCI-34051, SR-4370, givinostat, tubacin, AR-42, parthenolide, tubastatin A HC1, resminostat, CUDC-907, M344, tacedinaline, romidepsin, 4-phenylbutyric acid, sinapinic acid, sulforaphane, UFO 10, suberohydroxamic acid, NKL22, ITSA-1, KA2507, isoguanosine, raddeanin A, BRD3308, TH34, and tinostamustine.

[0230] Aspect 170: The method of Aspect 1, wherein a culture method provides the pluripotent stem cells with one or a plurality of culture media or conditions for generatinga mesenchymal stem cells, and wherein the soluble factors released by the mesenchymal stem cells comprise exosomes.

[0231] Aspect 171 : The method of Aspect 170, wherein the exosomes are administered to the subject systemically.

[0232] Aspect 172: The method of Aspect 170, wherein the mesenchymal stem cells are further modified by transfection with a growth factor.

[0233] Aspect 173: The method of Aspect 172, wherein the growth factor comprises one or a plurality of the following: HGF-1, FGF-1, EGF-1, angiopoietin, placental growth factor, adiponectin, vasoactive intestinal peptide precursor, endoglin, myostatin, TGF- beta, VEGF, GDF-11, GDF-15, hyaluronic acid synthase, IL-33, osteosarcoma-derived growth factor, midkine, PDGF-BB, IGF-1, nerve growth factor, brain derived neurotrophic factor, and apelin.

[0234] Aspect 174: The method of Aspect 76, wherein the regulatory T cell comprises an effector regulatory T cell, a memory regulatory T cell, or a chimeric antigen receptor (CAR) regulatory T cell.

[0235] Aspect 175: The method of Aspect 174, wherein the CAR regulatory T cell is generated by genetically modifying the pluripotent stem cell to express the CAR.

[0236] Aspect 176: The method of Aspect 174, wherein the CAR regulatory T cell is generated by differentiating the pluripotent stem cell into a T cell using artificial thymic organoids.

[0237] Aspect 177: The method of Aspect 174, wherein the CAR regulatory T cell is generated by coculture with stromal cells that express the Notch ligand.

[0238] Aspect 178: The method of Aspect 174, wherein the regulatory T cell produces immune modulatory cytokines.

[0239] Aspect 179: The method of Aspect 174, wherein the regulatory T cell is subsequently treated in culture with one or a plurality of injury-associated antigens.

[0240] Aspect 180: The method of Aspect 179, wherein the one or plurality of injury-associated antigens comprise thrombin, chondroitin sulfate, NOGO, calreticulin, heat shock protein, vimentin, epidermal growth factor receptor, IL- 10 receptor, TLR4, IL- 4 receptor, IL- 12 receptor, IL- 15 receptor, IL- 17 receptor, IL- 18 receptor, IL-27 receptor, IL-33 receptor, TLR5, TLR7, TLR9, TNF-alpha receptor p55, and TNF-alpha receptor p75.

[0241] Aspect 181 : The method of Aspect 179, wherein the CAR regulatory T cell is administered to the subject.

[0242] Aspect 182: The method of Aspect 179, wherein one or a plurality of soluble factors are isolated from the culture following treatment with the one or plurality of injury-associated antigens.

[0243] Aspect 183: The method of Aspect 182, wherein the one or plurality of soluble factors are administered to the subject.

[0244] Aspect 184: The method of Aspect 80, wherein the mesenchymal stem cell is treated with one or a plurality of stressing agents.

[0245] Aspect 185: The method of Aspect 184, wherein the stressing agent comprises cobalt chloride, interferon gamma, TNF-alpha, poly(I:C), or a combination thereof.

[0246] Aspect 186: The method of Aspect 184, wherein the one or plurality of stressing agents are added to the induce or enhance the production of one or a plurality of soluble factors by the mesenchymal stem cell.

[0247] Aspect 187: The method of Aspect 186, wherein the one or plurality of soluble factors comprise vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and ciliary neurotrophic factor (CTNF).

[0248] Aspect 188: The method of Aspect 187, wherein at least 10 ng / mL, at least 50 ng / mL, or at least 100 ng / mL of VEGF are produced in cultures of approximately 1 million mesenchymal stem cells

[0249] Aspect 189: The method of Aspect 187, wherein at least 50 pg / mL, at least 100 pg / mL, or at least 200 pg / mL of EGF are produced in cultures of approximately 1 million mesenchymal stem cells

[0250] Aspect 190: The method of Aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of BDNF are produced in cultures of approximately 1 million mesenchymal stem cells

[0251] Aspect 191 : The method of Aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of NGF are produced in cultures of approximately 1 million mesenchymal stem cells

[0252] Aspect 192: The method of Aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of CTNF are produced in cultures of approximately 1 million mesenchymal stem cells

[0253] Aspect 193: The method of Aspect 80, wherein the mesenchymal stem cell is cultured with allogeneic stem cells prior to administration to the subject.

[0254] Aspect 194: The method of Aspect 193, wherein the allogeneic stem cells are induced pluripotent stem cells.

[0255] Aspect 195: The method of Aspect 1, wherein the primary somatic cell is harvested from an allogeneic tissue or organ source, and wherein the allogeneic tissue or organ source comprises umbilical cord tissue, umbilical cord blood, peripheral blood. Wharton’s jelly, bone marrow, adipose tissue, endometrium, muscle, skin, bone, cardiac tissue, renal tissue, hepatic tissue, and neural tissue.

[0256] Aspect 196: The method of Aspect 1, wherein the primary somatic cell is harvested from an autologous tissue or organ.

[0257] Aspect 197: The method of Aspect 1, wherein the one or plurality of differentiated somatic cell types are administered to the subject in combination with an angiogenic agent.

[0258] Aspect 198: The method of Aspect 197, wherein the angiogenic agent is administered to the subject prior to, following, or concurrent with the administration of the one or plurality of differentiated somatic cell types, or at a combination of time points.

[0259] Aspect 199: The method of Aspect 1, wherein the one or plurality of soluble factors are administered to the subject in combination with an angiogenic agent.

[0260] Aspect 200: The method of Aspect 199, wherein the angiogenic agent is administered to the subject prior to, following, or concurrent with the administration of the one or plurality of soluble factors, or at a combination of time points.

[0261] Aspect 201 : The method of Aspect 197, wherein the angiogenic agent comprises one or a plurality of factors comprising activin A, adrenomedullin, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, endothelin-1, endothelial differentiation gene 1 (EDG1), ephrin, erythropoietin, beta thymosin 4, PDGF, PDGF-BB, IL-8, IL-20, IL-22, IL-38, fibrin fragment E, factor X, HB-EGF, HBNF, KFGF, leukemia inhibitory factor, leiomyoma-derived growth factor, macrophage-derived growth factor, MCP-1, MD-EC1, GC-MAF, MECIF, MMP2, MMP3, MMP9, urokinase plasminogen activator, neuropilin, neurothelin, notch, occludin, zona occludins, oncostatin M, PDECGF, placental factor 4, Pl GF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-164, VEGF-1, EC-VEGF, transferrin, thrombospondin, and urokinase.

[0262] Aspect 202: The method of Aspect 200, wherein the angiogenic agent comprises one or a plurality of factors comprising activin A, adrenomedullin, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4,endothelin-1, endothelial differentiation gene 1 (EDG1), ephrin, erythropoietin, beta thymosin 4, PDGF, PDGF-BB, IL-8, IL-20, IL-22, IL-38, fibrin fragment E, factor X, HB-EGF, HBNF, KFGF, leukemia inhibitory factor, leiomyoma-derived growth factor, macrophage-derived growth factor, MCP-1, MD-EC1, GC-MAF, MECIF, MMP2, MMP3, MMP9, urokinase plasminogen activator, neuropilin, neurothelin, notch, occludin, zona occludins, oncostatin M, PDECGF, placental factor 4, Pl GF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-164, VEGF-1, EC-VEGF, transferrin, thrombospondin, and urokinase.

[0263] Aspect 203: The method of Aspect 197, wherein the angiogenic agent is delivered to the subject in a specific form, and wherein the form comprises a protein, a protein mimetic, a receptor agonist, a DNA plasma, a microRNA, an mRNA, an active peptide, in a transfected mesenchymal stem cell, a transfected monocyte, a transfected B cell, transfected Bl cell, a transfected neural progenitor cell, a transfected radial cell, a transfected oligodendrocyte, a transfected astrocyte, a transfected fibroblast, or combinations thereof.

[0264] Aspect 204: The method of Aspect 199, wherein the angiogenic agent is delivered to the subject in a specific form, and wherein the form comprises a protein, a protein mimetic, a receptor agonist, a DNA plasma, a microRNA, an mRNA, an active peptide, in a transfected mesenchymal stem cell, a transfected monocyte, a transfected B cell, transfected Bl cell, a transfected neural progenitor cell, a transfected radial cell, a transfected oligodendrocyte, a transfected astrocyte, a transfected fibroblast, or combinations thereof.

[0265] Aspect 205: The method of Aspect 1, wherein the one or plurality of differentiated somatic cell types are administered to the subject in combination with a stem cell chemoattracting cytokine.

[0266] Aspect 206: The method of Aspect 205, wherein the stem cell chemoattracting cytokine is administered to the subject prior to, following, or concurrent with the administration of the one or plurality of differentiated somatic cell types, or at a combination of time points.

[0267] Aspect 207: The method of Aspect 205, wherein the stem cell chemoattracting cytokine comprises one or a plurality of factors comprising CXCL12, MIP-1 alpha, MIP-1 beta, VEGF, and RANTES.

[0268] Aspect 208: The method of Aspect 1, wherein the one or plurality of soluble factors are administered to the subject in combination with a stem cell chemoattracting cytokine.

[0269] Aspect 209: The method of Aspect 208, wherein the stem cell chemoattracting cytokine is administered to the subject prior to, following, or concurrent with the administration of the one or plurality of soluble factors, or at a combination of time points.

[0270] Aspect 210: The method of Aspect 208, wherein the stem cell chemoattracting cytokine comprises one or a plurality of factors comprising CXCL12, MIP-1 alpha, MIP-1 beta, VEGF, and RANTES.

[0271] Aspect 211 : The method of Aspect 1, wherein a culture medium is provided for differentiating pluripotent stem cells into hematopoietic stem cells.

[0272] Aspect 212: The method of Aspect 211, wherein the hematopoietic stem cells release one or a plurality of soluble factors into culture.

[0273] Aspect 213: The method of Aspect 212, wherein the soluble factors comprise exosomes.

[0274] Aspect 214: The method of Aspect 211, wherein the hematopoietic stem cells are defined by expression of one or a plurality of markers comprising CD34, CD133, c-kit, stem cell factor receptor, CD127, fas ligand, GITR ligand, STAT4, SCA-1, hTERT, NANOG, IL-6 receptor, TGF-beta receptor, autocrine TGF-beta, and trk75.

[0275] Aspect 215: The method of Aspect 211, wherein the hematopoietic stem cells are defined by lack of expression or low expression of one or a plurality of markers comprising CD38, CD16, CD14, and CD45.

[0276] Aspect 216: The method of Aspect 213, wherein the exosomes are defined by expression of one or a plurality of markers comprising CD34, c-kit, stem cell factor receptor, CD 127, fas ligand, GITR ligand, STAT4, SCA-1, hTERT, NANOG, IL-6 receptor, TGF-beta receptor, autocrine TGF-beta, and trk75.

