de-differentiation system

Abstract

Means for rejuvenating a subject by exposure to soluble factors or acellular means, wherein the soluble factors are secreted by immature cells or tolerogenic cells, and wherein the soluble factors include peptides, proteins, nucleic acids, microvesicles, nanovesicles, apoptotic bodies, and exosomes. Administration of the soluble factors is regenerative and used to treat or prevent diseases associated with aging. Ultrasound can be utilized to enhance uptake, wherein the ultrasound can be delivered by extracorporeal shock wave therapy or other means. Cell secretions including soluble factors are collected from bioreactors in which immature cells are viable under basal conditions or under stimulation by inflammatory or age-related stimuli.

Description

[0001] Cross-reference to related applications This application claims priority and benefit to U.S. Provisional Application No. 63 / 548,725, filed February 1, 2024, entitled “Systematic Dedifferentiation Methods,” the entire contents of which are expressly incorporated herein by reference. Technical Field

[0002] This invention relates to the field of regenerative medicine; more specifically, this invention relates to stimulating regeneration / dedifferentiation processes using cell derivatives without relying on the application of cells. Background Technology

[0003] The basic concept of cell therapy has been discussed since the dawn of medicine. Unfortunately, outside the field of hematology, the clinical implementation of cell therapy has remained relatively stagnant. Despite numerous successful Phase I and II clinical trials, stem cell-mediated regeneration experiments have yielded somewhat unpredictable results in Phase III trials. One reason is that allogeneic stem cells are often rejected by the host, making it impossible to implant a sufficient number of cells for a sufficiently long period. Furthermore, autologous therapies cannot provide enough stem cells due to limitations in starting materials.

[0004] This invention provides a method for treating age-related diseases and symptoms by applying therapeutic cells and their soluble products, overcoming the limitations of previous methods that failed to provide an effective source of cells.

[0005] This invention provides therapeutic cells derived from pluripotent stem cells and their derived soluble factors, as well as methods of using them to treat age-related diseases and symptoms. Various types of stem cells mediate therapeutic activity through the secretion of soluble factors such as cytokines, growth factors, exosomes, microvesicles, microRNAs (miRNAs), and various other proteins. This document discloses compositions of therapeutic factors (or soluble factors) derived from regenerating cells and methods of using them. Methods for treating subjects with said therapeutic factors are also disclosed, which can be used to enhance cell therapy. Summary of the Invention

[0006] The teachings of this article relate to methods for restoring an organism to a youthful state by exposing it to a concentration of a soluble factor composition or a cell-free method, said concentration being sufficient to induce one or more regenerative processes or functions in the body.

[0007] This invention discloses a method for treating a subject with paracrine factors (i.e., soluble factors or cell-free methods) generated from pluripotent stem cells and their derivatives, said paracrine factors being able to mimic the specific therapeutic benefits of regenerating cells. In one embodiment, the invention provides a therapeutic cell population that generates soluble factors, wherein said soluble factors can be co-administered with said therapeutic cells to a subject to treat age-related diseases, conditions, or symptoms. In another embodiment, the invention provides soluble factors derived from regenerating cells, which are administered to a subject to reduce some of the limitations and disadvantages associated with the clinical use of regenerating cells themselves.

[0008] In some embodiments, the soluble factor comprises one or more entities released by cells, wherein said one or more entities may be secreted by cells, cleaved from the cell surface, detached as cell debris, or be any cellular product released into the extracellular environment. Embodiments of the present invention provide methods for inducing or maintaining the release of one or more soluble factors from cells into a liquid or culture medium, for example, in a tissue culture plate / flask or bioreactor. In some embodiments, the soluble factor comprises proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, apoptotic bodies, DNA molecules, or RNA molecules such as microRNA (RNA), messenger RNA (mRNA), and other non-coding RNAs. In some embodiments, a tissue culture medium or conditioned medium is a source of one or more soluble factors, wherein said medium contains various entities, including but not limited to proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, apoptotic bodies, DNA molecules, or RNA molecules. In some embodiments, the conditioned medium may contain a variety of soluble factors released by a cell population. In some embodiments, the term "soluble factor" may refer to a biological factor containing an antigen; for example, a member of the "differentiation cluster" cell surface marker family. In some embodiments, soluble factors include cytokines, growth factors, adhesion molecules, addressins, chemokines, or another type of molecule that acts on cells or tissues. In some embodiments, soluble factors include molecules or antigens that float freely in a liquid such as a culture medium. In other embodiments, soluble factors include molecules or antigens that are components of extracellular vesicles, exosomes, or apoptotic bodies, wherein said molecules or antigens may be expressed on the surface of the vesicle, within the vesicle membrane, or both. In still other embodiments, the term "soluble factor" is used to describe vesicles containing one or more antigens or molecules, such as exosomes. In some embodiments, soluble factors are isolated or separated from cellular components in a liquid or culture medium to provide a cell-free composition reflecting the secretory group of the cell type(s) present in said liquid or culture.

[0009] In some embodiments, cell-free therapy is provided that utilizes the secretome of regenerating cells as a therapeutic modality to treat certain conditions, particularly age-related diseases and symptoms. In some embodiments, the secretome comprises one or more molecules, including proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, DNA molecules, or RNA molecules. The inventors have optimized methods for generating populations of regenerating cells and derivatives thereof as regenerative agents or senescent cell scavengers, wherein the secretome generated by the regenerating cell populations and derivatives thereof can be used to prevent age-related disorders and symptoms associated with one or more organ systems in the body. Therefore, cell populations as sources of the cell-free therapy of the present invention are disclosed herein. The present invention provides a method for treating subjects suffering from age-related diseases, conditions, or symptoms using therapeutic soluble factors derived from regenerating cells or their derivatives.

[0010] In one embodiment, a method is provided for treating a subject suffering from an age-related disease, condition, or symptom, the method comprising: a) identifying the subject affected by the age-related disease, condition, or symptom; b) isolating primary cells from a biological fluid or tissue of the subject; c) providing a first cell culture system in which the primary cells are exposed to culture conditions and treated to induce dedifferentiation into induced pluripotent stem cells; d) providing a second cell culture system in which the induced pluripotent stem cells are exposed to culture conditions to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions; e) harvesting or isolating the one or more soluble factors from the culture medium of the second culture system; and f) administering the one or more soluble factors to the subject, wherein the one or more soluble factors exert regenerative, immunomodulatory, or both functions. In some embodiments, immunomodulatory function refers to a function related to stimulating or suppressing the immune system. In some embodiments, additional growth factors, immunomodulatory proteins, or therapeutic agents may optionally be applied systemically or locally to the subject at tissue sites to enhance the effect of the applied soluble factors or to provide unique regenerative capacity to combat aging or age-related diseases. Other embodiments of the invention provide methods for preventing age-related diseases, disorders, or symptoms using the cell compositions of the invention, the soluble factor compositions of the invention, or combinations thereof. In this embodiment, subjects at risk of developing age-related diseases, conditions, or symptoms are selected to receive treatment with the methods disclosed herein, wherein the risk may be assessed based on the subject's age, environmental factors, genetic factors, symptoms exhibited by the subject, or a combination thereof. In some embodiments, primary autologous cells are isolated from subjects who have or are at risk of developing age-related conditions or diseases, wherein the autologous cells are treated with the methods of the invention. In other embodiments, the primary autologous cells are derived from an allogeneic donor who is not a recipient of the therapy disclosed herein.

[0011] In another embodiment, a method is provided for treating or preventing age-related diseases, disorders, or symptoms in a subject, the method comprising: a) identifying a subject affected by or at risk of developing an age-related disease, disorder, or symptom; b) obtaining primary somatic cells from a biological fluid or tissue of the subject; c) providing a first cell culture system wherein the primary somatic cells are provided with culture conditions to induce their dedifferentiation into pluripotent stem cells; d) providing a second cell culture system wherein the pluripotent stem cells are provided with culture conditions to induce their differentiation into somatic cells; e) providing a third cell culture system wherein the differentiated somatic cells are provided with culture conditions to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions; f) harvesting or isolating the one or more autologous soluble factors from the culture medium of the third culture system; and g) administering the one or more autologous soluble factors to the subject, wherein the one or more soluble factors exert regenerative, immunomodulatory, or both functions. In some embodiments, the soluble factor may be autologous or allogeneic. In some embodiments, the differentiated somatic cells from the second culture system are selected from the group comprising progenitor cells, tissue-specific progenitor cells, pluripotent cells, mesenchymal stem cells, hematopoietic stem cells, fibroblasts, granulocytes, regulatory T cells, B cells, T cells, neural lineage cells, or another cell type. Some embodiments of the invention also provide a method for generating multiple differentiated cell types from induced pluripotent stem cells. In this embodiment, the method provides multiple culture conditions in step d) to support the differentiation of multiple cell types from an induced pluripotent stem cell population. In one exemplary embodiment, an induced pluripotent stem cell population may be assigned to a first culture system for generating a first differentiated cell type (e.g., mesenchymal stem cells), and induced pluripotent stem cells may be seeded into a second culture system to generate a second differentiated cell type (e.g., regulatory T cells). Furthermore, multiple different cell culture conditions may be provided in step e) to induce 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 implementation, soluble factors are harvested from cultures of induced pluripotent stem cells and their derivatives (i.e., differentiated cells) for administration to subjects in need.

[0012] In another embodiment, a method is provided for treating or preventing age-related diseases, conditions, or symptoms in a subject, the method comprising: a) identifying a subject affected by or at risk of developing an age-related disease, condition, or symptom; b) obtaining somatic cells directly from the subject's biological fluid or tissue; c) providing a first cell culture system wherein the primary somatic cells are provided with culture conditions to induce their dedifferentiation into pluripotent stem cells; d) providing a second cell culture system wherein the pluripotent stem cells are provided with culture conditions to induce their differentiation into somatic cells; e) providing a third cell culture system wherein the differentiated somatic cells are provided with culture conditions to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions; f) harvesting or isolating the one or more autologous soluble factors from the culture medium of the third culture system; g) administering the one or more soluble factors to the subject; and h) optionally, administering the differentiated somatic cells to the subject. In some embodiments, other agents may be administered to the subject to enhance the regenerative capacity of the applied cells and / or soluble factors generated according to the method of the present invention. For example, agents that promote cell migration or homing (e.g., chemokines), immunosuppressants, and / or immunostimulants may be administered to the subject before, after, or simultaneously with the administration of the therapy of the present invention containing soluble factors and / or cells. In other embodiments, in addition to the therapies disclosed herein, therapeutic cell populations generated by other means may be co-administered to the subject.

[0013] In some embodiments, the invention teaches the selection of cell populations that produce one or more soluble factors, and the subsequent selection or harvesting of said one or more soluble factors that are clinically available for the treatment or prevention of age-related diseases, conditions, or symptoms. In one embodiment, the age-related diseases are selected from the group comprising autoimmune diseases, arthritis, cardiovascular disease, hypertension, stroke, heart failure, diabetes, hearing loss, osteoporosis, osteoarthritis, cataracts, pain, chronic obstructive pulmonary disease (COPD), dementia, Alzheimer's disease, Parkinson's disease, depression, and cancer. In one implementation, age-related diseases include one type of cancer selected from the following list: melanoma (e.g., metastatic malignant melanoma), kidney cancer, prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), breast cancer, colon cancer and non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, intracutaneous or ocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, Cancers of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia and chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma and T-cell lymphoma. Attached Figure Description

[0014] Figure 1 The bar chart shows the effect of serum-free medium extracted and added to pluripotent iPSC conditioned medium of doxorubicin-exposed fibroblasts on p16 expression.

[0015] Figure 2 The bar chart shows the effect of serum-free medium extracted and added at 5% v / v, 10% v / v and 15% v / v to pluripotent MSC conditioned medium exposed to doxorubicin on p16 expression.

[0016] Figure 3 The bar chart shows the effect of different concentrations of pluripotent stem cell conditioned medium on IL-11 expression in BALB / c mice.

[0017] Figure 4 The bar chart shows the effect of different concentrations of pluripotent stem cell-derived MSC conditioned medium on IL-11 expression in BALB / c mice. Detailed Implementation

[0018] The detailed embodiments described below are intended to depict various configurations of the present subject matter and are not intended to represent the only configuration in which the present subject matter can be implemented. The detailed embodiments include specific details intended to facilitate understanding of the present subject matter. It will be apparent to those skilled in the art that the present subject matter can be implemented without these specific details.

[0019] definition As used herein, when “a” and “an” are used in conjunction with the word “comprising / including” in this specification (including the claims), they mean “one / a type or a plurality of / a types”. Some embodiments of this disclosure may consist of one or more elements, method steps and / or methods of this disclosure, or substantially consist of one or more elements, method steps and / or methods of this disclosure. It will be understood that any method or composition described herein may be practiced relative to any other method or composition described herein, and different embodiments may be combined.

[0020] As used herein, the terms “or” and “and / or” are used to describe combinations of multiple components that exclude each other. 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 explicitly envisioned that x, y, or z may be specifically excluded from one implementation.

[0021] When describing the absolute value of a feature or property of a thing or behavior described herein, the terms “substantial,” “substantially / essentially,” “approximately,” “about,” and / or other terms or phrases relating to degree may be used without specifying a numerical range. When applied to a feature or property of a thing or behavior described herein, these terms refer to the range of that feature or property consistent with the desired function associated with achieving that feature or property.

[0022] When a single numerical value is given for a particular feature or property, it is intended to be interpreted as covering at least the deviation of that value from one significant number of the given value.

[0023] If a numerical value or range is provided to define a characteristic or property of the thing or behavior described herein, the document may also define a specific method for measuring that characteristic or property, regardless of whether the value or range is defined by a term indicating degree. If the document does not define a specific method for measuring that characteristic or property, and different generally accepted methods exist for measuring that characteristic or property, the measurement method should be interpreted as the method most likely to be used by a person skilled in the art based on the description and context of that characteristic or property.

[0024] As used herein, the terms “subject” or “patient” refer to a person or other mammal who receives the application or administration of the methods or therapies of the present invention.

[0025] As used in this article, "orientation" refers to cells that are considered to have been permanently oriented to a specific function. Oriented cells are also called "terminally differentiated cells".

[0026] As used herein, “cytoplasmic extract modification” refers to a process in which a cell extract, consisting of the cytoplasmic contents of a cell, is used to induce genomic changes in a donor cell or its nucleus, thereby enabling the donor cell or its nucleus to respond during maturation and to accept host cell cytoplasm.

[0027] As used in this article, the terms “dedifferentiation” or “reversal differentiation” can be used to refer to the reversal of cell differentiation, in which cells lose their morphological or functional specialization and become cells with a lower degree of orientation.

[0028] As used in this article, “differentiation” refers to the adaptation of a cell to a specific morphology or function. In cells, differentiation leads to an increased degree of cell orientation.