[0277] Aspect 217: The method of Aspect 213, wherein the exosomes are defined by lack of expression or low expression of one or a plurality of markers comprising CD38, CD 16, CD 14, and CD45.

[0278] Aspect 218: The method of Aspect 211, wherein the hematopoietic stem cells are allogeneic to the subject.

[0279] Aspect 219: The method of Aspect 211, wherein the hematopoietic stem cells are autologous to the subject.

[0280] Aspect 220: The method of Aspect 211, wherein the hematopoietic stem cells are xenogeneic to the subject.

[0281] Aspect 221 : The method of Aspect 211, wherein the pluripotent stem cell is derived from a dedifferentiated somatic cell, and wherein the somatic cell is extracted from a tissue comprising bone marrow, peripheral blood, mobilized peripheral blood, cord blood, or adipose tissue.

[0282] Aspect 219: The method of Aspect 1, wherein the third cell culture method provides a culture media comprising a cell stressor.

[0283] Aspect 220: The method of Aspect 219, wherein the cell stressor comprises one or a plurality of HMGB1, flagellin protein, imiquimod, poly(I:C), and Bacillus Calmette-Guerin (BCG).

[0284] Aspect 221 : The method of Aspect 219, wherein the cell stressor induces the generation of soluble factors.

[0285] Aspect 222: The method of Aspect 221, wherein the soluble factors comprise exosomes.

[0286] Aspect 223: The method of Aspect 1, wherein the third cell culture comprises hematopoietic stem cells, and wherein the culture media comprises a cell stressor.

[0287] Aspect 224: The method of Aspect 223, wherein the cell stressor comprises one or a plurality of HMGB1, flagellin protein, imiquimod, poly(I:C), and Bacillus Calmette-Guerin (BCG).

[0288] Aspect 225: The method of Aspect 223, wherein the cell stressor induces the generation of soluble factors.

[0289] Aspect 226: The method of Aspect 225, wherein the soluble factors comprise exosomes.

[0290] Aspect 227: The method of Aspect 223, wherein the hematopoietic stem cells express CD34.

[0291] Aspect 228: The method of Aspect 223, wherein the cell stressor is administered at a sufficient concentration and for a sufficient time to stimulate production of one or a plurality of factors comprising matrix metalloproteinase- 1 (MMP1), TGF- beta, brain-derived neurotrophic factor (BDNF), soluble PD-L1, irisin, and soluble HLA- G.

[0292] Aspect 229: The method of Aspect 228, wherein the cell stressor stimulates production of at least 10 ng / mL, at least 20 ng / mL, or at least 50 ng / mL of MMP from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0293] Aspect 230: The method of Aspect 228, wherein the cell stressor stimulates production of at least 50 ng / mL, at least 100 ng / mL, or at least 200 ng / mL of TGF-beta from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0294] Aspect 231 : The method of Aspect 228, wherein the cell stressor stimulates production of at least 1 ng / mL, at least 5 ng / mL, or at least 10 ng / mL of BDNF from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0295] Aspect 232: The method of Aspect 228, wherein the cell stressor stimulates production of at least 20 ng / mL, at least 40 ng / mL, or at least 80 ng / mL of soluble PD-L1 from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0296] Aspect 233: The method of Aspect 228, wherein the cell stressor stimulates production of at least 2 ng / mL, at least 4 ng / mL, or at least 8 ng / mL of irisin from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0297] Aspect 234: The method of Aspect 228, wherein the cell stressor stimulates production of at least 100 pg / mL, at least 200 pg / mL, or at least 400 pg / mL of soluble HLA-G from a culture comprising approximately 1 million CD34+ hematopoietic stem cells.

[0298] Aspect 235: The method of Aspect 1, wherein the subject is also administered one or a plurality of agents capable of increasing regulatory T cell numbers locally or systemically.

[0299] Aspect 236: The method of Aspect 235, wherein the one or plurality of agents capable of increasing regulatory T cell activity comprise IL-2, IL-10, TGF-beta, and a cell population associated with tolerance induction.

[0300] Aspect 237: The method of Aspect 236, wherein the cell population associated with tolerance induction comprises monocytes.

[0301] Aspect 238: The method of Aspect 237, wherein the monocytes are treated with one or a plurality of cytokines and growth factors comprising IL-4, IL-10, TGF-beta, VEGF, and an inhibitor of IFN-gamma.

[0302] Aspect 239: The method of Aspect 238, wherein the one or plurality of agents endow the monocytes with the ability to produce arginase and / or the ability to produce more arginase than nitric oxide synthase.

[0303] Aspect 240: The method of Aspect 237, wherein the monocytes are transfected with one or a plurality of the following molecules comprising CTLA-4, PD-1, VISTA, B7-H2, B7-H3, B7-H4, PD-L1, ICOS, HVEM / CD270, PD-L2, CD160, gp49B, PIR-B, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-1BB, OX-40, BTLA, CD47, CD48, CD244, ILT2, ILT4, TIGIT, A2aR, IL-1 receptor antagonist, HLA-G, fas ligand, TNF receptor p55, and TNF receptor p75.

[0304] Aspect 241 : The method of Aspect 236, wherein the cell population associated with tolerance induction comprises mesenchymal stem cells.

[0305] Aspect 242: The method of Aspect 241, wherein the mesenchymal stem cells are transfected with one or a plurality of the following molecules comprising CTLA-4, PD-1, VISTA, B7-H2, B7-H3, B7-H4, PD-L1, ICOS, HVEM / CD270, PD-L2, CD160, gp49B, PIR-B, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-1BB, OX-40, BTLA, CD47, CD48, CD244, ILT2, ILT4, TIGIT, A2aR, IL-1 receptor antagonist, HLA-G, fas ligand, TNF receptor p55, and TNF receptor p75.

[0306] Aspect 243: The method of Aspect 1, wherein extracorporeal pulsed ultrasound is administered prior to, following, or concurrent with the administration of the one or plurality of differentiated somatic cell types from the second culture method to the subject.

[0307] Aspect 244: The method of Aspect 243, wherein the differentiated somatic cell comprises a regulatory T cell.

[0308] Aspect 245: The method of Aspect 243, wherein the differentiated somatic cell comprises an oligodendrocyte progenitor cell.

[0309] Aspect 246: The method of Aspect 245, wherein a complement inhibitor is administered to the subject prior to, following, or concurrent with the administration of the oligodendrocyte progenitor cell.

[0310] Aspect 247: The method of Aspect 246, wherein the complement inhibitor comprises one or a plurality of agents comprising anti-CD5 antibody, anti-CD3 antibody, cobra venom factor, Factor H, CD55, CD37, and CD73.

[0311] Aspect 248: The method of Aspect 1, wherein an age-related disease, disorder, or symptom is associated with a decline in cytotoxic activity of T cells isolated from the subject.

[0312] Aspect 249: The method of Aspect 248, wherein cytotoxic activity of T cells is ascertained based on measuring perforin production, granzyme B production, expression of fas ligand, or a combination thereof.

[0313] Aspect 250: The method of Aspect 1, wherein an age-related disease, disorder, or symptom is associated with an increase in the production of one or a plurality of cytokines by cells isolated from a tissue of the subject.

[0314] Aspect 251 : The method of Aspect 250, wherein the cells are selected from the group comprising fibroblasts, mesenchymal stem cells, T cells, B cells, dendritic cells, neutrophils, monocytes, macrophages, or peripheral blood mononuclear cells.

[0315] Aspect 252: The method of Aspect 250, wherein the one or plurality of cytokines comprise C-reactive protein (CRP), interferon-gamma, TNF-alpha, IL-lalpha, IL-lbeta, IL-6, IL-12, IL-15, IL-17, IL-18, IL-27, G-CSF, GM-CSF, MCP-1, or a combination thereof.

[0316] Aspect 253 : The method of Aspect 250, wherein the increase in production of one or a plurality of cytokines is measured by comparing the levels of the one or plurality of cytokines in the subject to with age-related disease, disorder, or symptom to the levels of the same cytokine(s) in a second subject who is not afflicted with the age- related disease, disorder, or symptom.

[0317] Aspect 245: The method of Aspect 1, wherein the primary somatic cell is selected from the group consisting of a fibroblast, a T cell, a B cell, a myeloid cell, a neutrophil, an eosinophil, a basophil, a myeloblast, a monocyte, a macrophage, a mesenchymal stem cell, a keratinocyte, an epithelial cell, an exfoliated renal epithelial cell, an epithelial progenitor cell, a hematopoietic stem cell, a common myeloid progenitor cell, a natural killer (NK) cell, an NK T cell, a common lymphoid progenitor cell, an endothelial cell, an endothelial progenitor cell, a synovial cell, a pancreatic cell, a mobilized CD34+ peripheral blood mononuclear cell, an isolated blood mononuclear cell, or a tissue-specific stem or progenitor cell.

[0318] In certain embodiments, the invention provides a medical device allowing for perfusion of patient blood or plasma over regenerative cells in a manner in which said regenerative cells do not escape the therapeutic device.

[0319] In one embodiment, the invention provides a device which isolates said cells from contact with other cells but allows for production and excretion of soluble factors from such cells. In another embodiment said personalized regenerative cells, and / or cells derived from said cells are placed in a device which allows for passage of blood through said device while maintaining personalized regenerative cells in said device. One use of the invention is treatment of age-related pathology. In other embodiments, the invention provides means of treating inflammatory conditions and allowing for host generatedinflammatory mediators to interact with cells and stimulate regenerative cells to produce anti-inflammatory compounds in response to inflammatory agents provided.

[0320] In one embodiment the invention describes a system for maintaining immunological and / or regenerative cells alive and functioning in an extracorporeal manner, while allowing for contain of said cells with blood or plasma of the patient. The contact may be as a continuous loop, or may be in segmented intervals. The present invention discloses regenerative cells such as pluripotent stem cells used for production of growth factors and / or anti-inflammatory factors. The device disclosed allows for circulation of blood or plasma in a manner so that blood or plasma released materials are capable of interacting with regenerative cells and in some embodiments altering the functionality of said regenerative cells. The invention teaches that since a culture of regenerative cells can form a reticular structure in the form of a sheet, they conducted various researches on the shape of a module that can constitute an extracorporeal device in which such a reticular structure in the form of a sheet is maintained. As a result, the invention teaches culture vessel constituted by two chambers of a part in which plasma ingredients from a patient circulate, and a part for culturing regenerative cells separated by a separation membrane from the former part, found that such a culture vessel is suitable for culturing various regenerative cells and / or immunomodulatory cells.