[0029] As used herein, "donor cell" means any diploid (2N) cell derived from a pre-embryonic, embryonic, fetal, or postnatal multicellular organism, or a primordial germ cell that contributes its nuclear genetic material to a heterozygous stem cell. Donor cells are not limited to terminally differentiated cells or cells in the process of differentiation. For the purposes of this invention, "donor cell" refers to the entire cell or the nucleus alone.

[0030] As used in this article, "germ cell" refers to germ cells such as spermatocytes or oocytes, or cells that will develop into germ cells.

[0031] As used in this article, “host cell” means any pluripotent stem cell that contributes its cytoplasm to a heterozygous stem cell from a pre-embryonic, embryonic, fetal, or postnatal multicellular organism.

[0032] As used in this article, “host cell preparation” refers to the process of enucleating a host cell.

[0033] As used herein, “hybrid stem cell” means any pluripotent cell derived from an enucleated host cell or a donor cell or its nucleus from a multicellular organism. Hybrid stem cells are further disclosed in co-pending U.S. Patent Application Serial No. 10 / 864,788.

[0034] As used in this article, "karyoplast" refers to the cell nucleus surrounded by a thin layer of cytoplasm and plasma membrane, or the cell nucleus that has been removed from the cell.

[0035] As used herein, “nucleoplasm extract modification” refers to a process in which a cell extract consisting of the cell’s nuclear contents and lacking DNA is used to induce genomic changes in a donor cell or its nucleus, thereby enabling the donor cell or its nucleus to respond during maturation or to accept host cell cytoplasm.

[0036] As used in this article, “maturation” refers to the process of coordinating forward or backward steps in the differentiation pathway, and can refer to both differentiation and dedifferentiation. As used in this article, when applied to the process described herein, “maturation” is synonymous with “development”.

[0037] As used herein, a "modified germ cell" refers to a cell consisting of a host enucleated oocyte and a donor cell nucleus derived from a spermatogonium, oogonium, or primordial germ cell. The host enucleated oocyte and the donor cell nucleus may be from the same or different species. Modified germ cells may also be referred to as "heterozygous germ cells."

[0038] As used in this article, "pluripotent" refers to the ability of a cell to produce several other cell types, in a limited number. For example, a pluripotent cell can be a hematopoietic cell, that is, a blood stem cell that can develop into several types of blood cells but cannot develop into brain cells.

[0039] As used in this article, "pluripotent adult progenitor cells" refers to pluripotent cells isolated from bone marrow that have the potential to differentiate into mesenchymal lineage, endothelial lineage, and endoderm lineage cells.

[0040] As used in this article, "pre-embryo" refers to the fertilized egg in the early developmental stage before cell division. In the pre-embryo stage, the initial stage of cleavage is taking place.

[0041] As used in this article, "postnatal stem cells" refers to pluripotent cells derived from multicellular organisms after birth.

[0042] As used in this article, "pluripotency" refers to a state in which cells are able to produce any cell type other than placental cells or other supporting cells of the uterus.

[0043] As used in this article, "primordial sex cells" refer to diploid cells that originate from the mature or developing gonads of males or females, are capable of producing cells that enable the species to reproduce, and contain a diploid genome. Primordial sex cells can be quiescent or actively dividing. These cells include male sex primordia, female sex primordia, spermatogonial stem cells, ovarian stem cells, oogonia, type A spermatogonia, and type B spermatogonia. They are also known as germline stem cells.

[0044] As used in this article, "primordial germ cells" refers to cells that exist in the early stages of embryonic development and are destined to become germ cells.

[0045] As used in this article, "somatic stem cell" refers to diploid pluripotent or multipotent stem cells. Somatic stem cells are not totipotent stem cells. Stem cells are the original cells that produce other types of cells. Also called progenitor cells, they possess several types of stem cells. Totipotent cells are considered the body's "master" cells because they contain all the genetic information needed to produce all the cells in the body, as well as the placenta that nourishes the human embryo. Human cells possess this totipotency only during the first few divisions of the fertilized egg. After the totipotent cells divide three to four times, a series of increasingly specialized cell phases follow. The next stage of division produces pluripotent cells, which are highly plastic and capable of producing any cell type except placental cells or other uterine support tissue cells. In the next stage, the cells become pluripotent cells, meaning they are capable of producing several other cell types, but in limited numbers. At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells—cells that are considered permanently oriented to a specific function.

[0046] As used herein, when used in the context of cells, "reprogramming" refers to the process of transforming one cell type into another using reprogramming factors such as transcription factors. For example, "reprogramming" can include converting somatic cells into induced pluripotent stem cells. Cell reprogramming can involve introducing reprogramming factors into a cell type through genetic manipulation and / or the use of compounds. In some cases, reprogrammed cells can be used for implantation into a host to replace or repair diseased, damaged, defective, or genetically modified tissues.

[0047] As used in this article, "totipotent" is used to describe cells that contain all the genetic information needed to produce all the cells in the body, as well as the placenta. Human cells are totipotent only during the first few divisions of a fertilized egg.

[0048] As used herein, “whole-cell extract modification” refers to a process in which a cell extract, consisting of the cytoplasm and nuclear contents of a cell, is used to induce genomic changes in a donor cell or its nucleus, thereby enabling the donor cell or its nucleus to respond during maturation and to accept the cytoplasm of a host cell.

[0049] As used herein, “induced pluripotent stem cells” (“iPSCs”) are also referred to herein as “personalized regenerative cells”, meaning pluripotent stem cells that can be reprogrammed from adult somatic cells (e.g., primary cells isolated directly from skin, peripheral blood cells, bone marrow, or other tissue sources). For example, induced pluripotent stem cells can be obtained by reprogramming mouse adult fibroblasts under embryonic stem (ES) cell culture conditions by introducing specific genes encoding transcription factors Oct3 / 4, Sox2, c-Myc, and Klf4. Personalized regenerative cells can refer to pluripotent stem cells suitable for modification or differentiation to provide autologous therapeutic cells for treating the same subject. For example, in some embodiments, personalized progenitor cells are used to generate autologous cells suitable for genetic modification using the methods of the present invention, wherein said autologous cells may include progenitor cells, tissue-specific progenitor cells, pluripotent cells, mesenchymal cells, hematopoietic cells, fibroblasts, lymphocytes, or another cell type.

[0050] As used herein, “mesenchymal stem cells” or “MSCs” refers to cells that adhere to plastic, express CD73, CD90, and CD105 antigens, are negative for CD14, CD34, CD45, and HLA-DR, and possess the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. As used herein, “mesenchymal stromal cells” or “MSCs” can be derived from any tissue, including but not limited to bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissue, umbilical cord blood, Wharton's jelly, placenta, amniotic tissue, pluripotent stem cells, and teeth. As used herein, “mesenchymal stromal cells” or “MSCs” includes cells that are CD34-positive at initial tissue isolation but are phenotypically and functionally similar to the cells described above. As used herein, “MSC” includes cells isolated from tissues using cell surface markers selected from a list comprising 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 that meet ISCT criteria before or after expansion. As used herein, mesenchymal stem cells may include, as described in the literature, bone marrow stromal stem cells (BMSSC), bone marrow-derived adult pluripotent inducible cells (MIAMI) cells, pluripotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), Multistem cells, etc. ® Prochymal ® remestemcel-L, mesenchymal progenitor cells (MPC), dental pulp stem cells (DPSC), 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 regeneration cells (ERC), and adipose-derived stem cells and regeneration cells (ADRC).

[0051] The methods and compositions of the present invention This invention discloses a method for treating a subject using paracrine factors (i.e., soluble factors or cell-free methods) generated from pluripotent stem cells and their derivatives, said paracrine factors capable of mimicking the specific therapeutic benefits of regenerating cells. In one embodiment, the invention provides a therapeutic cell population that generates soluble factors, wherein said soluble factors can be co-administered with said therapeutic cells to a subject to treat age-related diseases, conditions, or symptoms. In another embodiment, the invention provides soluble factors derived from regenerating cells, which are administered to a subject to reduce some of the limitations and disadvantages associated with the clinical use of regenerating cells themselves. The methods of the invention are provided to promote tissue regeneration and cellular rejuvenation in one or more organ systems in a subject that may be affected by aging, to treat diseases associated with biological aging, and to correct or prevent cellular and molecular changes in vivo that contribute to age-related diseases.

[0052] In some embodiments, the soluble factor comprises one or more entities released by cells, said entities being secreted by cells, detached from the cell surface by cutting or budding, released as cell debris, or comprising any cell-derived product released into the extracellular environment, for example, through apoptosis or necrosis. This document discloses culture systems and methods for generating and maintaining conditioned media containing soluble factors derived from regenerating cell populations. In some embodiments, methods are provided for inducing or maintaining the release of one or more soluble factors from cells into a fluid or culture medium, for example, in a tissue culture plate / flask or bioreactor. In some embodiments, the soluble factor comprises proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, apoptotic bodies, DNA molecules, or RNA molecules such as microRNA (RNA), messenger RNA (mRNA), and other non-coding RNAs. In some embodiments, the tissue culture medium or conditioned medium is a source of one or more soluble factors, wherein the medium contains various entities, including but not limited to proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, apoptotic bodies, DNA molecules, or RNA molecules. In some embodiments, the conditioned medium may contain a variety of soluble factors released by a cell population. In some embodiments, the term "soluble factor" may refer to a biological factor containing an antigen; for example, a member of the "differentiation cluster" cell surface marker family. In some embodiments, soluble factors include cytokines, growth factors, adhesion molecules, addressins, chemokines, or another type of molecule that acts on cells or tissues. In some embodiments, soluble factors include molecules or antigens that float freely in a liquid such as a culture medium. In other embodiments, soluble factors include molecules or antigens that are components of extracellular vesicles, exosomes, or apoptotic bodies, wherein the molecules or antigens may be expressed on the surface of the vesicles or bodies, within their membranes, or both. In other embodiments, the term "soluble factor" is used to describe vesicles containing one or more antigens or molecules, such as exosomes. In some embodiments, the soluble factor is isolated or separated from the cellular components in the liquid or culture medium, thereby providing a cell-free composition reflecting the secretory group of the cell types present in said liquid or culture.

[0053] In some embodiments, cell-free therapy is provided that utilizes the secretome of regenerating cells as a therapeutic modality to treat certain disorders, particularly age-related diseases and conditions. In some embodiments, the secretome comprises one or more molecules, including proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, DNA molecules, or RNA molecules. The inventors have optimized methods for producing regenerating cell populations and derivatives thereof as regenerative agents or senescent cell scavengers, wherein the secretome generated by the regenerating cell populations and derivatives thereof can be used to prevent age-related conditions and symptoms associated with one or more organ systems in the body. Therefore, cell populations as sources of the cell-free therapy of the present invention are disclosed herein. The present invention provides a method for treating subjects suffering from age-related diseases, conditions, or symptoms using therapeutic soluble factors derived from stem cell populations or derivatives thereof.

[0054] In one embodiment, the therapeutic soluble factor is derived from autologous regenerative cells comprising induced pluripotent stem cells or derivatives thereof (i.e., their differentiated cell products), wherein the soluble factor is secreted by the cells or otherwise released into the extracellular environment. In another embodiment, the soluble factor for treating a subject is derived from allogeneic regenerative cells or derivatives thereof. In one embodiment, the soluble factor is derived from cell derivatives of induced pluripotent stem cells, i.e., cells generated or differentiated from induced pluripotent stem cells. In one embodiment, the therapeutic soluble factor is generated from cells comprising progenitor cells, tissue-specific progenitor cells, pluripotent cells, mesenchymal stem cells, hematopoietic cells, fibroblasts, lymphocytes, granulocytes, or another cell type. In some embodiments, the autologous regenerative cells comprising pluripotent stem cells are generated from somatic cells isolated directly from a subject's tissue source or biofluid, such as from bone marrow, blood, or adipose tissue. In some embodiments, somatic cells are isolated directly from a subject's tissue source or biofluid and cultured in vitro under specific conditions to generate autologous regenerative cells. In some embodiments, the regenerative cells comprising stem cells are further cultured in vitro under conditions of induced cell differentiation or reprogramming. In one specific embodiment, the regenerating cells comprise induced pluripotent stem cells (iPSCs), wherein the iPSCs are cultured in vitro to induce reprogramming, and wherein the reprogramming is achieved by introducing a specific gene encoding a transcription factor. In an exemplary embodiment, iPSCs are reprogrammed by introducing a specific gene encoding one or more transcription factors (including Oct3 / 4, c-Myc, and Klf4) into primary cells under embryonic stem cell culture conditions. In some embodiments, the regenerating cells are adapted for gene modification or treatment with one or more soluble factors. In some embodiments, the regenerating cells are adapted to differentiate in vitro into one or more cell types, wherein the differentiated cell types may include cells at different terminal differentiation stages, such as stem cells, progenitor cells, tissue-specific progenitor cells, pluripotent cells, and terminally differentiated cells. In one specific implementation, the autologous regenerating cells include pluripotent stem cells adapted to differentiate into the following cell types: mature dendritic cells, immature dendritic cells, lymphoid dendritic cells, myeloid dendritic cells, monocytes, macrophages, alternatively activated macrophages, mesenchymal stem cells, T cells, B cells, CD5-positive B cells, regulatory T cells, neutrophils, eosinophils, basophils, mast cells, myeloid cells, common myeloid progenitor cells, common lymphoid progenitor cells, hematopoietic stem cells, natural killer (NK) cells, NKT cells, fibroblasts, synovial cells, epithelial cells, epithelial progenitor cells, endothelial cells, endothelial progenitor cells, hepatocyte-like cells, hepatic stellate cells, cardiomyocytes, keratinocytes, kidney progenitor cells, neural stem cells, neural progenitor cells, neurons, or any tissue-specific stem cells or progenitor cells.

[0055] Embodiments of the present invention provide a method for producing one or more therapeutic factors from autologous regenerating cells or derivatives thereof, and a method for treating a subject with said one or more regenerating factors. In some embodiments, a conditioned medium containing one or more soluble factors is produced by culturing said autologous regenerating cells or derivatives thereof, said conditioned medium being usable as a cell-free therapeutic composition. In some embodiments, one or more soluble factors, such as proteins, glycoproteins, vesicles, or other molecules, are isolated from said conditioned medium to provide a therapeutic composition for treating a subject.