[0321] Accordingly, the present invention thus provides an extracorporeal rejuvenation / immune modulation device having at least one module comprising the followings: a) a plasma ingredient inlet port, and a plasma ingredient outlet port; b) a plasma ingredient circulation chamber having the plasma ingredient inlet port, and the plasma ingredient outlet port; and c) a culture chamber provided adjacently to the plasma ingredient circulation chamber and separated from the plasma ingredient circulation chamber with a separation membrane through which biological molecules can permeate, but cells cannot permeate. In some embodiments the cells utilized for insertion into the device are pretreated with molecules to enhance therapeutic activity of said cells. These molecules may be selected from a group comprising: cyclosporine, rapamycin, campath- 1H, ATG, Prograf, anti-IL-2r, MMF, FTY, LEA, cyclosporin A, diftitox, denileukin, levamisole, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, and trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, and thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine,emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, and tegafur) fluocinolone, triaminolone, anecortave acetate, fluoromethoIone, medrysone, prednislone, etc. In another embodiment, the use of stem cell conditioned media may be used to potentiate an existing anti-inflammatory agent. Anti-inflammatory agents may comprise one or more agents including NS AIDs, interleukin-1 antagonists, dihydroorotate synthase inhibitors, p38 MAP kinase inhibitors, TNF-a inhibitors, TNF-a sequestration agents, and methotrexate. More specifically, anti-inflammatory agents may comprise one or more of, e.g., anti-TNF-a, lysophylline, alpha 1-antitrypsin (AAT), interleukin- 10 (IL-10), pentoxyfilline, COX-2 inhibitors, 21 -acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluoromethoIone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, predni carb ate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (eg., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones(e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1 -naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lomoxicam, piroxicam, tenoxicam), epsilon. -acetamidocaproic acid, s- adenosylmethionine, 3-amino-4-hydroxybutyric .acid, amixetrine, bendazac, benzydamine, a-bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, zileuton, candelilla wax, alpha bisabolol, aloe vera, Manjistha, Guggal, kola extract, chamomile, sea whip extract, glycyrrhetic acid, glycyrrhizic acid, oil soluble licorice extract, monoammonium glycyrrhizinate, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate, 1-beta-glycyrrhetic acid, stearyl glycyrrh etinate, and 3-stearyloxy-glycyrrhetinic acid.

[0322] In some embodiments, growth factors are added to maintain the viability and / or functionality of regenerative cells ex vivo. Suitable growth factors useful for the practice of some methods disclosed herein include adrenomedullin (AM), angiopoietin (Ang), bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (Epo), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GNDF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth differentiation factor (GDF-9), hepatocyte growth factor (HGF), hepatoma derived growth factor (HDGF), insulin-like growth factor (IGF), migration-stimulating factor, myostatin (GDF-8), myelomonocytic growth factor (MGF), nerve growth factor (NGF), placental growth factor (P1GF), platelet-derived growth factor (PDGF), thrombopoietin (Tpo), transforming growth factor alpha (TGF-a), TGF-P, tumor necrosis factor alpha (TNF-a), vascular endothelial growth factor (VEGF), or a Wnt protein.

[0323] Certain embodiments of the invention provide a method for preventing, reducing, or ameliorating age-associated fibrosis. In certain embodiments, cytokines may be added to one or a plurality of the culture methods or steps disclosed herein to enhance regenerative factor production, said cytokines are required to endow properties onto thevarious cells to stimulate transcription and other factors associated with supporting viability and mitogenesis while suppressing fibrosis. In some embodiments MSC are treated first with cytokines before co-culture. Suitable cytokines for the practice of some methods disclosed herein are selected from the group consisting of AM, Ang, BMP, BDNF, EGF, Epo, FGF, GNDF, G-CSF, GM-CSF, GDF-9, HGF, HDGF, IGF, migration-stimulating factor, GDF-8, MGF, NGF, P1GF, PDGF, Tpo, TGF-a, TGF-P, TNF-a, VEGF, or a Wnt protein; an interleukin; a soluble receptor for IL- la, IL-ip, IL- 1F1, IL-1F2, IL-1F3, IL-1F4, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 35 kDa alpha subunit, IL-12 40 kDa beta subunit, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F isoform 1, IL-17F isoform 2, IL- 18, IL- 19, IL-20, IL-21, IL-22, IL-23 pl9 subunit, IL-23 p40 subunit, IL-24, IL-25, IL-26, IL-27B, IL-27-p28, IL-28A, IL-28B, IL-29, IL-30, IL- 31, IL-32, IL-33, IL-34, IL-35, IL-36a, IL-36P, IL-36y; an interferon (IFN); a soluble receptor for IFN-a, IFN-P, IFN-y, IFN-.lamda.1, IFN-.lamda.2, IFN-.lamda.3, IFN-K, IFN-8, IFN-K, IFN-T, IFN-5, IFN-^, IFNCO, or IFN-u; insulin or proinsulin; a receptor for insulin; leptin (LEP). In some embodiments, cytokines and / or growth factors are administered to said cells continually through a micropump connected to an encapsulation device that houses said tolerogenic cells. Particular encapsulation devices of interest may be obtained from U.S. Pat. Nos. 5,725,854; 5,849,285; 5,759,534; 5,843,430; 5,958,404; and 6,149,907.

[0324] Various cells may be utilized for the practice of the invention. In one embodiment the invention teaches phenotypically defined pluripotent stem cells which can be generated from isolated Wharton’s jelly of umbilical cord segments and defined morphologically and by cell surface markers. In some embodiments Wharton’s Jelly used as starting cells for dedifferentiation processes. By dissecting out the veins and arteries of cord segments and exposing the Wharton’s jelly, the cells of invention, of one embodiment of the invention, may be obtained. An approximately 1-5 cm cord segment is placed in collagenase solution (1 mg / ml, Sigma) for approximately 18 hrs at room temperature. After incubation, the remaining tissue is removed, and the cell suspension is diluted with PBS into two 50 ml tubes and centrifuged. Cells are then washed in PBS and counted using hematocytometer. 5-20. times.10. sup.6 cells were then plated in a 6 cm tissue culture plate in low-glucose DMEM (Gibco) with 10% FBS (Hyclone), 2 mM L- Glutamine (Gibco), 100 U / ml penicillin / 100 ug / ml streptomycin / 0.025 ug / ml amphotericin B (Gibco). At this step of the purification process, cells are exposed tohypoxia. The amount of hypoxia needed is the sufficient amount to induce activation of HIF-1 alpha. In one embodiment cells are cultured for 24 hours at 2% oxygen. After 48 hours, the cells are washed with PBS and given fresh media. Cells were given new media twice weekly. After 7 days, cells are approximately 70-80% confluent and are passed using HyQTase (Hyclone) into a 10 cm plate. Cells are then regularly passed 1 :2 every 7 days or upon reaching 80% confluence.

[0325] In certain embodiments, biologically useful stem cells such as autologous pluripotent stem cells are disclosed for application to the methods of the invention. These maybe be undifferentiated, differentiated into progenitor cells, or differentiated into mature or semi-mature cells. In some cases, said cells are of the mesenchymal or related lineages, which are therapeutically reprogrammed cells having minimal oxidative damage and telomere lengths that compare favorably with the telomere lengths of undamaged, pre-natal or embryonic stem cells (that is, the therapeutically reprogrammed cells of the present invention possess near prime physiological state genomes). Moreover, the therapeutically reprogrammed cells of the present invention are immunologically privileged and therefore suitable for therapeutic applications. Additional methods of the present invention provide for the generation of hybrid stem cells. Furthermore, the present invention includes related methods for maturing stem cells made in accordance with the teachings of the present invention into specific host tissues. For use in the current invention, the practitioner is thought that ontogeny of mammalian development provides a central role for stem cells. Early in embryogenesis, cells from the proximal epiblast destined to become germ cells (primordial germ cells) migrate along the genital ridge. These cells express high levels of alkaline phosphatase as well as expressing the transcription factor Oct4. Upon migration and colonization of the genital ridge, the primordial germ cells undergo differentiation into male or female germ cell precursors (primordial sex cells). For the purpose of this disclosure, only male primordial sex cells (PSC) will be discussed, but the qualities and properties of male and female primordial sex cells are equivalent, and no limitations are implied. During male primordial sex cell development, the primordial stem cells become closely associated with precursor Sertoli cells leading to the beginning of the formation of the seminiferous cords. When the primordial germ cells are enclosed in the seminiferous cords, they differentiate into gonocytes that are mitotically quiescent. These gonocytes divide for a few days followed by arrest at G0 / G1 phase of the cell cycle. In mice and rats these gonocytes resume division within a few days after birth to generate spermatogonial stem cells andeventually undergo differentiation and meiosis related to spermatogenesis. It is known that embryonic stem cells are cells derived from the inner cell mass of the preimplantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From a practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural epiblast environment, they only exist transiently during embryogenesis. Manipulation of embryonic stem cells in vitro leads to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage. Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and / or dedifferentiation. In one example of the maturation process, a cell, or group of cells, interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has progressed such that cells and appropriate cellular niches are present for the organism to function and survive postnatally. Developmental processes are highly conserved amongst the different species allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory. During the lifetime of an organism, the cellular composition of the organs and organs systems are exposed to a wide range of intrinsic and extrinsic factors that induce cellular or genomic damage. Ultraviolet light not only has an effect on normal skin cells but also on the skin stem cell population. Chemotherapeutic drugs used to treat cancer have a devastating effect on hematopoietic stem cells. Reactive oxygen species, which are the byproducts of cellular metabolism, are intrinsic factors that compromises the genomic integrity of the cell. In all organs or organ systems, cells are continuously being replaced from stem cell populations. However, as an organism ages, cellular damage accumulates in these stem cell populations. If the damage is inheritable, such as genomic mutations, then all progeny will be affected and thus compromised. A single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged. The body responds to a compromised stem cell by inducing apoptosis thereby removing it from thepool and preventing potentially dysfunctional or tumorigenic properties. Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homing mechanism of stem cells. Telomeres are the physical ends of chromosomes that contain highly conserved, tandemly repeated DNA sequences. Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre-determined threshold, a signal is activated to initiate cellular senescence. Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress. In one teaching, or embodiment, of the invention, therapeutically reprogrammed cells, in some embodiments mesenchymal stem cells, are provided. Therapeutic reprogramming refers to a maturation process wherein a stem cell is exposed to stimulatory factors according the teachings of the present invention to yield enhanced therapeutic activity. In some embodiments, enhancement of therapeutic activity may be increase proliferation, in other embodiments, it may be enhanced chemotaxis. Other therapeutic characteristics include ability to under resistance to apoptosis, ability to overcome senescence, ability to differentiate into a variety of different cell types effectively, and ability to secrete therapeutic growth factors which enhance viability / activity, of endogenous stem cells. In order to induce therapeutic reprogramming of cells, in some cases, as disclosed herein, of Wharton’s jelly originating cells, the invention teaches the utilization of stimulatory factors, including without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells. These stimulatory factors induce, among other results, genomic methylation changes in the donor DNA. Embodiments of the present invention include methods for preparing cellular extracts from whole cells, cytoplasts, and karyoplasts, although other types of cellular extracts are contemplated as being within the scope of the present invention. In a non-limiting example, the cellular extracts of the present invention are prepared from stem cells, specifically embryonic stem cells. Donor cells are incubated with the chemicals, biochemicals or cellular extracts for defined periods of time, in a nonlimiting example for approximately one hour to approximately two hours, and those reprogrammed cells that express embryonic stem cell markers, such as Oct4, after a culture period are then ready for transplantation, cry opreservation or further maturation.In another embodiment of the present invention, hybrid stem cells are provided which can be used for cellular regenerative / reparative therapy. The hybrid stem cells of the present invention are pluripotent and customized for the intended recipient so that they are immunologically compatible with the recipient. Hybrid stem cells are a fusion product between a donor cell, or nucleus thereof, and a host cell. Typically, the fusion occurs between a donor nucleus and an enucleated host cell. The donor cell can be any diploid cell, including but not limited to, cells from pre-embryos, embryos, fetuses and post-natal organisms. More specifically, the donor cell can be a primordial sex cell, including but not limited to, oogonium or differentiated or undifferentiated spermatogonium, or an embryonic stem cell. Other non-limiting examples of donor cells are therapeutically reprogrammed cells, embryonic stem cells, fetal stem cells and multipotent adult progenitor cells. Preferably the donor cell has the phenotype of the intended recipient. The host cell can be isolated from tissues including, but not limited to, pre-embryos, embryos, fetuses and post-natal organisms and more specifically can include, but is not limited to, embryonic stem cells, fetal stem cells, multipotent adult progenitor cells and adipose-derived stem cells. In a non-limiting example, cultured cell lines can be used as donor cells. The donor and host cells can be from the same individual or different individuals. In one embodiment of the present invention, lymphocytes are used as donor cells and a two-step method is used to purify the donor cells. After the tissues was disassociated, an adhesion step was performed to remove any possible contaminating adherent cells followed by a density gradient purification step. The majority of lymphocytes are quiescent (in GO phase) and therefore can have a methylation status than conveys greater plasticity for reprogramming. Multipotent or pluripotent stem cells or cell lines useful as donor cells in embodiments of the present invention are functionally defined as stem cells by their ability to undergo differentiation into a variety of cell types including, but not limited to, adipogenic, neurogenic, osteogenic, chondrogenic and cardiogenic cell.