[0056] In one embodiment, a method is provided for treating a subject suffering from a biological aging disease, condition, or symptom, the method comprising: a) identifying the subject affected by the age-related disease, condition, or symptom; b) isolating primary cells from a biological fluid or tissue of the subject; c) providing a first culture system in which the primary cells are exposed to culture conditions and treatments to induce dedifferentiation into induced pluripotent stem cells; d) providing a second culture system in which the induced pluripotent stem cells are exposed to culture conditions and treatments to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions; e) harvesting or isolating the one or more soluble factors from the culture medium of the second culture system; and f) administering the one or more soluble factors to the subject, wherein the one or more soluble factors exert regenerative, immunomodulatory, or both functions. Optionally, additional growth factors, immunomodulatory proteins, or therapeutic agents may be administered to the subject systemically or locally at tissue sites to enhance the effect of the administered soluble factors or to provide unique regenerative capacity to combat aging. Other embodiments of the invention provide methods for preventing age-related diseases, disorders, or symptoms using the cell compositions, soluble factor compositions, or combinations thereof of the invention. In this embodiment, subjects at risk of developing age-related diseases, conditions, or symptoms are selected to receive treatment with the methods disclosed herein, wherein the risk may be assessed based on the subject's age, environmental factors, genetic factors, symptoms exhibited by the subject, or a combination thereof. In some embodiments, primary cells are isolated from subjects who have or are at risk of developing age-related conditions or diseases, wherein the autologous cells are treated with the methods of the invention. In other embodiments, the primary cells are derived from an allogeneic source, i.e., from subjects who are not recipients of the therapies disclosed herein.

[0057] In another embodiment, a method is provided for treating or preventing a disease, disorder, or symptom associated with biological aging in a subject, the method comprising: a) identifying a subject affected by or at risk of developing an age-related disease, disorder, or symptom; b) obtaining primary somatic cells from a biological fluid or tissue of the subject; c) providing a first cell culture system in which the primary somatic cells are exposed to culture conditions and treatments to induce their dedifferentiation into induced pluripotent stem cells; d) providing a second culture system in which the induced pluripotent stem cells are exposed to culture conditions and treatments to induce their differentiation into somatic cells; e) providing a third culture system in which the differentiated somatic cells are exposed to culture conditions and treatments to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions; f) harvesting or isolating the one or more soluble factors from the culture medium of the third culture system; and g) administering the one or more soluble factors to the subject, wherein the one or more soluble factors exert regenerative, immunomodulatory, or both functions. In some embodiments, the soluble factor is of autologous or allogeneic origin. In one embodiment, the different culture systems disclosed herein include different cell culture media or different additives, wherein the additives are selected from the group consisting of carbon sources, nitrogen sources, phosphate sources, essential minerals, amino acids, vitamins, buffers, pH indicators, antibiotics, specific growth factors, cytokines, hormones, and other molecules. In other embodiments, the composition of the culture systems used in each different step of the method of the present invention overlaps. In some embodiments, the differentiated somatic cells from the second culture system are selected from the group consisting of: stem cells, progenitor cells, mature dendritic cells, immature dendritic cells, lymphoid dendritic cells, myeloid dendritic cells, monocytes, macrophages, alternatively activated macrophages, mesenchymal stem cells, T cells, B cells, CD5-positive B cells, regulatory T cells, neutrophils, eosinophils, basophils, mast cells, myeloid cells, common myeloid progenitor cells, common lymphoid progenitor cells, hematopoietic stem cells, natural killer (NK) cells, NKT cells, fibroblasts, epithelial cells, epithelial progenitor cells, endothelial cells, endothelial progenitor cells, hepatocyte-like cells, hepatic stellate cells, cardiomyocytes, keratinocytes, kidney progenitor cells, neural stem cells, neural progenitor cells, neurons, or tissue-specific stem cells or progenitor cells.

[0058] Some embodiments of the present invention also provide a method for generating multiple differentiated cell types from induced pluripotent stem cells. In this embodiment, the method provides multiple culture conditions in step d) to support the differentiation of multiple cell types from a population of induced pluripotent stem cells. In one exemplary embodiment, a population of induced pluripotent stem cells may be allocated to a first culture system for generating a first differentiated cell type (e.g., mesenchymal stem cells), and induced pluripotent stem cells may be seeded into a second culture system to generate a second differentiated cell type (e.g., regulatory T cells). Furthermore, multiple different cell culture conditions may be provided in step e) to induce 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 a culture of induced pluripotent stem cells and their derivatives (i.e., differentiated cells) for administration to a subject in need.

[0059] In another embodiment, a method is provided for treating or preventing age-related diseases, disorders, or symptoms in a subject, the method comprising: a) identifying a subject affected by or at risk of developing an age-related disease, disorder, or symptom; b) obtaining somatic cells directly from the subject's biological fluid or tissue; c) providing a first cell culture system wherein the primary somatic cells are provided with culture conditions to induce dedifferentiation into induced pluripotent stem cells; d) providing a second culture system wherein the induced pluripotent stem cells are provided with culture conditions to induce differentiation into somatic cells; e) providing a third culture system wherein the differentiated somatic cells are provided with culture conditions to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into a culture medium, and wherein the one or more soluble factors have regenerative or immunomodulatory functions; f) harvesting or isolating the one or more autologous soluble factors from the culture medium of the third culture system; g) administering the one or more autologous soluble factors to the subject; and h) optionally, administering the differentiated somatic cells to the subject. In some embodiments, other agents may be administered to the subject to enhance the regenerative capacity of the applied cells and / or soluble factors generated according to the method of the present invention. For example, agents that promote cell migration or homing (e.g., chemokines), immunomodulators, and / or immunostimulants may be administered to the subject before, after, or simultaneously with the administration of the therapy of the present invention containing soluble factors and / or cells. In other embodiments, in addition to the therapies disclosed herein, therapeutic cell populations generated by other means may be co-administered to the subject.

[0060] In some embodiments, the invention teaches the selection of cell populations that produce one or more soluble factors, and the selection or harvesting of said one or more soluble factors that are clinically useful for the treatment or prevention of age-related diseases, conditions, or symptoms exacerbated by underlying biological mechanisms of aging. In one embodiment, the age-related diseases are selected from the group comprising autoimmune diseases, arthritis, cardiovascular disease, hypertension, stroke, heart failure, diabetes, hearing loss, osteoporosis, osteoarthritis, cataracts, pain, chronic obstructive pulmonary disease (COPD), osteoporosis, dementia, Alzheimer's disease, Parkinson's disease, depression, and cancer. In one implementation, age-related diseases include cancer types selected from the following list: melanoma (e.g., metastatic malignant melanoma), kidney 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, head or neck cancer, intradermal or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, and so on. Cancers of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and T-cell lymphoma. Certain embodiments of the invention provide methods for addressing aging-related molecular changes occurring at the cellular or tissue level. In certain embodiments, methods are provided for reducing, improving, or preventing fibrosis associated with biological aging. In one embodiment, methods are provided for preventing or treating fibrotic diseases. In one specific embodiment, fibrotic diseases include one or more 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 implementation, methods are provided to alleviate one or more symptoms associated with fibrotic disease in a subject, said one or more symptoms including shortness of breath, fatigue, cough, muscle weakness, muscle stiffness, poor healing after injury, and involuntary weight loss. Some implementations also provide methods to reduce the accumulation of senescent cells in organs or tissues such as connective tissue or blood. Other implementations provide methods to improve the immune function of a subject.Biological aging can have progressively adverse effects on immune function, in which subjects exhibit one or more of the following: impaired immunity to certain infectious pathogens, dysregulation or hyperactivity of the immune response against certain infectious pathogens or their own tissues (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 present invention are enumerated in the following paragraphs: Aspect 1: A method for inducing one or more regenerative processes or functions in a subject suffering from a disease, disorder, or symptom associated with biological aging, the method comprising: a) identifying a subject suffering from an age-related disease, disorder, or symptom; b) harvesting primary somatic cells from the subject's tissue or blood or alternatively from an allogeneic source; c) performing a first cell culture method or step, wherein the primary somatic cells are cultured in a culture medium and conditions for inducing dedifferentiation into pluripotent stem cells; d) performing a second culture method or step, wherein the induced pluripotent stem cells are cultured in a culture medium and conditions for generating differentiated somatic cell types; e) optionally, performing additional culture methods or steps to generate one or more other regenerative processes or functions from the induced pluripotent stem cells. The differentiated somatic cell type; 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 a subsequent culture method are provided with a culture medium and conditions that induce or maintain the release of one or more soluble factors into the culture medium; g) harvesting or isolating the one or more soluble factors derived from the third cell culture method; h) administering the one or more soluble factors from the third cell culture method to the subject at a concentration and duration sufficient to induce one or more regenerative processes or functions in the subject; and i) optionally, also administering the one or more differentiated somatic cell types produced by the second culture method or another culture method to the subject.

[0062] Aspect 2: The method according to aspect 1, wherein the harvested one or more soluble factors comprises a cell-free composition for administration to a subject.

[0063] Aspect 3: The method according to aspect 2, wherein the cell-free composition comprises conditioned medium, protein, nucleic acid, microRNA, apoptotic body, extracellular vesicle or exosome.

[0064] Aspect 4: According to the method described in aspect 1, wherein the disease, disorder or symptoms associated with biological aging are related to a decrease in endothelial function in an organ or tissue.

[0065] Aspect 5: The method of aspect 4, wherein endothelial function is assessed based on blood flow-mediated vasodilation, nitric oxide production, maintenance of anticoagulant 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.

[0066] Aspect 6: The method of aspect 5, wherein vasodilation in response to inflammation is caused by one or more of the following: prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), interleukin-1 beta (IL-1 beta), 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 damage, tissue factor, ferroptosis bodies.

[0067] Aspect 7: The method of aspect 5, wherein the adhesion molecules upregulated in response to inflammation include one or more 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-1 (TIM-1), TIM-3, galectin-1, galectin-3, galectin-5, galectin-9, LPR-1, neuropilin (NRP), CD40 ligand, and receptor for advanced glycation end products (RAGE).

[0068] Aspect 8: The method of aspect 4, wherein reduced endothelial function is determined based on a reduced ability of the endothelium to allow cells to exit from circulation and enter tissue.

[0069] Aspect 9: The method of aspect 8, wherein the cells exit from circulation and enter tissue via extravasation through high endothelial venules.

[0070] Aspect 10: The method of aspect 8, wherein the cells are stem cells.

[0071] Aspect 11: The method of aspect 10, wherein the stem cells are hematopoietic stem cells.

[0072] Aspect 12: The method of aspect 11, wherein the hematopoietic stem cells express one or more molecules including CD34, CD38, CXCR4, IL-1 receptor antagonist, c-kit, VEGF receptor, CD127, 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, Angiogenin receptor, FGF-1 receptor, FGF-2 receptor, FGF-5 receptor, MMP9, Thromboxane, SMAD2, and TGF-β.

[0073] Aspect 13: The method of aspect 11, wherein the hematopoietic stem cells are capable of inducing multilineage engraftment in an immunodeficient host.

[0074] Aspect 14: The method of aspect 10, wherein the stem cells are mesenchymal stem cells.

[0075] Aspect 15: The method of aspect 14, wherein the mesenchymal stem cells have a fibroblast-like morphology.

[0076] Aspect 16: The method of aspect 14, wherein the mesenchymal stem cells migrate toward an SDF-1 gradient.

[0077] Aspect 17: The method of aspect 14, wherein the mesenchymal stem cells express one or more molecules including CD37, CD133, CD74, CD90, CD105, c-kit, and PD-L1.

[0078] Aspect 18: The method of aspect 14, wherein the mesenchymal stem cells lack expression of one or more molecules including CD14, CD16, CD19, CD45, CD34, and HLA-G.

[0079] Aspect 19: The method of aspect 14, wherein the mesenchymal stem cells are capable of adhering to plastic.

[0080] Aspect 20: The method of aspect 14, wherein the mesenchymal stem cells are capable of inhibiting T cell proliferation.

[0081] Aspect 21: The method of aspect 20, wherein the T cell proliferation is caused by one or more mitogens stimulating the T cells.

[0082] Aspect 22: The method according to aspect 21, wherein the mitogen comprises one or more lectins, phytohemagglutinins, and concanavalin A.

[0083] Aspect 23: The method according to aspect 21, wherein the mitogen comprises one or more antibodies capable of cross-linking T cell receptors.

[0084] Aspect 24: The method according to aspect 21, wherein the mitogen comprises one or more antibodies capable of cross-linking T cell receptors and one or more antibodies capable of cross-linking co-stimulatory receptors.

[0085] Aspect 25: The method according to aspect 24, wherein the antibody capable of crosslinking the T cell receptor includes one or more of anti-CD3 antibody, anti-T cell receptor (TCR) antibody and anti-CD52 antibody.

[0086] Aspect 27: The method according to aspect 24, wherein the antibody capable of crosslinking the co-stimulatory receptor includes one or more of anti-CD40 antibody, anti-CD5 antibody, anti-CD25 antibody and anti-ICAM antibody.

[0087] Aspect 28: The method according to aspect 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0088] Aspect 29: According to the method of aspect 1, wherein the one or more soluble factors are obtained from induced pluripotent stem cells.

[0089] Aspect 30: The method according to aspect 28, wherein induced pluripotent stem cells include dedifferentiated monocytes.

[0090] Aspect 31: According to the method of aspect 30, dedifferentiated monocytes are generated by exposure to a conditioned medium containing induced pluripotent stem cells.

[0091] Aspect 32: The method according to aspect 31, wherein induced pluripotent stem cells are cultured in a liquid culture medium in embryonic form.

[0092] Aspect 33: The method according to aspect 32, wherein the liquid culture medium includes Iscove's medium, DMEM medium, OptiMEM medium, EMEM medium, RPMI-1640 medium, or AIM-V medium.

[0093] Aspect 34: The method described in aspect 32, wherein the embryonic body is disintegrated or separated every 2 days, every 5 days, or every 10 days.

[0094] Aspect 35: The method according to aspect 32, wherein the embryonic body comprises induced pluripotent stem cells and monocytes.

[0095] Aspect 36: The method according to aspect 35, wherein the monocytes are exposed to a stimulant or stressor before being mixed with induced pluripotent stem cells to generate a conditioned medium.

[0096] Aspect 37: The method according to aspect 36, wherein the stressor includes one or more of the following: hypoxia, hyperosmolarity, hypoosmolarity, hyperthermia, serum starvation, mTOR inhibition, AMPK activation, or activation of inflammatory pathways.

[0097] Aspect 38: The method according to aspect 37, wherein the inflammatory pathway includes one or more of the following pathways: MAP kinase, Janus-activated kinase, signal transduction and transcription activator-3 (STAT3), STAT5, STAT6, TLR2, TLR3 and TLR4.

[0098] Aspect 39: The method according to aspect 38, wherein the TLR2 pathway is activated via peptidoglycan.

[0099] Aspect 40: The method according to aspect 38, wherein TLR3 is activated by one or more of the following: double-stranded RNA, polyinosinic acid-polycytidine (poly(I:C), polyinosinic acid-polycytidine-poly-L-lysine-carboxymethyl cellulose (poly-ICLC), and inactivated influenza virus.

[0100] Aspect 41: The method according to aspect 38, wherein the TLR4 pathway is activated by one or more of the following: neutrophil extracellular traps, β-glucan, HMGB1, lipopolysaccharide, yeast cell wall extract, or anti-TLR4 antibody.

[0101] Aspect 42: The method according to aspect 31, wherein the induced pluripotent stem cells comprise genetically engineered cells that express a TGF-β inhibitor.

[0102] Aspect 43: The method according to aspect 42, wherein the TGF-β inhibitor comprises an antibody molecule.

[0103] Aspect 44: The method according to aspect 43, wherein the antibody includes camelid antibodies, microantibodies, or aptamers.

[0104] Aspect 45: The method according to aspect 42, wherein the TGF-β inhibitor includes a molecule that induces RNA interference.

[0105] Aspect 46: The method according to aspect 45, wherein the molecules that induce RNA interference include short interfering RNA, short hairpin RNA, microRNA, or long noncoding RNA.

[0106] Aspect 47: According to the method of aspect 1, wherein the first culture method provides an agent for inducing primary somatic cell dedifferentiation or reprogramming and promoting stem cell pluripotency.

[0107] Aspect 48: The method according to aspect 47, wherein the agent comprises one or more of the following: GDF-11, GDF-15, amniotic fluid, umbilical cord plasma and BMP2.

[0108] Aspect 49: The method according to aspect 47, wherein the agent inhibits the activation of nuclear factor-κB.

[0109] Aspect 50: The method according to aspect 47, wherein the agent increases the activation of nuclear factor erythroid 2-associated factor 2 (NRF2).

[0110] Aspect 51: The method according to aspect 47, wherein the agent increases the activation of heme oxygenase-1.

[0111] Aspect 52: The method according to aspect 47, wherein the agent increases the activation of Bcl-2.

[0112] Aspect 53: The method according to aspect 47, wherein the agent increases the activation of Bcl-2XL.

[0113] Aspect 54: The method according to aspect 47, wherein the agent increases the activation of Survivin.

[0114] Aspect 55: The method according to aspect 47, wherein the agent increases the activation of Livin.

[0115] Aspect 56: The method according to aspect 47, wherein the agent is an HDAC inhibitor.

[0116] Aspect 57: The method according to aspect 56, wherein the HDAC inhibitor comprises one or more of the following: valproic acid, trichostatin A, sodium phenylbutyrate, and butyric acid.

[0117] Aspect 58: The method according to aspect 47, wherein the agent comprises a GSK-3 inhibitor.

[0118] Aspect 59: The method according to aspect 58, wherein the GSK inhibitor comprises lithium.

[0119] Aspect 60: The method according to aspect 47, wherein the agent comprises PIM1.

[0120] Aspect 61: The method according to aspect 47, wherein the agent comprises an immortal oncogene.

[0121] Aspect 62: The method according to aspect 61, wherein the oncogene includes one or more of the following: SV40 large T antigen, abl1, 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, MAS1, MDM2, MOS, MYH11, MYB, MYCN, PAX5, RAF, RAS, REL, ROS1, SKI (PDGF-BB), SET, SRC, TAL1, TAN1, TIAN, TSC2, and TRK.

[0122] Aspect 63: The method according to aspect 47, wherein the agent causes a change in the genotype of pluripotent stem cells.

[0123] Aspect 64: The method according to aspect 47, wherein the agent can be conditionally deactivated.

[0124] Aspect 65: According to the method described in aspect 1, wherein the disease, disorder or symptom associated with biological aging is related to a decline in immune function.

[0125] Aspect 66: According to the method described in aspect 65, the decline in immune function is associated with the loss of thymus volume.

[0126] Aspect 67: According to the method described in aspect 65, the decline in immune function is associated with the loss of T cell activity.

[0127] Aspect 68: The method according to aspect 1, wherein the first somatic cell is reprogrammed using one or more retroviral expression vectors, the vectors being designed to deliver one or more specific genes to cause the first somatic cell to dedifferentiate into pluripotent stem cells.

[0128] Aspect 69: The method according to aspect 68, wherein one or more specific genes encode one or more transcription factors.

[0129] Aspect 70: The method according to aspect 68, wherein the retroviral expression vector includes one or more of the following vectors: pMX vector, lentiviral vector, adenovirus vector, pMX adeno-associated vector, and herpesvirus vector.

[0130] Aspect 71: The method according to aspect 68, wherein the retroviral expression vector includes one or more of the following types of vectors: plasmid-based vectors, RNA-based vectors, mRNA-based vectors, and microRNA-based vectors.

[0131] Aspect 72: The method according to aspect 47, wherein the first cell culture system further comprises one or more of the following molecules or compounds: ascorbic acid, RAS pathway stimulant, Myc pathway stimulant, PIM1 pathway stimulant, and Janus activated kinase pathway stimulant.

[0132] Aspect 73: According to the method described in aspect 1, wherein the disease, disorder or symptom associated with biological aging is related to fibrosis in an organ or tissue.

[0133] Aspect 74: A method for treating or preventing age-related fibrosis, the method comprising: a) identifying a subject who exhibits signs of age-related fibrosis in an organ or tissue or is at risk of developing age-related fibrosis; b) harvesting first somatic cells from the subject's tissue or blood; c) providing a first cell culture system for reprogramming the first somatic cells into pluripotent stem cells; d) providing a second cell culture system for inducing the pluripotent stem cells to differentiate into regulatory T cells; e) providing a third cell culture system for inducing or maintaining the production of one or more soluble factors by the regulatory T cells produced in the second cell culture system; f) harvesting the one or more soluble factors from the third cell culture system; and g) administering the one or more soluble factors from step f) to the subject at a concentration and duration sufficient to induce one or more regenerative processes or functions in the subject.

[0134] Aspect 75: A method for treating age-related fibrosis, the method comprising: a) identifying a subject exhibiting signs of age-related fibrosis in an organ or tissue; b) harvesting first somatic cells from the subject's tissue or blood; c) providing a first cell culture system for reprogramming the first somatic cells into pluripotent stem cells; d) providing a second cell culture system for inducing the pluripotent stem cells to differentiate into mesenchymal stem cells; e) providing a third cell culture system for inducing or maintaining the production of one or more soluble factors from the mesenchymal stem cells produced by the second cell culture system; f) harvesting the one or more soluble factors from the third cell culture system; g) administering the one or more soluble factors from step f) to the subject at a concentration and duration sufficient to induce one or more regenerative processes in the subject.

[0135] Aspect 76: According to the method described in aspect 1, wherein the second culture method generates a differentiated somatic cell type including regulatory T cells.

[0136] Aspect 77: The method described in aspect 76, wherein regulatory T cells produce one or more soluble factors.

[0137] Aspect 78: According to the method of aspect 77, one or more soluble factors induce one or more regenerative processes after being administered to the subject.

[0138] Aspect 79: The method according to aspect 78, wherein the regeneration process includes reducing or improving fibrosis in an organ or tissue.

[0139] Aspect 80: According to the method of aspect 1, wherein the second culture method generates differentiated somatic cell types including mesenchymal stem cells.

[0140] Aspect 81: According to the method described in aspect 80, mesenchymal stem cells produce one or more soluble factors.

[0141] Aspect 82: According to the method of aspect 81, the one or more soluble factors induce one or more regenerative processes after being administered to a subject.

[0142] Aspect 83: The method according to aspect 82, wherein the regeneration process includes reducing or improving fibrosis in an organ or tissue.

[0143] Aspect 84: According to the method of aspect 1, wherein the second culture method generates multiple differentiated somatic cell types, wherein the differentiated somatic cell types include regulatory T cells and mesenchymal stem cells.

[0144] Aspect 85: According to the method of aspect 84, wherein the second culture method provides a first culture medium or conditions for regulatory T cell differentiation.

[0145] Aspect 86: According to the method of aspect 85, the first culture medium or condition comprises one or more cytokines, chemokines, and growth factors.

[0146] Aspect 87: According to the method of aspect 84, wherein the second culture method provides a second culture medium or conditions for the differentiation of mesenchymal stem cells.

[0147] Aspect 88: According to the method of aspect 87, the second culture medium or condition includes one or more cytokines, chemokines, and growth factors.

[0148] Aspect 89: According to the method of aspect 84, the regulatory T cell is capable of producing one or more soluble factors.

[0149] Aspect 90: According to the method described in aspect 84, mesenchymal stem cells are capable of producing one or more soluble factors.

[0150] Aspect 91: The method according to aspect 1, wherein the one or more differentiated somatic cell types administered to the subject include 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.

[0151] Aspect 92: According to the method of aspect 91, wherein the first differentiated somatic cell type administered to the subject includes mesenchymal stem cells and the second differentiated somatic cell type administered to the subject includes regulatory T cells.

[0152] Aspect 93: The method according to aspect 92, wherein mesenchymal stem cells are 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.

[0153] Aspect 94: The method according to aspect 89, wherein the one or more soluble factors are administered to the subject, and the one or more soluble factors induce a regenerative process in the subject.

[0154] Aspect 95: The method according to aspect 90, wherein the one or more soluble factors are administered to the subject, and the one or more soluble factors induce a regenerative process in the subject.

[0155] Aspect 96: The method according to aspect 80, wherein the mesenchymal stem cells produce soluble factors including brain-derived neurotrophic factor.

[0156] Aspect 97: According to the method described in aspect 96, mesenchymal stem cells are selected because they express CD56.

[0157] Aspect 98: The method described in aspect 96, wherein brain-derived neurotrophic factor is generated in response to molecular signals associated with tissue damage.

[0158] Aspect 99: The method according to aspect 98, wherein at least 10 pg, at least 25 pg, or at least 100 pg of brain-derived neurotrophic factor is produced by mesenchymal stem cells upon poly(I:C) stimulation.

[0159] Aspect 100: The method according to aspect 80, wherein mesenchymal stem cells produce a soluble factor containing ciliary neurotrophic factor.

[0160] Aspect 101: The method according to aspect 100, wherein ciliary neurotrophic factor is generated in response to molecular signals associated with tissue damage.

[0161] Aspect 102: According to the method described in aspect 80, mesenchymal stem cells are selected because they express CD73.

[0162] Aspect 103: The method according to aspect 101, wherein at least 4 pg, at least 8 pg, or at least 12 pg of ciliary neurotrophic factor is produced by mesenchymal stem cells upon poly(I:C) stimulation.

[0163] Aspect 104: According to the method of aspect 80, mesenchymal stem cells produce soluble factors containing basic fibroblast growth factor.

[0164] Aspect 105: The method according to aspect 104, wherein basic fibroblast growth factor is generated in response to molecular signals associated with tissue damage.

[0165] Aspect 106: The method according to aspect 80, wherein the mesenchymal stem cells are selected for expressing CD105.

[0166] Aspect 107: The method according to 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 cells upon stimulation with poly(I:C).

[0167] Aspect 108: According to the method of aspect 1, wherein the second culture method generates a differentiated somatic cell type comprising regulatory T cells, and wherein the regulatory T cells produce one or more soluble factors.

[0168] Aspect 109: The method according to aspect 1, wherein the one or more soluble factors are administered before or after plasmin administration to the subject.

[0169] Aspect 110: The method according to aspect 109, wherein the plasmin comprises a matrix metalloproteinase.

[0170] Aspect 111: The method according to aspect 110, wherein the matrix metalloproteinase comprises MMP-3, MMP-5, MMP-6, MMP-9 or MMP-12.

[0171] Aspect 112: According to the method of aspect 1, wherein the one or more soluble factors are administered before or after the NOGO inhibitor is administered to the subject.

[0172] Aspect 113: The method according to aspect 112, wherein the NOGO inhibitor comprises one or more of the following molecules or compounds: antisense oligonucleotides, short interfering RNA, short hairpin RNA, small molecule inhibitors, antibodies, aptamers, SOMAmer®, bispecific antibodies, ribozymes, antibodies, microantibodies, hammerhead ribozymes, and soluble receptors.

[0173] Aspect 114: According to the method of aspect 1, the differentiated somatic cell type from the second culture method and applied to the subject includes myeloid lineage cells.

[0174] Aspect 115: The method according to aspect 114, wherein the myeloid lineage cells include myeloid-derived dendritic cells.

[0175] Aspect 116: The method according to aspect 115, wherein the myeloid-derived dendritic cells are immature.

[0176] Aspect 117: The method according to aspect 115, wherein the myeloid-derived dendritic cells are generated from monocytes under specific in vitro culture conditions.

[0177] Aspect 118: The method according to aspect 117, wherein the mononuclear cells are isolated from peripheral blood.

[0178] Aspect 119: The method according to aspect 118, wherein the number of monocytes in peripheral blood is first increased using a mobilizing agent, and wherein the mobilizing agent is selected from the group comprising G-CSF, GM-CSF, M-CSF, IL-3, IL-10, β-glucan and interferon γ.

[0179] Aspect 120: The method according to aspect 117, wherein the mononuclear cells are isolated from sources including menstrual blood, umbilical cord blood, Wharton's jelly, bone marrow, adipose tissue, and omentum.

[0180] Aspect 121: The method according to aspect 117, wherein the monocytes are cultured with one or more agents to induce a tolerance phenotype before being differentiated into myeloid dendritic cells in vitro.

[0181] Aspect 122: The method according to aspect 117, wherein exosomes are isolated or harvested from myeloid-derived dendritic cells generated in vitro.

[0182] Aspect 123: According to the method of aspect 121, one or more agents in the culture include 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-κB bait oligonucleotide, nuclear factor κB kinase subunit β (Ikk-β) bait oligonucleotide inhibitor, IL-35, TGF-β, ascorbic acid, N-acetylcysteine, α-lipoic acid, methylene blue, and valproic acid.

[0183] Aspect 124: The method according to aspect 121, wherein one or more agents in the culture induce monocytes to produce IL-10.

[0184] Aspect 125: The method according to aspect 124, wherein at least 10 pg / mL, at least 20 pg / mL or at least 40 pg / mL of IL-10 is produced by monocytes in a culture, and the monocytes in the culture contain at least 1 million cells, at least 2 million cells or at least 5 million cells.

[0185] Aspect 126: The method according to aspect 116, wherein immature dendritic cells express or produce one or more molecules, said molecules including IL-10, IL-4, IL-13, IL-20, IL-22, IL-35, IL-37, IL-38, TGF-β, IL-20, IL-22, IL-35, IL-37, IL-38, endothelin, HLA-G, and IL-12 p40 homodimer.

[0186] Aspect 127: The method according to aspect 116, wherein immature dendritic cells have increased phagocytic activity compared to mature dendritic cells.

[0187] Aspect 128: The method according to aspect 116, wherein immature dendritic cells have increased activity toward a chemotactic gradient in vitro compared to mature dendritic cells.

[0188] Aspect 129: According to the method of aspect 128, the chemotactic gradient is induced by one or more chemokines, the chemokines including IL-8, MIP-1 α, MIP-1 beta, CXCL12, MCP-1, TNF-α, lymphotoxin, hyaluronic acid degradation products and HMGB1.