[0326] In some embodiments, host cell enucleation for the generation of hybrid stem cells according to the teachings of the present invention can be conducted using a variety of means. In a non-limiting example, ADSCs were plated onto fibronectin coated tissue culture slides and treated with cells with either cytochalasin D or cytochalasin B. After treatment, the cells can be trypsinized, re-plated and are viable for about 72 hours post enucleation. Host cells and donor nuclei can be fused using one of a number of fusion methods known to those of skill in the art, including but not limited to electrofusion,microinj ection, chemical fusion or virus-based fusion, and all methods of cellular fusion are envisioned as being within the scope of the present invention. The hybrid stem cells made according to the teachings of the present invention possess surface antigens and receptors from the enucleated host cell but has a nucleus from a developmentally younger cell. Consequently, the hybrid stem cells of the present invention will be receptive to cytokines, chemokines and other cell signaling agents, yet possess a nucleus free from age-related DNA damage. The therapeutically reprogrammed cells and hybrid stem cells made in accordance with the teachings of the present invention are useful in a wide range of therapeutic applications for cellular regenerative / reparative therapy. For example, and not intended as a limitation, the therapeutically reprogrammed cells and hybrid stem cells of the present invention can be used to replenish stem cells in animals whose natural stem cells have been depleted due to age or ablation therapy such as cancer radiotherapy and chemotherapy. In another non-limiting example, the therapeutically reprogrammed cells and hybrid stem cells of the present invention are useful in organ regeneration and tissue repair. In one embodiment of the present invention, therapeutically reprogrammed cells and hybrid stem cells can be used to reinvigorate damaged muscle tissue including dystrophic muscles and muscles damaged by ischemic events such as myocardial infarcts. In another embodiment of the present invention, the therapeutically reprogrammed cells and hybrid stem cells disclosed herein can be used to ameliorate scarring in animals, including humans, following a traumatic injury or surgery. In this embodiment, the therapeutically reprogrammed cells and hybrid stem cells of the present invention are administered systemically, such as intravenously, and migrate to the site of the freshly traumatized tissue recruited by circulating cytokines secreted by the damaged cells. In another embodiment of the present invention, the therapeutically reprogrammed cells and hybrid stem cells can be administered locally to a treatment site in need of repair or regeneration.

[0327] In one embodiment, a source of regenerative cells comprises umbilical cord samples were obtained following the delivery of normal term babies. Methods of the invention are provided for processing the umbilical cord for cell isolation. By way of example, a portion of the umbilical cord is cut into approximately 3 cm long segments. Each umbilical cord segment is dissected to remove the umbilical vein and both umbilical arteries. The remaining segment is sutured inside out and incubated in PBS, antibiotic, and collagenase at room temperature. After 16-18 hours, the remaining suture and connective tissue is removed and discarded. The cell suspension is then subjected to redblood cell lysis and the cells are plated in the appropriate medium for supporting the growth of umbilical cord mesenchymal stem cells.

[0328] One object of the invention is to provide a bioreactor and method that will allow the growth of three-dimensional tissue, wherein said bioreactor is perfused and said perfusate is utilized for stimulation of regeneration and / or immune modulation. The foregoing and other objects are achieved by this invention which provides a system for maintaining and culturing three-dimensional tissues in vitro. In one embodiment, the system comprises a bioreactor that has a tissue disposed between two media flows. The tissue is cultured on a porous substrate. A first media flow having a first concentration of solutes is in direct contact with one side of the tissue, and a second media flow having a second concentration of solutes is in direct contact with the opposing side of the tissue. In one embodiment, the first concentration of solutes is different than the second concentration of solutes, creating a diffusional or osmotic pressure gradient which causes movement of solutes, including nutrients, through the tissue. The media flows may also be used to supply gases and growth factors to the cells in varying concentrations, depending on the needs of the particular tissue being cultured. Metabolic waste products, including carbon dioxide, are secreted into the media flows and carried away. In an alternative embodiment, a semi-permeable membrane can be disposed intermediate the tissue and each of the two media flows. A membrane that allows the passage of desired solutes and waste products should be utilized.

[0329] In another embodiment, the disclosure provides a system comprising a bioreactor that has a tissue, cultured on a porous membrane, disposed within a housing, with a first space between the tissue and a top portion of the housing, a second space between the opposing side of the tissue and a bottom portion of the housing, media inlet ports for providing a first media flow into the first space and a second media flow into the second space, and at least one media outlet port. As described above, the concentrations of solutes, including nutrients, growth factors and gases, may be different for the first and second media flows such that the solutes, nutrients, growth factors and / or gases move through the tissue by a diffusion or osmotic pressure gradient.

[0330] The invention provides methods for generating cell-free therapeutic means comprising soluble factors from pluripotent stem cells and their derivatives. In certain embodiments, a pluripotent stem cell is an induced pluripotent stem cell that originates from an already-differentiated somatic cell that has been isolated directly from a tissue or organ of the subject, i.e., a primary somatic cell. In certain embodiments, the primarysomatic cell is converted to an induced pluripotent stem cell by the process of dedifferentiation or reprogramming using reprogramming transcription factors. In certain embodiments, the reprogramming factors comprise Oct3 / 4, Sox2, Klf4, and C-myc. Means of dedifferentiating cells are known in the art and include treatment of cells with agents that act epigenetically, which may be utilized alone or in combination with inducible pluripotent stem cell techniques known in the art. In one embodiment, somatic cells are dedifferentiated by treatment with a DNA methyltransferase inhibitor said inhibitors include nucleoside analogues and non-nucleoside analogues. Exemplary nucleoside analogs include 5-Azacytidine (which may be used at a concentration of 100 nM to 10 pM), 5-Aza-2'-deoxy cytidine (which may be used at a concentration of 100 nM to 10 pM), 5-Fluoro-2'-deoxy cytidine (which may be used at a concentration of 100 nM to 10 pM), 5,6-Dihydro-5-azacytidine (which may be used at a concentration of 100 nM to 10 pM), and Zebularine (which may be used at a concentration of 1 pM to 10 mM). Exemplary non-nucleoside analogues include Hydralazine (which may be used at a concentration of 100 nM to 10 pM), Procainamide (which may be used at a concentration of 1000 nM to 10 pM), EGCG (which may be used at a concentration of 100 nM to 10 pM), Psammaplin A (which may be used at a concentration of 100 nM to 10 pM), MG98 (which may be used at a concentration of 100 nM to 10 pM), and RG108 (which may be used at a concentration of 100 nM to 10 pM).

[0331] Exemplary histone-deacetylase inhibitors include short chain fatty acids, hydroxamic acids, cyclic benzamides, cyclic tetrapeptides, and benzamides. Exemplary short chain fatty acids include Butyrate (which may be used at a concentration of 1 pM to 10 mM) and Valproic acid (which may be used at a concentration of 1 pM to 10 mM). Exemplary hydroxamic acids include m-carboxy cinnamic acid bishydroxamic acid (CBHA) (which may be used at a concentration of 100 nM to 10 pM), Oxamflatin (which may be used at a concentration of 100 nM to 10 pM), PDX 101 (which may be used at a concentration of 100 nM to 10 pM), Pyroxamide (which may be used at a concentration of 1 nM to 10 pM), Scriptaid (which may be used at a concentration of 100 nM to 10 pM), Suberoylanilide hydroxamic acid (SAHA) (which may be used at a concentration of 100 nM to 10 pM), Trichostatin A (TSA) (which may be used at a concentration of 1 nM to 10 pM), LBH589 (which may be used at a concentration of 1 nM to 10 pM), and NVP- LAQ824 (which may be used at a concentration of 1 nM to 10 pM). Exemplary cyclic tetrapeptides and benzamides include Apicidin (which may be used at a concentration of 1 nM to 10 pM), Depsipeptide (which may be used at a concentration of 100 nM to 10pM), TPX-HA analogue (CHAP) (which may be used at a concentration of 1 nM to 10 pM), and Trapoxin (which may be used at a concentration of 1 nM to 10 pM). Exemplary Benzamides include CI-994 (N-acetyldinaline) (which may be used at a concentration of 100 nM to 10 pM) and MS-275 (which may be used at a concentration of 100 nM to 10 pM).

[0332] In one embodiment, the permeabilized cell is incubated with an interphase reprogramming media. Preferably, the nucleus in the permeabilized cell remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with the interphase reprogramming media. In another preferred embodiment, a chromatin mass is formed from incubation of the permeabilized cell in a mitotic reprogramming media. In yet another preferred embodiment, the reprogrammed cell is incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to being administered to the mammal. Preferably, the permeabilized cell is from the mammal in need of that cell type. In another preferred embodiment, the permeabilized cell is formed by incubating an intact cell with a detergent, such as digitonin, or a bacterial toxin, such as Streptolysin O.

[0333] In some embodiments, at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, or more mRNA or protein molecules are expressed in the reprogrammed cell that are not expressed in the donor or permeabilized cell. In another preferred embodiment, the number of mRNA or protein molecules that are expressed in the reprogrammed cell, but not expressed in the donor or permeabilized cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300, inclusive. Preferably, at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, or more mRNA or protein molecules are expressed in the donor or permeabilized cell that are not expressed in the reprogrammed cell. In yet another preferred embodiment, the number of mRNA or protein molecules that are expressed in the donor or permeabilized cell, but not expressed in the reprogrammed cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300, inclusive. Preferably, the mRNA or protein molecules are specific for the cell type of the donor, permeabilized, or reprogrammed cell, such that the molecules are only expressed in cells of that particular cell type. In still another preferred embodiment, these mRNA or protein molecules are expressed in both the donor cell (i.e., the donor or permeabilized starting cell) and the reprogrammed cell, but the expression levels in these cells differ by at least 2, 5, 10, or 20-fold, as measured using standard assays. In one embodiment, the size of the donor orpermeabilized cell differs from that of the reprogrammed cell by at least 10, 20, 30, 50, 75, or 100%, as measured using standard methods. In another preferred embodiment, the volume of cytoplasm in the donor or permeabilized cell differs from that in the reprogrammed cell by at least 10, 20, 30, 50, 75, or 100%, based on standard methods. In yet another preferred embodiment, the reprogrammed cell has gained or lost an activity relative to the donor or permeabilized cell, such as production of matrix metalloproteases, angiogenic activity or chemotactic activity. In still other preferred embodiments, the reprogramming media is an interphase reprogramming media, such as an extract formed from cells synchronized in one or more of the phases of the cell cycle. In another embodiment, the reprogramming media is an extract formed from cells synchronized in mitosis or from unsynchronized cells. The reprogramming media is an extract from the cell type one wishes the donor or permeabilized cell to become, or the reprogramming media is a solution containing factors specific for the cell type one wishes the donor or permeabilized cell to become.

[0334] In one embodiment, the reprogramming media is modified by the enrichment or depletion of a factor, such as a DNA methyltransferase, histone deacetylase, histone, nuclear lamin, transcription factor, activator, repressor, growth factor, hormone, or cytokine. The reprogramming media may or may not contain exogenous nucleotides. In other embodiments, a chromatin mass in a reprogramming media or formed in a permeabilized cell is contacted with a vector having a nucleic acid encoding a gene of interest under conditions that allow homologous recombination between the nucleic acid in the vector and the corresponding nucleic acid in the genome of the chromatin mass, resulting in the alteration of the genome of the chromatin mass. Due to the lack of an intact plasma membrane and the lack of a nuclear membrane, a chromatin mass in a permeabilized cell may be easier to genetically modify than a naturally occurring cell. Preferably, the chromatin mass or nucleus is purified from the reprogramming media prior to insertion into the recipient cell or cytoplast, or the reprogrammed cell is purified prior to administration into the mammal. Preferably, the donor or permeabilized cell is haploid (DNA content of n), diploid (2n), or tetrapioid (4n), and the recipient cell is hypodiploid (DNA content of less than 2n), haploid, or enucleated.

[0335] Generation of pluripotent cells by other means are known in the art. In some embodiments, pluripotent stem cells are treated to differentiate into thymic medullary epithelial cells. Said differentiation is accomplished by culture in approximately 10 pg / mL collagen IV precoated 6-well culture plates containing differentiation medium(mESC medium without leukemia inhibitory factor) and human fibroblast growth factor 7 at a concentration of approximately 20 ng / mL, human fibroblast growth factor 10 at a concentration of approximately 20 ng / mL, human bone morphogenetic protein 4 at a concentration of approximately 20 ng / mL, and human epithelial growth factor at a concentration of approximately 50 ng / mL. The medium and growth factors were changed every 3-4 days. Differentiated pluripotent cells are harvested at day 10 by treating the cells with approximately 2 mg / mL collagenase IV. Said differentiated thymic medullary epithelial cells may be utilized to induce transplant tolerance or reduction in transplant associated immune suppression in conditions of solid organ transplantation, in which case donor derived, or partially donor derived cells are utilized, or in the context of hematopoietic stem cell transplant in which prevention of graft versus host disease is required and as a resulted utilization of recipient derived, or partially recipient derived. In the context of autoimmunity thymic medullary epithelial cells are made to express autoantigens so as to promote tolerance to autoantigens stimulating autoimmunity. Autoantigens may be genetically engineered into pluripotent stem cells used to generate thymic medullary epithelial cells, or engineered directly into differentiated thymic medullary epithelial cells. In addition to genetically engineered, said autoantigens may be incorporated by means of protein delivery through protein transduction domains, by administering mRNA encoding for autoantigens, or by addition of autoantigens or peptides derived thereof so that pinocytosis may allow for uptake of said autoantigens.

[0336] In some embodiments, combinations of immature dendritic cells together with mesenchymal stem cells are utilized to generate a tolerogenic milieu in order to treat or prevent autoimmunity. Said tolerogenic milieu may be generated in vivo or may be created by coculture of said immature dendritic cells and mesenchymal stem cells in vitro. In some embodiments cells of the tissue to which prevention or inhibition of autoimmunity is desired are added to the culture, said cells include endothelial cells, epithelial cells, dermal cells, endodermal cells, mesodermal cells, fibroblasts, osteocytes, chondrocytes, natural killer cells, dendritic cells, hepatic cells, pancreatic cells, stromal cells, salivary gland mucous cells, salivary gland serous cells, von Ebner’s gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, gland of Moll cells, sebaceous gland cells. Bowman’s gland cells, Brunner’s gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin’s gland cells, gland of Littre cells, uterus endometrium cells, isolated goblet cells, stomach lining mucous cells, gastricgland zymogenic cells, gastric gland oxyntic cells, pancreatic acinar cells, paneth cells, type II pneumocytes, clara cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cells, magnocellular neurosecretory cells, gut cells, respiratory tract cells, thyroid epithelial cells, parafollicular cells, parathyroid gland cells, parathyroid chief cell, oxyphil cell, adrenal gland cells, chromaffin cells, Leydig cells, theca interna cells, corpus luteum cells, granulosa lutein cells, theca lutein cells, juxtaglomerular cell, macula densa cells, peripolar cells, mesangial cell, blood vessel and lymphatic vascular endothelial fenestrated cells, blood vessel and lymphatic vascular endothelial continuous cells, blood vessel and lymphatic vascular endothelial splenic cells, synovial cells, serosal cell (lining peritoneal, pleural, and pericardial cavities), squamous cells, columnar cells, dark cells, vestibular membrane cell (lining endolymphatic space of ear), stria vascularis basal cells, stria vascularis marginal cell (lining endolymphatic space of ear), cells of Claudius, cells of Boettcher, choroid plexus cells, pia-arachnoid squamous cells, pigmented ciliary epithelium cells, nonpigmented ciliary epithelium cells, corneal endothelial cells, peg cells, respiratory tract ciliated cells, oviduct ciliated cell, uterine endometrial ciliated cells, rete testis ciliated cells, ductulus efferens ciliated cells, ciliated ependymal cells, epidermal keratinocytes, epidermal basal cells, keratinocyte of fingernails and toenails, nail bed basal cells, medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular hair root sheath cells, hair root sheath cells of Huxley’s layer, hair root sheath cells of Henle’s layer, external hair root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal cell of epithelia, urinary epithelium cells, auditory inner hair cells of organ of Corti, auditory outer hair cells of organ of Corti, basal cells of olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, touch-sensitive primary sensory neurons, type I carotid body cells, type II carotid body cell (blood pH sensor), type I hair cell of vestibular apparatus of ear (acceleration and gravity), type II hair cells of vestibular apparatus of ear, type I taste bud cells cholinergic neural cells, adrenergic neural cells, peptidergic neural cells, inner pillar cells of organ of Corti, outer pillar cells of organ of Corti, inner phalangeal cells of organ of Corti, outer phalangeal cells of organ of Corti, border cells of organ of Corti, Hensen cells of organ of Corti, vestibular apparatussupporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, astrocytes, neurons, oligodendrocytes, spindle neurons, anterior lens epithelial cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white fat cells, brown fat cells, liver lipocytes, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, kidney distal tubule cells, kidney collecting duct cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gall bladder epithelial cells, ductulus efferens nonciliated cells, epididymal principal cells, epididymal basal cells, ameloblast epithelial cells, planum semilunatum epithelial cells, organ of Corti interdental epithelial cells, loose connective tissue fibroblasts, corneal keratocytes, tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblast / cementocytes, odontoblasts, odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, hyalocytes, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stellate cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, nuclear bag cells of muscle spindle, nuclear chain cells of muscle spindle, satellite cells, ordinary heart muscle cells, nodal heart muscle cells, Purkinje fiber cells, smooth muscle cells, myoepithelial cells of iris, myoepithelial cell of exocrine glands, reticulocytes, megakaryocytes, monocytes, connective tissue macrophages. Epidermal Langerhans cells, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cell, helper T cells, suppressor T cells, cytotoxic T cell, natural Killer T cells, B cells, natural killer cells, melanocytes, retinal pigmented epithelial cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoa, ovarian follicle cells, Sertoli cells, thymus epithelial cell, and / or interstitial kidney cells.

[0337] In certain embodiments, one or a plurality of the culture methods or systems of the invention utilize a bioreactor for generating or cultivating cells and for inducing or maintaining the generation of one or a plurality of soluble factors from the cells at various stages of dedifferentiation or differentiation. In one embodiment, a culture system is provided comprising a bioreactor, wherein a tissue is cultured on a porous membrane, located around the inner circumference of a cylindrical housing, with a first space between the tissue and cylindrical housing, a second center space within the cylindrical housing bounded by the opposing side of the tissue, media inlet ports for providing a firstmedia flow to the first space and a second media flow to the second space, and at least one media outlet port. The first and second media flows may have different concentrations of solutes, including nutrients, growth factors and gases, as described above.

[0338] In certain embodiments, the method of growing a tissue involves seeding structural support cells and parenchymal cells on a porous substrate such as a mesh to form a co-culture that will generate a tissue in vitro. The mesh is preferably made of nylon or a biodegradable material and is disposed in a preferably sterile container having openings at each end for media flows. A first media flow, with predetermined levels of concentration of solutes such as various nutrients, gases, and growth factors, flows in direct contact with the cells on one side of the mesh portion. A second media flow, with different concentrations of typically different solutes such as nutrients, gases, and growth factors flows in direct contact with the opposing side of said mesh portion. The differing concentrations of solutes in each of the media flows creates a diffusion or osmotic pressure gradient that drives diffusion of the various requirements for cell growth through the tissue stratum. Simultaneously, metabolic waste products diffuse into the media stream, and are carried away. In another mode of use, one medium stream can be pressurized to produce hydrostatic pressure differences to counteract the osmotic pressure and to provide further control for nutrient delivery. The present invention offers advantages over previous tissue culturing systems in that nutrient delivery is directed inward from two opposing planes, permitting the creation of, and maintaining the viability of, a thicker tissue substratum. Thus, the diffusion gradient bioreactor of the invention simulates in vitro the mechanisms by which nutrients are delivered to, and waste products are removed from tissues in vivo, allowing for a wide variety of three- dimensional tissues to be cultured.