[0189] Aspect 130: According to the method of aspect 116, the immature dendritic cells have reduced expression of one or more molecules or antigens compared to mature dendritic cells, said molecules or antigens including HLA I, HLA II, CD1a, CD40, CD80, CD86, IL-15 receptor, interferon-γ, IL-18 receptor, progesterone receptor and c-kit.

[0190] Aspect 131: According to the method of aspect 116, wherein the immature dendritic cells induce the generation of regulatory T cells when co-cultured with allogeneic immature T cells.

[0191] Aspect 132: According to the method of aspect 116, wherein the immature dendritic cells upregulate the expression of autoimmune regulatory factors (AIRE) upon contact with allogeneic immature T cells.

[0192] Aspect 133: The method according to aspect 132, wherein AIRE upregulation depends on the production of one or more molecules including IL-10, TGF-beta, HLA-G, endothelial linkerin, FGF-1, FGF-2, FGF-5 and NOTCH.

[0193] Aspect 134: According to the method of aspect 114, wherein myeloid lineage cells include immature neutrophils.

[0194] Aspect 135: According to the method of aspect 134, the immature neutrophils are neutrophil progenitor cells.

[0195] Aspect 136: According to the method of aspect 134, immature neutrophils express one or more molecules, said molecules including PU1.1, G-CSF receptor, stem cell factor receptor, c-met, M-CSF receptor and GM-CSF receptor.

[0196] Aspect 137: The method according to aspect 134, wherein immature neutrophils produce IL-10 upon stimulation by a TLR-4 agonist.

[0197] Aspect 138: The method according to aspect 137, wherein the TLR-4 agonist is selected from the group comprising β-glucan, hyaluronic acid degradation products, HMGB1 and histones.

[0198] Aspect 139: The method according to aspect 114, wherein myeloid lineage cells include myeloid suppressor cells.

[0199] Aspect 140: The method according to aspect 139, wherein myeloid suppressor cells are able to differentiate into monocytes upon treatment with a molecule selected from the group consisting of all-trans retinoic acid, vitamin D3, M-CSF and GM-CSF.

[0200] Aspect 141: The method according to aspect 139, wherein myeloid suppressor cells are able to suppress T cell proliferation by producing molecules selected from the group consisting of 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.

[0201] Aspect 142: According to the method of aspect 1, wherein the first differentiated somatic cell type administered to the subject includes oligodendrocyte lineage cells and the second differentiated somatic cell type administered to the subject includes regulatory T cells.

[0202] Aspect 143: The method according to aspect 142, wherein oligodendrocyte lineage cells are derived from induced pluripotent stem cells.

[0203] Aspect 144: According to the method of aspect 1, pluripotent stem cells are generated by transfecting somatic cells with cytoplasm from oocytes.

[0204] Aspect 145: The method according to aspect 144, wherein the cytoplasm is transfected by one or more methods, said methods including electroporation, cell fusion, generation of transient pores in the cytoplasm of recipient cells by streptococcal hemolysin O, and generation of transient pores in the cytoplasm of recipient cells by cell-penetrating peptides.

[0205] Aspect 146: The method according to aspect 1, wherein the pluripotent stem cells are induced pluripotent stem cells, wherein the induced pluripotent stem cells are generated by transfecting somatic cells with pluripotency inducing factors to induce stem cell reprogramming, and wherein the pluripotency inducing factors include one or more of OCT4, c-Myc, PIM-1, NANOG, c-met, hTERT, KLF4, RAS, NOTCH, BMP2, BMP4 and AIRE.

[0206] Aspect 147: The method according to aspect 76, wherein regulatory T cells are treated with one or more histone deacetylase inhibitors.

[0207] Aspect 148: The method according to aspect 1, wherein the soluble factor comprises one or more entities or molecules, said entities or molecules comprising exosomes, and said exosomes express one or more antigens or molecules.

[0208] Aspect 149: The method according to aspect 148, wherein the one or more antigens or molecules are selected from the group comprising CD4, CD8, CD63, CD9, CD81, Tsg101, Alix, flotillin, QSOX1, IFN-γ, TGF-beta, IL-6, IL-8, MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2, MCP-1, MIP-1, MIP-2, VEGF, growth regulator protein α (GRO-α), collagen α1 (COL1A1), 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.

[0209] Aspect 150: The method according to aspect 1, wherein the soluble factor comprises exosomes, and wherein the exosomes express phosphatidylserine on their outer membrane.

[0210] Aspect 151: According to the method of aspect 1, the soluble factor is selected from the group comprising exosomes, apoptotic bodies and extracellular vesicles, and the soluble factor is present in a conditioned medium.

[0211] Aspect 152: The method according to aspect 1, wherein the third culture step includes a culture medium and conditions, wherein the cells are exposed to one or more agents including cellular stressors.

[0212] Aspect 153: According to the method of aspect 152, one or more agents including cellular stressors induce the production of one or more soluble factors in the third cell culture step.

[0213] Aspect 154: According to the method of aspect 153, one or more cellular stressors include molecules or agents that induce inflammation.

[0214] Aspect 155: The method according to aspect 154, wherein the inflammatory molecules activate one or more danger-related receptors in cultured cells.

[0215] Aspect 156: According to the method of aspect 155, one or more risk-related receptors include Toll-like receptors.

[0216] Aspect 157: The method according to aspect 156, wherein the Toll-like receptor is activated by one or more of the following compounds or methods: β-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 exposing cells to hyperthermic exposure.

[0217] Aspect 158: The method described in aspect 157, wherein hyperthermia exposes cells to express hsp90.

[0218] Aspect 159: The method according to aspect 158, wherein the expression of hsp90 is at least 50% higher than the expression of hsp90 on untreated cells that are not exposed to hyperthermia and are of the same cell type.

[0219] Aspect 160: The method according to aspect 1, wherein one or more soluble factors administered to the subject are derived from regulatory T cells, and wherein a histone deacetylase inhibitor is administered to the subject before, after, or both of the administration of the one or more soluble factors.

[0220] Aspect 161: The method according to aspect 160, wherein a histone deacetylase inhibitor is systemically administered to the subject.

[0221] Aspect 162: The method according to aspect 160, wherein the histone deacetylase inhibitor is directly applied to the organ or tissue of the subject.

[0222] Aspect 163: The method according to aspect 162, wherein the histone deacetylase inhibitor induces one or more regenerative processes in the subject.

[0223] Aspect 164: The method according to aspect 1, wherein the regenerative process or function includes one or more of the following effects on cells, organs and tissues in the subject: reversing or preventing epigenetic changes, stopping or reducing telomere depletion, inducing telomere elongation, improving genome stability by promoting the detoxification of DNA damaging agents, reducing or improving protein homeostasis, regulating nutrient signaling pathways, preventing or reversing mitochondrial dysfunction and damage, preventing cellular senescence, counteracting stem cell depletion and loss of function, and producing anti-inflammatory effects.

[0224] Aspect 165: The method according to aspect 160, wherein the regulatory T cells express one or more of the following molecules or antigens, said molecules or antigens including: CD4, CD8, CD25, FoxP3 and CD103.

[0225] Aspect 166: The method according to aspect 160, wherein one or more soluble factors derived from regulatory T cells are directly applied to the organ or tissue of the subject.

[0226] Aspect 167: The method according to aspect 160, wherein one or more soluble factors derived from regulatory T cells are systematically administered.

[0227] Aspect 168: The method according to aspect 167, wherein systemic administration involves administration via an intravenous route.

[0228] Aspect 169: The method according to aspect 160, wherein the histone deacetylase inhibitor comprises one or more of the following dosage forms or compounds: CXD101, HDAC10-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. (butyrate), PCI-34051, SR-4370, givinostat, tubacin, AR-42, parthenolide, tubastatin A hydrochloride, resminostat, CUDC-907, M344, tacedinaline, romidepsin, 4-phenylbutyric acid, sinapinic acid, sulforaphane, UF010, suberohydroxamic acid, NKL22, ITSA-1, KA2507, isoguanosine, raddeanin A, BRD3308, TH34, tinostamustine.

[0229] Aspect 170: The method according to aspect 1, wherein the culture method provides one or more culture media or conditions for pluripotent stem cells to generate mesenchymal stem cells, and wherein the soluble factors released by the mesenchymal stem cells include exosomes.

[0230] Aspect 171: The method according to aspect 170, wherein exosomes are systematically administered to the subject.

[0231] Aspect 172: According to the method described in aspect 170, the mesenchymal stem cells are further modified by transfection with growth factors.

[0232] Aspect 173: The method according to aspect 172, wherein the growth factor comprises one or more of the following: HGF-1, FGF-1, EGF-1, angiopoietin, placental growth factor, adiponectin, vasoactive intestinal peptide precursor, endothelin, myostatin, TGF-β, 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.

[0233] Aspect 174: The method according to aspect 76, wherein the regulatory T cell includes effector regulatory T cells, memory regulatory T cells or chimeric antigen receptor (CAR) regulatory T cells.

[0234] Aspect 175: The method according to aspect 174, wherein the CAR regulatory T cell is generated by genetically modifying pluripotent stem cells to express CAR.

[0235] Aspect 176: The method according to aspect 174, wherein the CAR regulatory T cell is generated by differentiating pluripotent stem cells into T cells using an artificial thymus organoid.

[0236] Aspect 177: The method according to aspect 174, wherein the CAR regulatory T cells are generated by co-culturing with stromal cells expressing Notch ligands.

[0237] Aspect 178: The method according to aspect 174, wherein the regulatory T cells produce immunomodulatory cytokines.

[0238] Aspect 179: The method according to aspect 174, wherein the regulatory T cells are subsequently treated with one or more damage-associated antigens in the culture.

[0239] Aspect 180: The method according to aspect 179, wherein the one or more damage-associated antigens include 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-α receptor p55, and TNF-α receptor p75.

[0240] Aspect 181: The method according to aspect 179, wherein CAR regulatory T cells are administered to the subject.

[0241] Aspect 182: The method according to aspect 179, wherein one or more soluble factors are isolated from the culture after treatment with one or more damage-associated antigens.

[0242] Aspect 183: The method according to aspect 182, wherein one or more soluble factors are administered to the subject.

[0243] Aspect 184: The method according to aspect 80, wherein mesenchymal stem cells are treated with one or more stress agents.

[0244] Aspect 185: The method according to aspect 184, wherein the stress agent comprises cobalt chloride, interferon γ, TNF-α, poly(I:C) or a combination thereof.

[0245] Aspect 186: The method according to aspect 184, wherein one or more stressors are added to induce or enhance the production of one or more soluble factors by mesenchymal stem cells.

[0246] Aspect 187: According to the method of aspect 186, one or more soluble factors include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor (NGF), and ciliary neurotrophic factor (CTNF).

[0247] Aspect 188: The method according to aspect 187, wherein VEGF is produced in a culture of approximately 1 million mesenchymal stem cells at a concentration of at least 10 ng / mL, at least 50 ng / mL, or at least 100 ng / mL.

[0248] Aspect 189: The method according to aspect 187, wherein at least 50 pg / mL, at least 100 pg / mL, or at least 200 pg / mL of EGF is produced in approximately 1 million mesenchymal stem cell cultures.

[0249] Aspect 190: The method according to aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of BDNF is produced in approximately 1 million mesenchymal stem cell cultures.

[0250] Aspect 191: The method according to aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of NGF is produced in approximately 1 million mesenchymal stem cell cultures.

[0251] Aspect 192: The method according to aspect 187, wherein at least 500 pg / mL, at least 1000 pg / mL, or at least 2000 pg / mL of CTNF is produced in approximately 1 million mesenchymal stem cell cultures.

[0252] Aspect 193: According to the method of aspect 80, mesenchymal stem cells are cultured together with allogeneic stem cells before being administered to a subject.

[0253] Aspect 194: The method described in aspect 193, wherein the allogeneic stem cells are induced pluripotent stem cells.

[0254] Aspect 195: The method according to aspect 1, wherein the primary cells are harvested from allogeneic tissue or organ sources, and wherein the allogeneic tissue or organ sources include umbilical cord tissue, umbilical cord blood, peripheral blood, Wharton's jelly, bone marrow, adipose tissue, endometrium, muscle, skin, bone, heart tissue, kidney tissue, liver tissue, and nerve tissue.

[0255] Aspect 196: The method according to aspect 1, wherein the primary cells are harvested from autologous tissues or organs.

[0256] Aspect 197: The method according to aspect 1, wherein the one or more differentiated somatic cell types are combined with an angiogenic agent and administered to a subject.

[0257] Aspect 198: The method according to aspect 197, wherein the angiogenic agent is administered to the subject before, after, or simultaneously with the administration of the one or more differentiated somatic cell types, or in combination at different time points.

[0258] Aspect 199: The method according to aspect 1, wherein the one or more soluble factors are combined with an angiogenic agent and administered to the subject.

[0259] Aspect 200: The method according to aspect 199, wherein the angiogenic agent is administered to the subject before, after, or simultaneously with the administration of the one or more soluble factors, or in combination at multiple time points.

[0260] Aspect 201: The method according to aspect 197, wherein the angiogenic agent comprises one or more factors, said factors including Activin A, adrenomedullin, ALK1, ALK5, ANF, angiogenesis, angiogenesis-1, angiogenesis-2, angiogenesis-3, angiogenesis-4, endothelin-1, endothelial differentiation gene 1 (EDG1), hepatocyte glycosides, erythropoietin, β-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, macrophages Cell-derived growth factor, MCP-1, MD-EC1, GC-MAF, MECIF, MMP2, MMP3, MMP9, urokinase-type plasminogen activator, neurocilia protein, neuromembrane protein, Notch, closure protein, closure band protein, oncogene M, PDECGF, placental factor 4, P1GF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-164, VEGF-1, EC-VEGF, transferrin, platelet-reactive protein, urokinase.

[0261] Aspect 202: The method according to aspect 200, wherein the angiogenic agent comprises one or more factors, said factors including activin A. A) Adrenal medullaris, ALK1, ALK5, ANF, angiopoietin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, endothelin-1, endothelial differentiation gene 1 (EDG1), ephrin, erythropoietin, β-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), oncogene M, PDECGF, placental factor 4, P1GF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-164, VEGF-1, EC-VEGF, transferrin, thromboretin, urokinase.

[0262] Aspect 203: The method of aspect 197, wherein the angiogenic agent is delivered to a subject in a specific form, and wherein the form comprises a protein, a protein mimic, a receptor agonist, a DNA plasmid, microRNA, mRNA, an active peptide, in transfected mesenchymal stem cells, transfected monocytes, transfected B cells, transfected B1 cells, transfected neural progenitor cells, transfected radial cells, transfected oligodendrocytes, transfected astrocytes, transfected fibroblasts, or combinations thereof.

[0263] Aspect 204: A method of aspect 199, wherein the angiogenic agent is delivered to a subject in a specific form, and wherein the form comprises a protein, a protein mimic, a receptor agonist, a DNA plasmid, microRNA, mRNA, an active peptide, in transfected mesenchymal stem cells, transfected monocytes, transfected B cells, transfected B1 cells, transfected neural progenitor cells, transfected radial cells, transfected oligodendrocytes, transfected astrocytes, transfected fibroblasts, or combinations thereof.