[0339] The bioreactor and resulting cultures have a variety of applications ranging from in vivo transplantation or implantation of tissues grown in the bioreactor, the continuous production of biological materials in vitro, and as an extracorporeal organ assist device for patients suffering from organ failure. In addition, tissue growth in the reactor can be used for in vitro toxicology testing either after removal to a static environment or as assessed by other means such as the metabolism of drugs or the release of key proteins into the effluent stream from the diffusion gradient bioreactor system. The bioreactor can be used to grow three-dimensional tissues for the production of a wide variety of biological materials and drugs, including but not limited to, monoclonalantibodies, viral vaccines, hormones, lymphokines, enzymes, tumor-specific antigens, and viral insecticides. To grow a tissue, structural support cells are seeded onto the mesh and provided with media flows to deliver the required nutrients and simultaneously remove waste products produced by the cells. Once the structural support cells have become established, such as, for example, when the structural support cells have grown to confluence, tissue-specific or parenchymal cells are added to the mesh. The content of one or both of the media flows may be altered at this point to meet the needs of the tissuespecific cells or the whole organism. Once the cells have grown into a three-dimensional tissue, the media flow may be stopped and the tissue can be used or preserved for future use. To preserve the tissue, cryopreservative is added, and the tissue is frozen, preferably in the same container that held the tissue in the bioreactor. The tissue can be later defrosted and-used as a bioreactor to produce biological materials. To use the tissue, a first media flow having predetermined concentrations of solutes such as nutrients, growth factors and gases flows in direct contact with one side of the tissue. A second media flow having different concentrations of solutes flows in direct contact with the opposing side of the tissue. Mass transfer within the bioreactor is achieved via a diffusion gradient. The concentrations of solutes and nutrients provided are sufficient to allow the three- dimensional tissue to produce the desired biological product. The product is withdrawn from the bioreactor via the first or second media flow, or both media flows. The three- dimensional tissue can be used or cryo-preserved for future use as described above. The tissue can be later defrosted and used as an extracorporeal liver device. To use the tissue, blood is withdrawn from a patient with a malfunctioning liver. The plasma can be separated from the blood if desired. The blood or plasma is then passed through the container with the liver tissue contacting one side of the tissue, while a media flow passes by the opposing side of the tissue. The tissue will act as a functioning liver on the blood or plasma, which can then be returned to the patient.

[0340] In some embodiments, the inventors disclose not only extracorporeal devices possessing regenerative and / or immune modulatory cells, but also implantable devices. In some embodiments, the implantable device includes a therapeutic cell population, a matrix capable of maintaining viability and function of said therapeutic cell population, and a layer of material with selective permeability surrounding said matrix capable of maintaining viability and function of said therapeutic cell population. In some embodiments, the therapeutic cell population comprises donor cells or recipient cells, and wherein the therapeutic cell population possesses an ability to induce apoptosis orinactivation of cells that are self-reactive. In some embodiments, the therapeutic cell population is selected from the group consisting of a differentiated cell expressing high growth factor production activity, a stem cell, and a cell or variety of cells expressing immune modulatory factors. In some embodiments, the differentiated cell expressing high growth factor production activity is endowed with an ability to produce high levels of growth factors by ex vivo manipulation. In some embodiments, the differentiated cell expressing high growth factor production activity naturally possesses the ability to express high levels of growth factors. In some embodiments, the stem cell is selected from the group consisting of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neuronal stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the cell or variety of cells expressing immune modulatory factors resemble thymic cell populations.

[0341] In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is a macroporous polymer scaffold comprising porous walls that are essentially non-membranous, said porous walls consisting of microporous polymer struts defining macropores which are interconnected by macroporous passageways, said microporous polymer struts containing microporous passageways extending through said microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through said given microporous polymer strut, said macropores having a mean diameter in a range from about 0.5 to about 3.5 mm, and said macroporous polymer scaffold having a porosity of at least 50%. In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is selected from the group consisting of irradiated bone, decellularized bone, decellularized placenta stroma, decellularized thymus, and decellularized organs. In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is made of a material in part or in whole selected from a group of materials comprised of glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulphone, polyvinyl alcohol, acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene,polypentane, nylon, magnetite, natural polysaccharide, modified polysaccharide, collagen, gelatin and modified gelatin.

[0342] In some embodiments, the layer of material with selective permeability is manufactured using biocompatible materials allowing for free diffusion of particles small than 1 micrometer. In some embodiments, the layer of material with selective permeability is manufactured using biocompatible materials that do not allow for substantial immune recognition of said cells inside said material.

[0343] In some embodiments, the implantable device may contain a porous substrate structure to which therapeutic cells are adherent, which can be implanted and explanted from a patient. In another embodiment, a medical device includes a porous substrate seeded with therapeutic cells, said device comprising of an additional layer allowing free exchange of molecules without cell escape. Said medical device is seeded with autologous or allogeneic pluripotent stem cells. In conditions where tolerance to alloreactive donor cells is desired, the thymic medullary epithelial cells or progenitors thereof are derived from recipient cells. In conditions where tolerance to alloreactive recipient cells is desired, the thymic medullary epithelial cells or progenitors thereof are derived from donor cells.

[0344] Some embodiments disclosed herein relate to a method of treating an inflammatory condition. In some embodiments, the method includes selecting a population of cells producing soluble anti-inflammatory mediators, culturing said cells producing soluble anti-inflammatory mediators with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, the inflammatory condition is characterized by elevated levels of inflammatory mediators, said mediators selected from a group comprising of IL-1, IL-2, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL- 21, IL-23, TNF-alpha, Interferon-gamma, and TRANCE. In some embodiments, the inflammatory condition is selected from the group consisting of autoimmunity, transplant rejection, and aging. In some embodiments, the population of cells producing soluble anti-inflammatory mediators are selected from the group consisting of immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow mononuclear cells, mesenchymal stem cells, T regulatory cells, NKT cells, hematopoietic stem cells, cord matrix mononuclear cells, adipose tissue mononuclear cells, placentalmatrix mononuclear cells, cord blood mononuclear cells, and CD5 positive B cells. In some embodiments, the cell populations are autologous, allogeneic, or xenogeneic. In some embodiments, the cells are derived from one or a plurality of cell lines. In some embodiments, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner allowing for subsequent explantation.

[0345] Some embodiments disclosed here relate to a method of modifying an immune response. In some embodiments, the method includes selecting a population of cells producing soluble immune modulators, culturing said cells producing soluble immune modulators with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, the immune response is an antibody mediated immune response or a cell mediated immune response.

[0346] In some embodiments disclosed herein related to an implantable device including a therapeutic cell population, a matrix capable of maintaining viability and function of said therapeutic cell population, and a layer of material with selective permeability surrounding said matrix capable of maintaining viability and function of said therapeutic cell population. Said cells implanted include regenerative cells and regenerative cells that have been primed for specific functions. For example, if they patient to be treated is suspected of sepsis, the regenerative cells in the device can be primed with sepsis associated molecular factors such as TLR4 agonists. In some embodiments, the therapeutic cell population is selected from the group consisting of a differentiated cell expressing high growth factor production activity, a stem cell, and a cell or variety of cells expressing immune modulatory factors. In some embodiments, the differentiated cell expressing high growth factor production activity is endowed with ability to produce high levels of growth factors by ex vivo manipulation. In some embodiments, the differentiated cell expressing high growth factor production activity naturally possesses the ability to express high levels of growth factors. In some embodiments, the stem cell is selected from the group of stem cells consisting of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neuronal stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stemcells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the cell or variety of cells expressing immune modulatory factors resemble thymic cell populations. In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is a macroporous polymer scaffold including porous walls that are essentially non-membranous, the porous walls including of microporous polymer struts defining macropores which are interconnected by macroporous passageways. In some embodiments, the microporous polymer struts contain microporous passageways extending through the microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through the given microporous polymer strut. In some embodiments, the macropores have a mean diameter in a range from about 0.5 to about 3.5 mm. In some embodiments, the macroporous polymer scaffold has a porosity of at least 50%. In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is selected from a group of matrices consisting of irradiated bone, decellularized bone, decellularized placenta stroma, decellularized thymus, and decellularized organs. In some embodiments, a source of matrices is selected from the group consisting of autologous, allogeneic, and xenogeneic. In some embodiments, the matrix capable of maintaining viability and function of a therapeutic cell population is made of a material in part or in whole selected from a group of materials consisting of glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulphone, polyvinyl alcohol, acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene, polypentane, nylon, magnetite, natural polysaccharide, modified polysaccharide, collagen, gelatin, and modified gelatin

[0347] In some embodiments, the layer of material with selective permeability is manufactured using biocompatible materials allowing for free diffusion of particles small than 1 micrometer. In some embodiments, the layer of material with selective permeability is manufactured using biocompatible materials that do not allow for substantial immune recognition of said cells inside said material. Some embodiments disclosed herein relate to a method of treating an inflammatory condition. In some embodiments, the method includes selecting a population of cells producing soluble antiinflammatory mediators, culturing the cells producing soluble anti-inflammatory mediators with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as toallow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, the inflammatory condition is characterized by elevated levels of inflammatory mediators selected from the group consisting of IL-1, IL-2, IL-5, IL-6, IL- 8, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, TNF-alpha, Interferon-gamma, and TRANCE. In some embodiments, the inflammatory condition is selected from the group consisting of autoimmunity, transplant rejection, and aging. In some embodiments, the population of cells producing soluble anti-inflammatory mediators are selected from the group consisting of immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow mononuclear cells, mesenchymal stem cells, T regulatory cells, NKT cells, hematopoietic stem cells, cord matrix mononuclear cells, adipose tissue mononuclear cells, placental matrix mononuclear cells, cord blood mononuclear cells, and CD5 positive B cells. In some embodiments, the cell populations are selected from the group of sources selected from autologous, allogeneic, and xenogeneic. In some embodiments, the cells are derived from one or a plurality of cell lines. In some embodiments, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner allowing for subsequent explantation.

[0348] Some embodiments disclosed herein relate to a method of modifying an antibody mediated immune response. In some embodiments, the method includes selecting a population of cells producing soluble immune modulators, culturing said cells producing soluble immune modulators with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, an immune suppressant is administered prior to, and / or concurrent with, and / or subsequently to implantation of said enclosed matrix containing therapeutic cells. In some embodiments, the immune suppressant selectively inhibits B cell responses. In some embodiments, the immune suppressant selectively inhibits T cell responses. In some embodiments, the cell population producing soluble anti-inflammatory mediators is selected from a group of cells consisting of immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow mononuclear cells, mesenchymal stem cells, T regulatory cells, NKT cells, hematopoietic stem cells, cord matrix mononuclear cells, adipose tissue mononuclear cells, placental matrix mononuclear cells, cord blood mononuclear cells, and CD5positive B cells. In some embodiments, the cell populations are selected from a group of sources selected from autologous, allogeneic, and xenogeneic. In some embodiments, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner allowing for subsequent explantation. Some embodiments disclosed herein relate to a method of modifying a cell mediated immune response. In some embodiments, the method includes selecting a population of cells producing soluble immune modulators, culturing said cells producing soluble immune modulators with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, an immune suppressant is administered prior to, and / or concurrent with, and / or subsequently to implantation of said enclosed matrix containing therapeutic cells. In some embodiments, the immune suppressant selectively inhibits B cell responses. In some embodiments, the immune suppressant selectively inhibits T cell responses. In some embodiments, the cell population producing soluble immune modulators is selected from a group of cells consisting of immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow mononuclear cells, mesenchymal stem cells, T regulatory cells, NKT cells, hematopoietic stem cells, cord matrix mononuclear cells, adipose tissue mononuclear cells, placental matrix mononuclear cells, cord blood mononuclear cells, and CD5 positive B cells. In some embodiments, the cell populations are selected from a group of sources selected from autologous, allogeneic, and xenogeneic. In some embodiments, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner allowing for subsequent explantation.

[0349] Some embodiments disclosed herein relate to a method of stimulating endogenous cellular repair processes. In some embodiments, the method includes selecting a population of cells producing soluble factors capable of augmenting endogenous cellular repair processes, culturing said cells producing soluble factors capable of augmenting endogenous cellular repair with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, the cells producing soluble factors capable of augmenting endogenous cellular repair processes are manipulated toenhance production of said soluble factors capable of augmenting endogenous cellular repair factors. In some embodiments, the cells producing soluble factors capable of augmenting endogenous cellular repair are selected from a group of cells consisting of a differentiated cell expressing high growth factor production activity, a stem cell, and a cell or variety of cells expressing immune modulatory factors. In some embodiments, the stem cells are selected from the group consisting of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neuronal stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the cell populations are selected from a group of sources selected from autologous, allogeneic, and xenogeneic. In some embodiments, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner allowing for subsequent explantation.

[0350] Some embodiments disclosed herein relate to a method of accelerating hematopoietic recovery subsequent to an insult to the hematopoietic system. In some embodiments, the method includes selecting a population of cells producing soluble factors capable of augmenting hematopoietic recovery, culturing said cells producing soluble factors capable of augmenting hematopoietic recovery with a matrix in vitro so as to allow adherence of said cells to said matrix, overlaying a material with selective permeability over said cells and said matrix so as to allow for diffusion of soluble factors through said material but not escape of cells, and implanting said enclosed matrix containing therapeutic cells into a patient in need. In some embodiments, the population of cells producing factors capable of augmenting hematopoietic recovery are selected from a group of cells consisting of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neuronal stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, sertoli cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells and side population stem cells. In some embodiments, the matrix is a macroporous polymer scaffold includes porous walls that are essentially non-membranous. In some embodiments, the porous walls include microporous polymer struts defining macropores which are interconnected bymacroporous passageways. In some embodiments, the microporous polymer struts containing microporous passageways extending through said microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through said given microporous polymer strut. In some embodiments, the macropores have a mean diameter in a range from about 0.5 to about 3.5 mm. In some embodiments, the macroporous polymer scaffold has a porosity of at least 50%. In some embodiments, the matrix is selected from a group of matrices consisting of irradiated bone, decellularized bone, decellularized placenta stroma, decellularized thymus, and decellularized organs. In some embodiments, the cells are derived from sources selected from a group of sources consisting of allogeneic, autologous, and xenogeneic. In some embodiments, a dose or plurality of doses of a factor capable of augmenting hematopoietic recovery is also administered. In some embodiments, the factor capable of augmenting hematopoietic recovery is selected from the group consisting of G-CSF, GM- CSF, human chorionic gonadotropin, an inhibitor of GSK-3beta, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, a hyaluronic acid fragment, IL-1, IL-3, IL- 7, thrombopoietin, and parathyroid hormone. In some embodiments, a permeable material as disclosed herein is not used so as to allow implanted cells to migrate out of said matrix. In some embodiments, the therapeutic cell population is derived from recipient cells in the context of a hematopoietic stem cell transplant where prevention of donor reactivity to recipient tissue is desired. In some embodiments, the therapeutic cell population is a cell population possessing ability to induce apoptosis or inactivation of cells that are self-reactive. In some embodiments, the self-reactive cells are lymphocytes recognizing recipient tissue in the context of a hematopoietic stem cell transplant. In some embodiments, the self-reactive cells are lymphocytes recognizing donor tissue in the context of a solid organ transplant.

[0351] Some embodiments disclosed herein relate to an implantable device including a therapeutic cell population, a matrix capable of maintaining viability and function of said therapeutic cell population, and a layer of material with selective permeability surrounding said matrix capable of maintaining viability and function of said therapeutic cell population. In some embodiments, the therapeutic cell population is derived from the steps of extracting an adult tissue, dedifferentiating the adult tissue to convey lineage plasticity, and differentiating said adult tissue into thymic medullary epithelial cells or progenitors thereof. In some embodiments, the adult tissue is skin. In some embodiments, the adult tissue possesses lineage plasticity. In some embodiments,the adult tissue possessing lineage plasticity is a stem cell. In some embodiments, the stem cell is selected from a group of cells consisting of embryonic stem cells, inducible pluripotent stem cells, nuclear transfer derived stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neuronal stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells and side population stem cells. In some embodiments, the embryonic stem cells are totipotent and express one or more antigens selected from the group consisting of stagespecific embryonic antigens (SSEA) 3, SSEA 4, Tra-1-60 and Tra-1-81, Oct-3 / 4, Cripto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), Rex-1, GCTM-2, Nanog, and human telomerase reverse transcriptase (hTERT). In some embodiments, the cord blood stem cells are multipotent and capable of differentiating into endothelial, smooth muscle, and neuronal cells. In some embodiments, the cord blood stem cells are identified based on expression of one or more antigens selected from the group consisting of S SEA-3, S SEA-4, CD9, CD34, c-kit, OCT-4, Nanog, and CXCR-4. In some embodiments, the cord blood stem cells do not express one or more markers selected from the group consisting of CD3, CD34, CD45, and CD1 lb. In some embodiments, the placental stem cells are isolated from the placental structure. In some embodiments, the placental stem cells are identified based on expression of one or more antigens selected from the group consisting of Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4 and Sox-2. In some embodiments, the bone marrow stem cells include bone marrow mononuclear cells. In some embodiments, the bone marrow stem cells are selected based on the ability to differentiate into one or more of endothelial cells, smooth muscle cells, and neuronal cells. In some embodiments, the bone marrow stem cells are selected based on expression of one or more of CD34, c-kit, flk-1, Stro-1, CD105, CD73, CD31, CD146, vascular endothelial-cadherin, CD133 and CXCR-4. In some embodiments, the placental stem cells are isolated from the Wharton’s Jelly. In some embodiments, the placental stem cells are mesenchymal in morphology. In some embodiments, the placental cell expresses one or more cytokines associated with regenerative activities. In some embodiments, the cells with lineage plasticity are differentiated into thymic medullary epithelial cells by culture in media lacking leukemia inhibitory factor and exposure to human fibroblast growth factor 7, human fibroblast growth factor 10, human bone morphogenetic protein 4, andhuman epithelial growth factor. In some embodiments, the concentration of human fibroblast growth factor 7 is 20 ng / mL. In some embodiments, the concentration of human fibroblast growth factor 10 is 20 ng / mL. In some embodiments, the concentration of human bone morphogenetic protein 4 is 20 ng / mL. In some embodiments, the concentration of human epithelial growth factor is 50 ng / mL.

[0352] In some embodiments, mesenchymal stem cells are dedifferentiated by processes selected from the group consisting of somatic cell nuclear transfer, cytoplasmic transfer, treatment with epigenetic acting agents, treatment with factors known to cause formation of inducible pluripotent stem cells, and treatment with acidic conditions. In some embodiments, the somatic cell nuclear transfer is performed by introducing nucleus of said donor mesenchymal stem cell into an enucleated recipient oocyte. In some embodiments, a process is performed to induce activation of said enucleated recipient oocyte. In some embodiments, pluripotent stem cells are derived from said activated recipient oocyte. In some embodiments, the pluripotent stem cells possess ability to selfrenew for more than one year. In some embodiments, the pluripotent stem cells possess expression of hTERT. In some embodiments, inhibition of spontaneous differentiation of said pluripotent stem cells is achieved by growth in media conditioned by feeder cells. In some embodiments, the inhibition of spontaneous differentiation of said pluripotent stem cells is achieved by growth on a feeder layer. In some embodiments, the feeder cells are fibroblasts. In some embodiments, the cytoplasmic transfer is performed using cytoplasm from a pluripotent stem cell into a mesenchymal stem cell derived from a donor of an allograft. In some embodiments, the pluripotent stem cell is selected from the group consisting of embryonic stem cells, parthenogenic stem cells, somatic cell nuclear transfer generated stem cells, and inducible pluripotent stem cells. In some embodiments, the cytoplasmic transfer is induced by permeabilization of said mesenchymal stem cell membrane utilizing an agent that induces pore formation in said membrane. In some embodiments, the agent capable of inducing pore formation in said membrane is streptolysin O. In some embodiments, induction of membrane permeability is performed using electrical current. In some embodiments, the epigenetic acting agents are DNA methyltransferase inhibitors. In some embodiments, the epigenetic acting agents are histone deacetylase inhibitors. In some embodiments, the DNA methyltransferase inhibitor is 5-azacytidine. In some embodiments, the histone deacetylase inhibitor is selected from the group consisting of valproic acid, trichostatin A, and sodium phenylbutyrate. In some embodiments, the induced pluripotent stem cell is generated byintroduction of genes selected from the group consisting of SOX-2, Oct4, c-Myc, Klf4, nanog, LIN28, and Glisl.Example 1: Reduction of Senescence by Culture with Pluripotent Cell Conditioned MediaPurification of Fibroblasts and Senescence Induction

[0353] A skin punch biopsy is obtained using standard procedure (e.g. obtained with 4mm round Visipunch instrument) and kept in complete DMEM 20% FBS media on ice.

[0354] The skin biopsy is subsequently placed on the bottom portion of a 10 cm dish is dissected in the dissecting microscope in the laminar flow hood. The dissection begins by cutting the 4 mm round skin biopsy into 12-15 evenly sized pieces with sharp edges by cutting pieces in equal halves using one scalpel to hold the biopsy in place and the second scalpel to cut with a rolling motion in one direction.

[0355] Using a pointed forceps 2-3 biopsy pieces are placed into each well of the prepared 6-well plate containing 800 pl of DMEM with 20% FCS and not on dry wells. A tapping or sliding motion is used to get the pieces to attach to the bottom of the well. At times the scalpel is use to remove any biopsy pieces from the forceps. Subsequently the 6 well plate is placed in a 37 °C incubator. Monitor daily to ensure there is a film of media coating the bottom of the well for the first week; add -200 pl every 2 days to replace any evaporated media.

[0356] After one week, the amount of media is increased to 2 ml of complete DMEM / 20%FBS and changed every 2-3 days. Once fibroblasts are confluent in each well to the point where the fibroblasts are reaching the edges of the well, the cells are trypsinzed and passaged from 6-well plate into 2X T75 flasks (passage 1). The tissue pieces can be transferred as well. They will not attach and be washed out during the next media change. Once fibroblasts are confluent, they are then transferred to 3X T175 flasks (passage 2), freeze in complete DMEM media plus 10% DMSO at 1x106 cells / ml per vial.