[0264] Aspect 205: The method of aspect 1, wherein one or more differentiated somatic cell types are combined with stem cell chemokines and administered to a subject.

[0265] Aspect 206: The method of aspect 205, wherein the stem cell chemokine is administered to the subject before, after, or simultaneously with the administration of the one or more differentiated somatic cell types, or in a combination of time points.

[0266] Aspect 207: The method of aspect 205, wherein the stem cell chemokines comprise one or more factors, the factors including CXCL12, MIP-1α, MIP-1β, VEGF and RANTES.

[0267] Aspect 208: The method of aspect 1, wherein the one or more soluble factors are combined with stem cell chemokines and administered to a subject.

[0268] Aspect 209: The method of aspect 208, wherein the stem cell chemokine is administered to the subject before, after, or simultaneously with the administration of one or more soluble factors, or in combination at different time points.

[0269] Aspect 210: The method of aspect 208, wherein the stem cell chemokines comprise one or more factors, the factors including CXCL12, MIP-1α, MIP-1β, VEGF and RANTES.

[0270] Aspect 211: The method of aspect 1, wherein a culture medium for differentiating pluripotent stem cells into hematopoietic stem cells is provided.

[0271] Aspect 212: The method of aspect 211, wherein the hematopoietic stem cells release one or more soluble factors into the culture.

[0272] Aspect 213: The method of aspect 212, wherein the soluble factor comprises exosomes.

[0273] Aspect 214: The method of aspect 211, wherein the hematopoietic stem cells are defined by expressing one or more markers, said markers including CD34, CD133, c-kit, stem cell factor receptor, CD127, fas ligand, GITR ligand, STAT4, SCA-1, hTERT, NANOG, IL-6 receptor, TGF-β receptor, autocrine TGF-β, and trk75.

[0274] Aspect 215: The method of aspect 211, wherein the hematopoietic stem cells are defined by lack of expression or low expression of one or more markers, including CD38, CD16, CD14 and CD45.

[0275] Aspect 216: The method of aspect 213, wherein the exosomes are defined by expressing one or more markers, said markers including CD34, c-kit, stem cell factor receptor, CD127, fas ligand, GITR ligand, STAT4, SCA-1, hTERT, NANOG, IL-6 receptor, TGF-β receptor, autocrine TGF-β, and trk75.

[0276] Aspect 217: The method of aspect 213, wherein the exosomes are defined by lack of expression or low expression of one or more markers, including CD38, CD16, CD14 and CD45.

[0277] Aspect 218: The method of aspect 211, wherein the hematopoietic stem cells are allogeneic to the subject.

[0278] Aspect 219: The method of aspect 211, wherein the hematopoietic stem cells are autologous to the subject.

[0279] Aspect 220: The method of aspect 211, wherein the hematopoietic stem cells are xenogeneic to the subject.

[0280] Aspect 221: The method of aspect 211, wherein the pluripotent stem cells are derived from dedifferentiated somatic cells, and wherein the somatic cells are extracted from tissues including bone marrow, peripheral blood, mobilized peripheral blood, umbilical cord blood or adipose tissue.

[0281] Aspect 219: The method of aspect 1, wherein the third cell culture method provides a culture medium containing a cell stressor.

[0282] Aspect 220: The method of aspect 219, wherein the cellular stressor includes one or more of HMGB1, flagellin, imiquimod, poly(I:C), and BCG.

[0283] Aspect 221: The method of aspect 219, wherein the cellular stressor induces the production of soluble factors.

[0284] Aspect 222: The method of aspect 221, wherein the soluble factor comprises exosomes.

[0285] Aspect 223: The method of aspect 1, wherein the third cell culture comprises hematopoietic stem cells, and wherein the culture medium comprises a cellular stressor.

[0286] Aspect 224: The method of aspect 223, wherein the cellular stressor includes one or more of HMGB1, flagellin, imiquimod, poly(I:C), and BCG.

[0287] Aspect 225: The method of aspect 223, wherein the cellular stressor induces the production of soluble factors.

[0288] Aspect 226: The method of aspect 225, wherein the soluble factor comprises exosomes.

[0289] Aspect 227: The method of aspect 223, wherein the hematopoietic stem cells express CD34.

[0290] Aspect 228: The method of aspect 223, wherein the cellular stressor is administered at a sufficient concentration and for a sufficient duration to stimulate the production of one or more factors, said factors including matrix metalloproteinase-1 (MMP1), TGFβ, brain-derived neurotrophic factor (BDNF), soluble PD-L1, irisin, and soluble HLA-G.

[0291] Aspect 229: The method of aspect 228, wherein the cellular stressor stimulation produces at least 10 ng / mL, at least 20 ng / mL, or at least 50 ng / mL of MMP from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0292] Aspect 230: The method of aspect 228, wherein the cellular stressor stimulation produces at least 50 ng / mL, at least 100 ng / mL, or at least 200 ng / mL of TGF-β from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0293] Aspect 231: The method of aspect 228, wherein the cellular stressor stimulation produces at least 1 ng / mL, at least 5 ng / mL, or at least 10 ng / mL of BDNF from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0294] Aspect 232: The method of aspect 228, wherein the cellular stressor stimulation produces at least 20 ng / mL, at least 40 ng / mL, or at least 80 ng / mL of soluble PD-L1 from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0295] Aspect 233: A method of aspect 228, wherein the cellular stressor stimulation produces at least 2 ng / mL, at least 4 ng / mL, or at least 8 ng / mL of irisin from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0296] Aspect 234: A method of aspect 228, wherein the cellular stressor stimulation produces at least 100 pg / mL, at least 200 pg / mL, or at least 400 pg / mL of soluble HLA-G from a culture containing approximately 1 million CD34+ hematopoietic stem cells.

[0297] Aspect 235: The method of aspect 1, wherein the subject is further administered one or more agents capable of locally or systematically increasing the number of regulatory T cells.

[0298] Aspect 236: The method of aspect 235, wherein one or more agents capable of increasing regulatory T cell activity include IL-2, IL-10, TGF-β and cell populations associated with tolerance induction.

[0299] Aspect 237: The method of aspect 236, wherein the cell population associated with tolerance induction includes monocytes.

[0300] Aspect 238: The method of aspect 237, wherein the monocytes are treated with one or more cytokines and growth factors, the cytokines and growth factors including IL-4, IL-10, TGF-β, VEGF and IFN-γ inhibitors.

[0301] Aspect 239: The method of aspect 238, wherein the one or more agents confer the ability of the monocyte to produce arginase and / or to produce more arginase than nitric oxide synthase.

[0302] Aspect 240: The method of aspect 237, wherein the monocytes are transfected with one or more of the following molecules, said molecules including: 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.

[0303] Aspect 241: The method of aspect 236, wherein the cell population associated with tolerance induction includes mesenchymal stem cells.

[0304] Aspect 242: The method of aspect 241, wherein the mesenchymal stem cells are transfected with one or more of the following molecules, said molecules including: 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.

[0305] Aspect 243: The method of aspect 1, wherein in vitro pulsed ultrasound is applied to the subject before, after, or simultaneously with the application of the one or more differentiated somatic cell types from the second culture method.

[0306] Aspect 244: The method of aspect 243, wherein the differentiated somatic cells include regulatory T cells.

[0307] Aspect 245: The method of aspect 243, wherein the differentiated somatic cells include oligodendrocyte progenitor cells.

[0308] Aspect 246: The method of aspect 245, wherein complement inhibitors are administered to the subject before, after or simultaneously with oligodendrocyte progenitor cell administration.

[0309] Aspect 247: The method of aspect 246, wherein the complement inhibitor comprises one or more agents, the agents comprising anti-CD5 antibody, anti-CD3 antibody, cobra venom factor, factor H, CD55, CD37 and CD73.

[0310] Aspect 248: The method of aspect 1, wherein age-related diseases, disorders, or symptoms are associated with a decrease in the cytotoxic activity of T cells isolated from the subject.

[0311] Aspect 249: The method described in aspect 248, wherein the cytotoxic activity of T cells is determined based on measurements of perforin production, granzyme B production, Fas ligand expression, or a combination thereof.

[0312] Aspect 250: The method of aspect 1, wherein age-related diseases, disorders, or symptoms are associated with an increase in the production of one or more cytokines from cells isolated from the subject's tissue.

[0313] Aspect 251: The method of aspect 250, wherein the cell is selected from the group consisting of fibroblasts, mesenchymal stem cells, T cells, B cells, dendritic cells, neutrophils, monocytes, macrophages or peripheral blood mononuclear cells.

[0314] Aspect 252: The method of aspect 250, wherein the one or more cytokines include C-reactive protein (CRP), interferon γ, TNF-α, IL-1α, IL-1β, IL-6, IL-12, IL-15, IL-17, IL-18, IL-27, G-CSF, GM-CSF, MCP-1, or combinations thereof.

[0315] Aspect 253: The method of aspect 250, wherein the increase in the production of said one or more cytokines is measured by comparing the levels of said one or more cytokines in a subject with an age-related disease, disorder, or symptom with the levels of the same cytokines in another subject without said age-related disease, disorder, or symptom.

[0316] Aspect 245: The method of aspect 1, wherein the primary somatic cells are selected from the group consisting of: fibroblasts, T cells, B cells, myeloid cells, neutrophils, eosinophils, basophils, myeloblasts, monocytes, macrophages, mesenchymal stem cells, keratinocytes, epithelial cells, exfoliated renal epithelial cells, epithelial progenitor cells, hematopoietic stem cells, common myeloid progenitor cells, natural killer (NK) cells, NK T cells, common lymphoid progenitor cells, endothelial cells, endothelial progenitor cells, synovial cells, pancreatic cells, mobilized CD34+ peripheral blood mononuclear cells, isolated blood mononuclear cells or tissue-specific stem cells or progenitor cells.

[0317] In some embodiments, the present invention provides a medical device that allows patient blood or plasma to be perfused onto regenerating cells in a manner that prevents the regenerating cells from escaping the treatment device.

[0318] In one embodiment, the invention provides a device that isolates the cells from other cells without contact, but allows the production and secretion of soluble factors by such cells. In another embodiment, the personalized regenerative cells and / or cells derived from the cells are placed in a device that allows blood to pass through while retaining the personalized regenerative cells within the device. One use of the invention is to treat age-related pathologies. In other embodiments, the invention provides means for treating inflammatory conditions and allowing host-produced inflammatory mediators to interact with cells and stimulating regenerative cells to produce anti-inflammatory compounds in response to provided inflammatory agents.

[0319] In one embodiment, the invention describes a system for maintaining the survival and function of immune and / or regenerative cells in vitro, while allowing the cells to come into contact with patient blood or plasma. This contact can be in the form of a continuous loop or in a segmented, spaced-out manner. The invention discloses regenerative cells, such as pluripotent stem cells, for producing growth factors and / or anti-inflammatory factors. The disclosed device allows blood or plasma to circulate in such a manner that materials released from the blood or plasma can interact with the regenerative cells and, in some embodiments, alter the function of the regenerative cells. The invention teaches that, because cultures of regenerative cells can form sheet-like mesh structures, various modular shapes for constructing in vitro devices in which such sheet-like mesh structures can be maintained have been studied. As a result, the invention teaches a culture vessel consisting of two chambers: a portion in which patient plasma components circulate and a portion for culturing regenerative cells, the latter separated from the former by a separating membrane, and finds that such a culture vessel is suitable for culturing a variety of regenerative cells and / or immunomodulatory cells.

[0320] Therefore, the present invention provides an in vitro revitalizing / immunomodulatory device having at least one module comprising: a) a plasma component inlet port and a plasma component outlet port; b) a plasma component circulation chamber having the plasma component inlet port and the plasma component outlet port; and c) a culture chamber disposed adjacent to the plasma component circulation chamber and separated from it by a septum membrane, wherein biomolecules can permeate through the septum membrane, but cells cannot permeate through it. In some embodiments, cells for insertion into the device are molecularly pretreated to enhance the therapeutic activity of the cells. These molecules can be selected from the group consisting of: cyclosporine, rapamycin, campath-1H, ATG, Prograf, anti-IL-2 receptor antibody, MMF, FTY, LEA, cyclosporine A, diffusionotropin, dinitroleukin, levamisole, azathioprine, buquina, gusperolimus, 6-mercaptopurine, imidazolidin, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., dinotrepinephrine, edaraxal, methotrexate, pirarubicin, pteroxetine, Tomud). ex® and trimesartan), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thimipramine, and thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, deoxyfluorouridine, imipramine, enoxabin, fluorouridine, fluorouracil, gemcitabine, and tegafur), fluocinolone, triaminolone, anocetate acetate, flumethrin, medroxyprogesterone, prednisone, etc. In another embodiment, the use of stem cell conditioned medium can be used to enhance the potency of existing anti-inflammatory agents. Anti-inflammatory agents may include one or more agents, including NSAIDs, interleukin-1 antagonists, dihydroorotate synthase inhibitors, p38 MAP kinase inhibitors, TNF-α inhibitors, TNF-α chelators, and methotrexate. More specifically,Anti-inflammatory agents may include one or more of the following substances: anti-TNF-α, lysoleothephylline, α1-antitrypsin (AAT), interleukin-10 (IL-10), pentoxifylline, COX-2 inhibitors, 21-acetoxypregnenolone, aclomethasone, alpha-pregnane, ancinonide, beclomethasone, betamethasone, budesonide, cloprednisolone, clobetasol, clobetasol, clocotropin, cloprednisolone, corticosteroids, cortisone, cotivazide, defcoccal, desonide, desoxymethasone, difluralasone, diflucortisone, difluprednisolone, glycyrrhetinic acid, fluzacrocin, fluclochloride, flumethasone, flunisolone, fluocinolone acetate, fluocinolone acetonide, fluocinolone acetonide, fluocinolone acetonide. Fluocinolone, Flumethonone, Flusperidone Acetate, Fluprednisolone Acetate, Fluprednisolone, Fluticasone Propionate, Formococcus, Halcinonide, Halometasone Propionate, Halometasone, Halometasone Acetate, Hydrocortisone, Hydrocortisone, Ecarboxyphenidyl, Maziprednisolone, Medroxyprogesterone, Methylprednisolone, Methylprednisolone, Mometasone Furoate, Peramisone, Prednicarbamate, Prednisolone, Prednisolone 25-diethylaminoacetate, Prednisolone Sodium Phosphate, Prednisolone, Prednisolone, Prednisolone, Rimesolone, Ticortisone, Triamcinolone, Triamcinolone Acetophenone, Triamcinolone Hexamethylenetetramine, Aminoaryl Carboxylic Acid Derivatives (e.g., Enphaniacin, Etofenamic Acid, Flufenamic Acid), Isonicin, meclofenamic acid, niflunic acid, tanylfluoxetine, terfenamic acid, tofenamic acid), aryl acetic acid derivatives (e.g., aceclofenac, asimexine, alclofenac, amifenac, ammoniatomycin, bromofenac, buprofen, methylphenidate, clopidogrel, diclofenac sodium, etodoxacin, biphenylacetic acid, fenclofenac, fentimic acid, glucosine, bufenic acid, indomethacin, isocolic acid, isoxic acid, lonazolic acid, methiocarboxylic acid, moxazolic acid, oxamexin, pyrazole acid, pyrimidine, sulinic acid, thiamethoxam, tometidine, triptoxin, zoleic acid), aryl butyric acid derivatives (e.g., bumadizoline, butebufen, fenbufen, biphenylbutyric acid), aryl carboxylic acids (e.g., chlorhexidine, butibufen, fenbufen, biphenylbutyric acid), Noradrenicol, ketorolac, tenoridone), aryl propionic acid derivatives (e.g., aminoxetine, benzoxetine, permoprofen, buccolic acid, carprofen, fenprofen, flunoprofen, flurbiprofen, ibuprofen, ibuprofen, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprazin, piketofen, pirprofen, praprofen, propionic acid, sulofen, tiprofen, cimolol, zaltoprofen), pyrazoles (e.g., diphenylamide pyrazole, ipilazole), pyrazole ketones (e.g., azaprozin, benpiridone, feprazolam, mofebuzoline, malazine, hydroxybuzoline, phenylbutazone, pibuzoline, isopropylantipyrine, reminalone, succinylbutazone, thiazolinone), salicylic acid derivatives (e.g.,Acetaminophen, aspirin, benorilate, bromosalicylic acid alcohol, calcium acetylsalicylate, diflunisal, italesyl, fendusal, gentianic acid, ethylene glycol salicylate, imidazole salicylate, lysine aspirin, mesalazine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, persamide, phenyl acetylsalicylate, phenyl salicylate, salamide, salicylamide-O-acetic acid, salicylsulfonic acid, disalicylate, sulfasalazine), thiazide carboxamides (e.g., ampicillin, droxoxiconazole, isoxixiconazole, lornoxicam, piroxicam, tenoxiconazole), ε-acetaminophen, S-adenosylmethionine, 3-amino-4-aminoacetic acid α-Hydroxybutyric acid, amicillin, benzoyl peroxide, benzalkonium chloride, α-bisabolol, bucolone, diphenylpyramide, ditazobazole, emmofazine, fepradiol, guaiacine, nabumetone, nimesulide, oxasiroxine, peranilidine, piperoxazole, proquinone, superoxide dismutase, tenidap, ziluotong, candelilla wax, α-bisabolol, aloe vera, madder root, cocaine extract, chamomile, sea whip extract, glycyrrhizic acid, glycyrrhizic acid, oil-soluble glycyrrhiza extract, monoammonium glycyrrhizate, monopotassium glycyrrhizate, dipotassium glycyrrhizate, 1-β-glycyrrhizic acid, stearyl glycyrrhizate, and 3-stearoxyglycyrrhizic acid.

[0321] In some implementations, growth factors are added to maintain the viability and / or function of regenerated cells in vitro. Suitable growth factors that can be used to implement some of the methods disclosed herein include adrenaline medullaris (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), hepatocellular carcinoma-derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor, myostatin (GDF-8), bone marrow monocyte growth factor (MGF), nerve growth factor (NGF), placental growth factor (PlGF), platelet-derived growth factor (PDGF), thrombopoietin (Tpo), transforming growth factor α (TGF-α), TGF-β, tumor necrosis factor α (TNF-α), vascular endothelial growth factor (VEGF), or Wnt protein.

[0322] Certain embodiments of the present invention provide methods for preventing, reducing, or improving age-related fibrosis. In some embodiments, cytokines may be added to one or more culture methods or steps disclosed herein to enhance the production of regenerative factors, said cytokines being essential for terminating various cell types to stimulate transcription and other factors associated with supporting viability and mitosis, while simultaneously inhibiting fibrosis. In some embodiments, MSCs are first treated with cytokines prior to co-culturing. Suitable cytokines that can be used to implement some of the 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, PlGF, PDGF, Tpo, TGF-α, TGF-β, TNF-α, VEGF, or Wnt protein; interleukins; IL-1α, IL-1β, 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 α subunit, IL-12 40 kDa β 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 p19 subunit, IL-23 Soluble receptors for 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-36α, IL-36β, and IL-36γ; interferon (IFN); soluble receptors for IFN-α, IFN-β, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-K, IFN-ε, IFN-κ, IFN-τ, IFN-δ, IFN-ζ, IFNω, or IFN-υ; insulin or proinsulin; insulin receptor; leptin (LEP). In some embodiments, cytokines and / or growth factors are continuously administered to the cells via micropumps connected to an encapsulation device containing the tolerant cells.The specific encapsulation devices of interest are available from US Pat. Nos. 5,725,854; 5,849,285; 5,759,534; 5,843,430; 5,958,404; and 6,149,907.

[0323] Various cell types can be used to implement this invention. In one embodiment, the invention teaches phenotypically defined pluripotent stem cells that can be generated from isolated Wharton's jelly of umbilical cord segments and defined by morphology and by cell surface markers. In some embodiments, Wharton's jelly is used as the initiating cell for the dedifferentiation process. The cells of the invention, i.e., the cells of one embodiment of the invention, can be obtained by dissecting the veins and arteries of the umbilical cord segment and exposing the Wharton's jelly. Umbilical cord segments of approximately 1–5 cm are placed in collagenase solution (1 mg / ml, Sigma) for approximately 18 hours at room temperature. After incubation, residual tissue is removed, and the cell suspension is diluted with PBS into two 50 ml tubes and centrifuged. The cells are then washed in PBS and counted using a hemocytometer. Then, 5–20 × 10⁶ cells are counted. 6 Cells were seeded into 6 cm tissue culture plates in low-glucose DMEM (Gibco), which contained 10% FBS (Hyclone), 2 mM L-glutamine (Gibco), 100 U / ml penicillin / 100 ug / ml streptomycin / 0.025 ug / ml amphotericin B (Gibco). During this purification step, the cells were exposed to hypoxia. The required amount of hypoxia was sufficient to induce HIF-1α activation. In one embodiment, cells were cultured at 2% oxygen for 24 hours. After 48 hours, the cells were washed with PBS and given fresh medium. The medium was changed twice weekly. After 7 days, when the cells were approximately 70-80% confluenced, they were passaged into 10 cm plates using HyQTase (Hyclone). Cells were then passaged regularly at a 1:2 ratio every 7 days or when 80% confluence was reached.

[0324] In some embodiments, biologically useful stem cells, such as autologous pluripotent stem cells, are disclosed for use in the methods of the present invention. These cells may be undifferentiated, differentiated into progenitor cells, or differentiated into mature or semi-mature cells. In some cases, the cells belong to a mesenchymal lineage or related lineage, which are therapeutically reprogrammed cells with minimal oxidative damage and whose telomere length is advantageous compared to that of undamaged prenatal or embryonic stem cells (i.e., the therapeutically reprogrammed cells of the present invention have a genome close to optimal physiological state). Furthermore, the therapeutically reprogrammed cells of the present invention are immune-exempt and therefore suitable for therapeutic applications. Other methods of the present invention provide for the generation of hybrid stem cells. Furthermore, the present invention includes related methods for maturing stem cells prepared according to the teachings of the present invention into specific host tissues. For applications in the present invention, practitioners believe that ontogenesis in mammalian development provides a central role for stem cells. In early embryogenesis, cells from the proximal epiblast destined to become germ cells (primordial germ cells) migrate along the gonadal ridge. These cells express high levels of alkaline phosphatase and also express the transcription factor Oct4. After migrating and colonizing the genital ridge, primordial germ cells differentiate into male or female germ cell precursors (primordial sex cells). For the purposes of this disclosure, only male primordial sex cells (PSCs) are discussed, but the quality and properties of male and female primordial sex cells are equivalent and are not intended to be limiting. During the development of male primordial sex cells, primordial stem cells are closely associated with supporting cell precursors, leading to the initiation of spermatogenic cord formation. As primordial germ cells are enclosed within the spermatogenic cord, they differentiate into mitotically quiescent sex cells. These sex cells divide for several days and then arrest in the G0 / G1 phase of the cell cycle. In mice and rats, these sex cells resume division within days after birth to generate spermatogonial stem cells and eventually undergo differentiation and meiosis associated with spermatogenesis. Embryonic stem cells are known to originate from cells in the inner cell mass of the preblastocyst embryo and possess the greatest differentiation potential, capable of generating cells found in all three germ layers of the embryo. From a practical perspective, embryonic stem cells are products of cell culture because they exist only briefly during embryogenesis in their natural epiblastic environment. Manipulating embryonic stem cells in vitro leads to the generation and differentiation of various cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerve cells, skeletal muscle cells, chondrocytes, adipocytes, liver cells, and pancreatic islets. Co-culturing embryonic stem cells with mature cells can influence and initiate differentiation of embryonic stem cells into specific lineages. Maturation is a process of coordinated forward or backward steps in the differentiation pathway and can refer to both differentiation and / or dedifferentiation. In one instance of the maturation process, cells or cell populations interact with their cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches, and these niches, or microenvironments, contain stem cells that guide and regulate organogenesis.At birth, maturation has progressed to the point where cells and appropriate cellular niches exist to enable the organism to function and survive after birth. Developmental processes are highly conserved across species, allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory. Throughout an organism's life, the cellular composition of organs and organ systems is exposed to a variety of intrinsic and extrinsic factors that induce cellular or genomic damage. Ultraviolet radiation affects not only normal skin cells but also skin stem cell populations. Chemotherapy drugs used to treat cancer have a destructive effect on hematopoietic stem cells. Reactive oxygen species (ROS), byproducts of cellular metabolism, are intrinsic factors that impair the integrity of the cellular genome. In all organs and organ systems, cells are constantly replaced by stem cell populations. However, as an organism ages, cellular damage accumulates in these stem cell populations. If the damage is heritable, such as a genomic mutation, then all offspring will be affected and thus impaired. A single stem cell clone can continuously generate lineages such as lymphoid and myeloid cells for over a year, therefore, if that stem cell is damaged, it has the potential to propagate mutations. The body responds to damaged stem cells by inducing apoptosis, thereby removing them from the pool and preventing potential dysfunction or tumorigenic properties. Apoptosis removes damaged cells from the population, but it also reduces the number of future usable stem cells. Therefore, the number of stem cells decreases as an organism ages. In addition to the loss of the stem cell pool, there is evidence that aging reduces the efficiency of stem cell homing mechanisms. Telomeres are the physical ends of chromosomes containing highly conserved tandemly repeating DNA sequences. Telomeres are involved in the replication and stability of linear DNA molecules and act as a counting mechanism in cells; with each cell division, telomere length shortens and activates signals at predetermined thresholds to initiate cellular senescence. Stem cells and somatic cells produce telomerase, which inhibits telomere shortening, but their telomeres still gradually shorten during aging and cellular stress. In one teaching or embodiment of the invention, therapeutically reprogrammed cells are provided, in some embodiments of mesenchymal stem cells. Therapeutic reprogramming refers to a maturation process in which stem cells are exposed to stimulating factors according to the teachings of the invention to produce enhanced therapeutic activity. In some embodiments, the enhancement of therapeutic activity may be increased proliferation; in other embodiments, it may be enhanced chemotaxis. Other therapeutic properties include resistance to apoptosis, the ability to overcome aging, the ability to effectively differentiate into multiple different cell types, and the ability to secrete therapeutic growth factors that enhance the viability / activity of endogenous stem cells. To induce therapeutic reprogramming of cells, in some cases, as disclosed herein, to induce therapeutic reprogramming of Wharton's gum-derived cells, this invention teaches the use of stimulating factors, including but not limited to chemicals, biochemicals, and cell extracts, to alter the epigenetic programming of cells. These stimulating factors, among other results, induce changes in genomic methylation in the donor DNA.Embodiments of the present invention include methods for preparing cell extracts from whole cells, cytoplasms, and nucleoplasms, although other types of cell extracts are also contemplated within the scope of the invention. In one non-limiting example, the cell extracts of the present invention are prepared from stem cells, particularly embryonic stem cells. Donor cells are incubated with chemicals, biochemicals, or cell extracts for a defined period of time, in one non-limiting example, from about one hour to about two hours, and those reprogrammed cells expressing embryonic stem cell markers such as Oct4 after the culture period are then ready for transplantation, cryopreservation, or further maturation. In another embodiment of the invention, hybrid stem cells are provided for use in cell regeneration / repair therapies. The hybrid stem cells of the present invention are pluripotent and tailored to the intended recipient, such that they are immunocompatible with the recipient. Hybrid stem cells are the product of a fusion between a donor cell or its nucleus and a host cell. Typically, the fusion occurs between the donor cell nucleus and the enucleated host cell. Donor cells can be any diploid cell, including but not limited to cells from pre-embryonic, embryonic, fetal, and postnatal organisms. More specifically, donor cells can be primordial cells, including but not limited to oogonia or differentiated or undifferentiated spermatogonia, or embryonic stem cells. Other non-limiting examples of donor cells are therapeutic reprogrammed cells, embryonic stem cells, fetal stem cells, and pluripotent adult progenitor cells. Preferably, the donor cells have the phenotype of the intended recipient. Host cells can be isolated from tissues including, but not limited to, pre-embryonic, embryonic, fetal, and postnatal organisms, and more specifically, may include, but not limited to, embryonic stem cells, fetal stem cells, pluripotent adult progenitor cells, and adipose-derived stem cells. In one non-limiting example, a cultured cell line can be used as the donor cell. The donor cells and host cells can be derived from the same individual or different individuals. In one embodiment of the invention, lymphocytes are used as donor cells, and the donor cells are purified using a two-step method. After tissue dissociation, an adhesion step is performed to remove any potentially contaminating adherent cells, followed by a density gradient purification step. Most lymphocytes are quiescent (in the G0 phase) and therefore can have a methylated state that confers greater reprogramming plasticity. Pluripotent or multipotent stem cells or cell lines that can be used as donor cells in embodiments of the present invention are functionally defined as stem cells by their ability to differentiate into multiple cell types, including but not limited to adipogenic, neurogenic, osteogenic, chondrogenic and cardiogenic cells.