[0357] To induce senescence, 24 h after plating on 6 well plates cells were treated with doxorubicin at a concentration 50 nm for 48 h and then cultured in fresh complete medium for three days. The control was represented by cells incubated for 48 h in complete medium with 0.0005 % of DMSO (used as a diluent for doxorubicin) and thenmaintained in fresh complete medium for three days. The appearance of the senescence phenotype was confirmed by expression of pl 6.Suppression of Senescence Acquisition

[0358] Pluripotent iPSC conditioned media was extracted in serum free media and added at 5% v / v; 10% v / v; and 15% v / v to fibroblasts exposed to doxorubicin. As seen in the figure below, a reduction of senescent cell generation was observed based on pl6 expression by conditioned media from pluripotent stem cells. The conditioned media was generated after 48 hour culture of pluripotent stem cells. The results are shown in FIG. 1.Example 2: Reduction of Senescence by Culture with Pluripotent Cell Derived Mesenchymal Stem Cell Conditioned Media

[0359] Mesenchymal stem cells derived from pluripotent stem cells were created by utilization of the “MSC Switch” technology. Briefly, a confluent 6-cm plate of iPSCs was trypsinized for 3 minutes at 37°C and placed on a gelatinized 10-cm dish containing knockout Dulbecco modified Eagle’s medium (DMEM GIBCO) supplemented with 10% serum replacement medium (GIBCO), 10 ng / mL basic fibroblast growth factor (bFGF; GIBCO), 10 ng / mL platelet-derived growth factor AB (Peprotech, Rocky Hill, NH), and 10 ng / mL epidermal growth factor (Peprotech) for enrichment of MSC outgrowth. After 1 week, differentiating iPSCs were harvested and incubated with CD24-phycoerythrin (PE) and CD105-FITC (BD PharMingen, San Diego, Calif). Sorting for CD24-CD105+ was performed by a fluorescence-activated cell sorting (FACS) system. The CD24-CD105+ cells were seeded in a 6-well plate beginning with 10 000 cells per well under knockout DMEM plus 10% FCS (GIBCO), bFGF (5 ng / mL), platelet-derived growth factor AB (10 ng / mL), and epidermal growth factor (10 ng / mL). When CD24-CD105+ cultures were confluent, one quarter of the cells were split for pLL3.7-green fluorescence proteinpositive (GFP+) labeling, followed by limiting dilution (0.5 cell per well in a 96-well plate). We selected wells containing a single cell visualized under fluorescent microscopy and excluded those containing more. When the clones derived from a single cell were grown up to 60% to 70% of confluence, the cells from each well were reseeded into 1 well of 6-well plates and serially reseeded thereafter in 25-, 75-, and 175-cm2 tissueculture flasks at a density of 1 x 104 / mL. When cells were confluent in 175-cm2 tissue culture flasks, they were set as passage 1 and frozen down as cell stocks.

[0360] MSC age was reduced by culture in valproic acid 5 nm per ml after thawing. Cells were grown in valproic acid containing media (10% FBS DMEM). Conditioned media was obtained after 48 hour culture in serum free DMEM without FBS.Conditioned media was used to inhibit senescence induction, which was induced as in Example 1.

[0361] Pluripotent MSC conditioned media was extracted in serum free media and added at 5% v / v; 10% v / v; and 15% v / v to fibroblasts exposed to doxorubicin. As seen in Figure 1 are reduction of senescent cell generation was observed based on pl6 expression by conditioned media from pluripotent stem cells. The conditioned media was generated after 48 hour culture of pluripotent stem cells. The results are shown in FIG. 2.Example 3: In Vivo Reduction of Senescence after Administration of Pluripotent Stem Cell Conditioned Media

[0362] Groups of 10 mice were divided into a) control (media concentrate with no cells); b) pluripotent stem cell conditioned media concentrate (1 microgram per mouse); c) pluripotent stem cell conditioned media concentrate (10 microgram per mouse); and d) pluripotent stem cell conditioned media concentrate (100 microgram per mouse).Administration was performed 3 times per week for 3 weeks.

[0363] Conditioned media was generated by culture of 1 million iPSC cells per ml in serum free DMEM media for 48 hours. Media was collected and filtered through a 0.2-pm filter to remove cellular debris. The mediat was concentrated ~25-fold, using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3, Millipore, Billerica, MA).

[0364] Administration of control or active conditioned media was performed in 20 month old BALB / c mice. Assessment of age-related IL-11 was performed before intervention, at completion of 3 weeks of treatment, and 3 weeks after completion of treatments. The results are shown in FIG. 3.Example 4: In Vivo Reduction of Senescence after Administration of Pluripotent Stem Cell Derived Mesenchymal Stem Cell Conditioned Media

[0365] Groups of 10 mice were divided into a) control (media concentrate with no cells); b) pluripotent stem cell derived MSC conditioned media concentrate (1 microgram per mouse); c) pluripotent stem cell derived MSC conditioned media concentrate (10 microgram per mouse); and d) derived MSC pluripotent stem cell conditioned media concentrate (100 microgram per mouse). Administration was performed 3 times per week for 3 weeks.

[0366] Conditioned media was generated by culture of 1 million iPSC cells per ml in serum free DMEM media for 48 hours. Media was collected and filtered through a 0.2-pm filter to remove cellular debris. The media was concentrated ~25-fold, using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3, Millipore, Billerica, MA).

[0367] Administration of control or active conditioned media was performed in 20 month old BALB / c mice. Assessment of age-related IL-11 was performed before intervention, at completion of 3 weeks of treatment, and 3 weeks after completion of treatments. The results are shown in FIG. 4.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a subject with a disease, condition, or symptom of biological aging, the method comprising: a) identifying a subject with an age-related disease, condition, or symptom; b) isolating a primary somatic cell from a biological fluid or tissue of the subject; c) providing a first cell culture system, wherein the primary somatic cell is provided with culture conditions and treatments to induce dedifferentiation into an induced pluripotent stem cell; d) providing a second cell culture system; wherein the induced pluripotent stem cell is provided with culture conditions to induce its differentiation into a somatic cell; e) providing a third cell culture system, wherein the differentiated somatic cell is provided with culture conditions to elicit or maintain the production of one or a plurality of soluble factors, wherein the one or plurality of soluble factors are secreted into the culture medium, and wherein the one or plurality of soluble factors possess regenerative functions, immune modulatory functions, or both; f) harvesting or isolating the one or plurality of soluble factors from the culture medium of the third culture system; and g) administering the one or plurality of soluble factors to the subject, wherein the one or plurality of soluble factors exert regenerative functions, immune modulatory functions, or both.

2. The method of Claim 1, wherein the differentiated somatic cell is selected from the group consisting of a mature dendritic cell, an immature dendritic cell, a lymphoid dendritic cell, a myeloid dendritic cell, a monocyte, a macrophage, an alternatively activated macrophage, a mesenchymal stem cell, a T cell, a B cell, a CD5-positive B cell, a T regulatory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloblast, a common myeloid progenitor, a common lymphoid progenitor, a hematopoietic stem cell, a natural killer (NK) cell, an NKT cell, a fibroblast, an epithelial cell, an epithelial progenitor cell, an endothelial cell, an endothelial progenitor cell, a hepatocyte-like cell, a hepatic stellate cell, a cardiomyocyte, a keratinocyte, arenal progenitor cell, a neural stem cell, a neural progenitor cell, a neuron, or any tissue-specific stem or progenitor cell.

3. The method of Claim 1, wherein the age-related disease comprises at least one disease selected from the group consisting of autoimmunity, arthritis, cardiovascular disease, hypertension, stroke, heart failure, diabetes mellitus, hearing loss, osteoporosis, osteoarthritis, cataracts, pain, chronic obstructive pulmonary disease (COPD), osteoporosis, dementia, Alzheimer’s disease, Parkinson’s disease, depression, and cancer.

4. The method of Claim 1, wherein the disease, disorder, or symptom of biological aging is associated with decreased endothelial function in an organ or tissue.

5. The method of Claim 1, wherein the disease, disorder, or symptom of biological aging is associated with fibrosis in an organ or tissue.

6. The method of Claim 1, wherein the disease, disorder, or symptom of biological aging is associated with a decline in T cell cytotoxic activity in the subject.

7. The method of Claim 6, wherein the cytotoxic activity of T cells is measured based on perforin production, granzyme B production, expression of Fas ligand, or a combination thereof.

8. The method of Claim 1, wherein the disease, disorder, or symptom of biological aging is associated with an increase in the production of one or a plurality of cytokines by cells isolated from a tissue of the subject.

9. The method of Claim 8, wherein the cells are selected from the group comprising fibroblasts, mesenchymal stem cells, T cells, B cells, dendritic cells, neutrophils, monocytes, macrophages, or peripheral blood mononuclear cells.

10. The method of Claim 8, wherein the one or plurality of cytokines comprise C- reactive protein (CRP), interferon-gamma, TNF-alpha, IL-lalpha, IL-lbeta, IL-6, IL-12, IL-15, IL-17, IL-18, IL-27, G-CSF, GM-CSF, and MCP-1.

11. The method of Claim 1, wherein the primary somatic cell is selected from the group consisting of a fibroblast, a T cell, a B cell, a myeloid cell, a neutrophil, an eosinophil, a basophil, a myeloblast, a monocyte, a macrophage, a mesenchymal stem cell, a keratinocyte, an epithelial cell, an exfoliated renal epithelial cell, an epithelial progenitor cell, a hematopoietic stem cell, a common myeloid progenitor cell, a natural killer (NK) cell, an NK T cell, a common lymphoid progenitor cell, an endothelial cell, an endothelial progenitor cell, a synovial cell, a pancreatic cell, a mobilized CD34+ peripheral blood mononuclear cell, an isolated blood mononuclear cell, or a tissue-specific stem or progenitor cell.

12. The method of Claim 1, wherein the soluble factor is selected from the group consisting of a protein, a glycoprotein, a lipid, a glycolipid, a carbohydrate, an antigen, an epitope, an antibody, an extracellular vesicle, an exosome, an apoptotic body, a DNA molecule, or an RNA molecule.

13. The method of Claim 1, wherein the regenerative functions comprise one or a plurality of the following effects on cells, organs and / or tissues in the subject: reversing or preventing epigenetic alterations, halting or reducing telomere attrition, inducing telomere lengthening, improving genomic stability by promoting detoxification of DNA damaging agents, reducing or ameliorating proteostasis, modulating nutrient signaling pathways, preventing or reversing mitochondrial dysfunction and damage, preventing the cellular senescent state, combating stem cell exhaustion and loss of function, and exerting antiinflammatory effects.

14. The method of Claim 2, wherein the second culture system generates a differentiated somatic cell type comprising a mesenchymal stem cell, and wherein the mesenchymal stem cell produces one or a plurality of factors from the group consisting of brain-derived neurotrophic factor, ciliary neurotrophic growth factor, and basic fibroblast growth factor.

15. The method of Claim 2, the second culture system generates a regulatory T cell, and wherein the regulatory T cell comprises an effector regulatory T cell, a memory regulatory T cell, or a chimeric antigen receptor (CAR) regulatory T cell.

16. The method of Claim 1, wherein the induced pluripotent stem cell is produced by transfection of a somatic cell with pluripotency inducing factors to induce stem cell reprogramming, and wherein the pluripotency inducing factors comprise one or a plurality of OCT4, c-Myc, PIM-1, NANOG, c-met, hTERT, KLF4, RAS, NOTCH, BMP2, BMP4, and AIRE.

17. The method of Claim 1, wherein the third cell culture system comprises culture conditions in which cells are exposed to one or a plurality of agents that comprise cell stressors.

18. The method of Claim 17, wherein the one or plurality of agents that comprise cell stressors elicit or maintain the one or plurality of soluble factors in the third cell culture system.

19. The method of Claim 17, wherein the one or plurality of cell stressors comprise molecules or agents that induce inflammation.

20. The method of Claim 19, wherein the molecules or agents that induce inflammation activate one or a plurality of toll-like receptors, and wherein the molecules or agents are selected from the group consigning of beta glucan, poly(I:C), CpG DNA, BCG, flagellin, LPS, low molecular weight hyaluronic acid, free histone, isoxazolo[5,4 -d]pyrimidine, imiquimod, poly(A:U), MPL, poly G3, poly GIO, or hyperthermia exposed cells.-SO-

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