[0325] In some embodiments, the enucleation of host cells used to generate hybrid stem cells according to the teachings of the present invention can be performed using a variety of methods. In one non-limiting example, ADSCs are seeded onto fibronectin-coated tissue culture slides and treated with cytochalasin D or cytochalasin B. After treatment, the cells can be digested with trypsin, reseeded, and are viable for approximately 72 hours after enucleation. The host cell and donor cell nuclei can be fused using one of a variety of fusion methods known to those skilled in the art, including but not limited to electrofusion, microinjection, chemical fusion, or virus-based fusion, and all cell fusion methods are contemplated to be within the scope of the invention. Hybrid stem cells prepared according to the teachings of the present invention have surface antigens and receptors from enucleated host cells but have nuclei from developmentally younger cells. Therefore, the hybrid stem cells of the present invention will be receptive to cytokines, chemokines, and other cell signaling agents but possess nuclei free from age-related DNA damage. Therapeutic reprogrammed cells and hybrid stem cells prepared according to the teachings of the present invention can be used for a wide range of therapeutic applications in cell regeneration / repair therapies. For example, and not intended to be limiting, the therapeutic 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 therapies such as cancer radiotherapy and chemotherapy. In another non-limiting example, the therapeutic reprogrammed cells and hybrid stem cells of the present invention can be used for organ regeneration and tissue repair. In one embodiment of the invention, the therapeutic reprogrammed cells and hybrid stem cells can be used to revitalize damaged muscle tissue, including malnourished muscle and muscle damaged by ischemic events such as myocardial infarction. In another embodiment of the invention, the therapeutic reprogrammed cells and hybrid stem cells disclosed herein can be used to improve scar formation in animals (including humans) following traumatic injury or surgery. In this embodiment, the therapeutic reprogrammed cells and hybrid stem cells of the present invention are systemically administered, such as intravenously, and recruited by circulating cytokines secreted by damaged cells to the site of recent trauma. In another embodiment of the invention, the therapeutic reprogrammed cells and hybrid stem cells can be locally administered to treatment sites requiring repair or regeneration.

[0326] In one embodiment, the source of regenerated cells includes umbilical cord samples obtained after delivery of a normal full-term infant. The present invention provides a method for processing the umbilical cord for cell isolation. As an example, a portion of the umbilical cord is cut into segments approximately 3 cm long. Each umbilical cord segment is dissected to remove the umbilical vein and two umbilical arteries. The remaining segments are sutured inside out and incubated at room temperature in PBS, antibiotics, and collagenase. After 16–18 hours, the remaining sutures and connective tissue are removed and discarded. The cell suspension is then subjected to erythrocyte lysis, and the cells are plated in a suitable culture medium supporting the growth of umbilical cord mesenchymal stem cells.

[0327] One object of the present invention is to provide a bioreactor and method that allows the growth of three-dimensional tissues, wherein the bioreactor is perfused and the perfusion solution is used to stimulate regeneration and / or immunomodulation. The foregoing and other objects are achieved by the present invention, which provides a system for maintaining and culturing three-dimensional tissues in vitro. In one embodiment, the system includes a bioreactor having a tissue disposed between two streams of culture medium. The tissue is cultured on a porous substrate. A first stream of culture medium having a first solute concentration is in direct contact with one side of the tissue, and a second stream of culture medium having a second solute concentration is in direct contact with the opposite side of the tissue. In one embodiment, the first solute concentration differs from the second solute concentration, thereby creating a diffusion or osmotic pressure gradient that causes solutes, including nutrients, to move through the tissue. The culture medium streams can also be used to supply gases and growth factors to cells at varying concentrations according to the needs of the specific tissue being cultured. Metabolic waste, including carbon dioxide, is secreted into the culture medium streams and carried away. In an alternative embodiment, a semi-permeable membrane may be disposed between the tissue and each of the two streams of culture medium. A membrane that allows the desired solutes and waste to pass through should be used.

[0328] In another embodiment, this disclosure provides a system comprising: a bioreactor having tissue cultured on a porous membrane disposed within a housing, a first space between the tissue and a top portion of the housing, and a second space between the tissue on an opposite side and a bottom portion of the housing; a culture medium inlet port for providing a first culture medium stream into the first space and a second culture medium stream into the second space; and at least one culture medium outlet port. As described above, the solute concentrations, including nutrients, growth factors, and gases, may differ for the first and second culture medium streams, such that the solutes, nutrients, growth factors, and / or gases pass through the tissue via diffusion or an osmotic pressure gradient.

[0329] This invention provides a method for generating cell-free therapeutic agents comprising soluble factors derived from pluripotent stem cells and their derivatives. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells derived from differentiated somatic cells, i.e., primary somatic cells, which have been directly isolated from a subject's tissue or organ. In some embodiments, the primary somatic cells are converted into induced pluripotent stem cells through a dedifferentiation or reprogramming process using reprogramming transcription factors. In some embodiments, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and C-myc. Means of dedifferentiating cells are known in the art and include treating cells with an agent that acts epigenetically, said agent may be used alone or in combination with induced pluripotent stem cell technologies known in the art. In one embodiment, somatic cells are dedifferentiated by treatment with a DNA methyltransferase inhibitor, said inhibitor comprising nucleoside analogs and non-nucleoside analogs. Exemplary nucleoside analogues include 5-azacitidine (which can be used at concentrations from 100 nM to 10 μM), 5-aza-2′-deoxycytidine (which can be used at concentrations from 100 nM to 10 μM), 5-fluoro-2′-deoxycytidine (which can be used at concentrations from 100 nM to 10 μM), 5,6-dihydro-5-azacitidine (which can be used at concentrations from 100 nM to 10 μM), and zebularine (which can be used at concentrations from 1 μM to 10 mM). Exemplary non-nucleoside analogues include hydralazine (which can be used at concentrations from 100 nM to 10 μM), procainamide (which can be used at concentrations from 1000 nM to 10 μM), EGCG (which can be used at concentrations from 100 nM to 10 μM), Psammaplin A (which can be used at concentrations from 100 nM to 10 μM), MG98 (which can be used at concentrations from 100 nM to 10 μM), and RG108 (which can be used at concentrations from 100 nM to 10 μM).

[0330] Exemplary histone deacetylase inhibitors include short-chain fatty acids, hydroxamic acid, cyclic benzamide, cyclic tetrapeptide, and benzamide. Exemplary short-chain fatty acids include butyrate (which can be used at concentrations from 1 μM to 10 mM) and valproic acid (which can be used at concentrations from 1 μM to 10 mM). Exemplary hydroxamic acids include bis(oxo)carboxycinnamic acid (CBHA) (which can be used at a concentration of 100 nM to 10 μM), Oxamflatin (which can be used at a concentration of 100 nM to 10 μM), PDX 101 (which can be used at a concentration of 100 nM to 10 μM), Pyroxamide (which can be used at a concentration of 1 nM to 10 μM), Scriptaid (which can be used at a concentration of 100 nM to 10 μM), succinyl aniline hydroxamic acid (SAHA) (which can be used at a concentration of 100 nM to 10 μM), trachostatin A (TSA) (which can be used at a concentration of 1 nM to 10 μM), LBH589 (which can be used at a concentration of 1 nM to 10 μM), and NVP-LAQ824 (which can be used at a concentration of 1 nM to 10 μM). Exemplary cyclic tetrapeptides and benzamides include Apidin (which can be used at concentrations from 1 nM to 10 μM), Depsipeptide (which can be used at concentrations from 100 nM to 10 μM), TPX-HA analogues (CHAP) (which can be used at concentrations from 1 nM to 10 μM), and Trapoxin (which can be used at concentrations from 1 nM to 10 μM). Exemplary benzamides include CI-994 (N-acetylbardinalin) (which can be used at concentrations from 100 nM to 10 μM) and MS-275 (which can be used at concentrations from 100 nM to 10 μM).

[0331] In one embodiment, the permeabilized cells are incubated with interphase reprogramming medium. Preferably, the nuclei of the permeabilized cells remain membrane-enclosed, and the chromosomes in the nuclei do not clump together during incubation with the interphase reprogramming medium. In another preferred embodiment, chromatin clusters are formed by incubating the permeabilized cells in mitotic reprogramming medium. In yet another preferred embodiment, the reprogrammed cells are incubated under conditions that allow the membranes of the reprogrammed cells to re-close before application to mammals. Preferably, the permeabilized cells are derived from mammals that require this cell type. In yet another preferred embodiment, the permeabilized cells are formed by incubating intact cells with a detergent (e.g., digitalis saponins, or bacterial toxins, such as streptococcal hemolysin O).

[0332] 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 cells but not in the donor or permeable cells. In another preferred embodiment, the number of mRNA or protein molecules expressed in the reprogrammed cells but not in the donor or permeable cells 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, including the extreme values. 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 permeable cells but not in the reprogrammed cells. In yet another preferred embodiment, the number of mRNA or protein molecules expressed in donor or permeabilized cells but not in the reprogrammed cells 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, including the extreme values. Preferably, the mRNA or protein molecules are cell type specific to the donor, permeabilized, or reprogrammed cells, such that the molecules are expressed only in cells of that specific cell type. In yet another preferred embodiment, these mRNA or protein molecules are expressed both in donor cells (i.e., donor or permeabilization initiating cells) and in the reprogrammed cells, but the expression levels in these cells differ by at least 2, 5, 10, or 20 times, as measured using standard assays. In one embodiment, the size of the donor or permeabilized cells differs from the size of the reprogrammed cells by at least 10%, 20%, 30%, 50%, 75%, or 100%, as measured using standard methods. In another preferred embodiment, the volume of the cytoplasm in the donor or permeabilized cells differs from the volume of the cytoplasm in the reprogrammed cells by at least 10%, 20%, 30%, 50%, 75%, or 100%, based on standard methods. In yet another preferred embodiment, the reprogrammed cells gain or lose an activity relative to the donor or permeabilized cells, such as the production of matrix metalloproteinases, angiogenesis activity, or chemotactic activity. In still some preferred embodiments, the reprogramming medium is an interphase reprogramming medium, such as an extract formed from cells synchronized at one or more stages of the cell cycle. In another embodiment, the reprogramming medium is an extract formed from cells synchronized during mitosis or from unsynchronized cells. The reprogramming medium is an extract from the cell type to which the donor or permeabilized cells are desired to become, or the reprogramming medium is a solution containing factors specific to the cell type to which the donor or permeabilized cells are desired to become.

[0333] In one embodiment, the reprogramming medium is modified by enriching or removing a factor, such as DNA methyltransferases, histone deacetylases, histones, laminins, transcription factors, activators, repressors, growth factors, hormones, or cytokines. The reprogramming medium may or may not contain exogenous nucleotides. In other embodiments, chromatin clusters in the reprogramming medium or formed in permeabilized cells are contacted with a vector containing nucleic acids encoding a target gene under conditions that allow homologous recombination between the nucleic acids in the vector and the corresponding nucleic acids in the genome of the chromatin cluster, resulting in alterations to the genome of the chromatin cluster. Due to the lack of an intact plasma membrane and a nuclear membrane, chromatin clusters in permeabilized cells may be more susceptible to genetic modification than naturally occurring cells. Preferably, the chromatin clusters or nuclei are purified from the reprogramming medium before insertion into recipient cells or cytoplasms, or the reprogrammed cells are purified before administration to mammals. Preferably, the donor or permeabilized cell is haploid (DNA content of n), diploid (2n), or tetraploid (4n), and the recipient cell is sub-diploid (DNA content less than 2n), haploid, or enucleated.

[0334] Generating pluripotent cells through other means is known in the art. In some embodiments, pluripotent stem cells are treated to differentiate into thymic medullary epithelial cells. This differentiation is achieved by culturing in 6-well plates pre-coated with approximately 10 μg / mL type IV collagen, the plates containing differentiation medium (mESC medium without leukemia inhibitory factor) and approximately 20 ng / mL human fibroblast growth factor 7, approximately 20 ng / mL human fibroblast growth factor 10, approximately 20 ng / mL human bone morphogenetic protein 4, and approximately 50 ng / mL human epithelial growth factor. The medium and growth factors are changed every 3–4 days. On day 10, the differentiated pluripotent cells are harvested by treating the cells with approximately 2 mg / mL type IV collagenase. The differentiated thymic medullary epithelial cells can be used to induce transplant tolerance or reduce transplant-related immunosuppression in the case of solid organ transplantation (where donor-derived or partially donor-derived cells are used), or in cases requiring prevention of graft-versus-host disease and therefore using recipient-derived or partially recipient-derived hematopoietic stem cell transplantation. In the case of autoimmunity, thymic medullary epithelial cells are made to express autoantigens, thereby promoting tolerance to autoantigens that stimulate autoimmunity. Autoantigens can be genetically engineered into pluripotent stem cells used to generate thymic medullary epithelial cells, or directly into differentiated thymic medullary epithelial cells. In addition to genetic engineering, the incorporation of the autoantigen can be achieved through protein delivery via a protein transduction domain, by administering mRNA encoding the autoantigen, or by adding the autoantigen or a peptide derived therefrom, thereby allowing pinocytosis to allow the uptake of the autoantigen.

[0335] In some embodiments, a combination of immature dendritic cells and mesenchymal stem cells is used to generate a tolerable environment to treat or prevent autoimmunity. This tolerable environment can be generated in vivo or produced by co-culturing the immature dendritic cells and mesenchymal stem cells in vitro. In some embodiments, cells from tissues requiring prevention or suppression of autoimmunity are added to the culture, including endothelial cells, epithelial cells, dermal cells, endoderm cells, mesodermal cells, fibroblasts, osteocytes, chondrocytes, natural killer cells, dendritic cells, hepatocytes, pancreatic cells, stromal cells, salivary gland mucus cells, salivary gland serous cells, von Ebner gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland dark cells, eccrine sweat gland light cells, apocrine sweat gland cells, molar gland cells, sebaceous gland cells, Bowman gland cells, Brenner gland cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, and others. Teresa cells, endometrial cells, isolated goblet cells, gastric mucus cells, gastric zymogen cells, gastric acid-secreting cells, pancreatic acinar cells, Panthen cells, type II alveolar cells, Clara cells, growth hormone cells, prolactin cells, thyroid-stimulating hormone cells, gonadotropin cells, adrenocorticotropic hormone cells, middle pituitary cells, large neurosecreting cells, intestinal cells, respiratory cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, Ledich cells, inner theca cells, luteal cells, granulosa luteal cells, theca luteal cells, juxtaglobin cells, and macula densa. Cells, peripheral cells, mesangial cells, pore-forming cells of vascular and lymphatic endothelium, continuous cells of vascular and lymphatic endothelium, spleen cells of vascular and lymphatic endothelium, synovial cells, serous cells (lining the peritoneal cavity, pleural cavity, and pericardial cavity), squamous cells, columnar cells, dark cells, vestibular cells (lining the endolymphatic space of the ear), basal cells of the stria vascularis, marginal cells of the stria vascularis (lining the endolymphatic space of the ear), Claudis cells, Berthes cells, choroid plexus cells, pia mater-arachnoid squamous cells, pigmented ciliary body epithelial cells, non-pigmented ciliary body epithelial cells, corneal endothelial cells, embolus cells, respiratory tract ciliated cells, fallopian tube ciliated cells, endometrial ciliated cells, reticulum testis Ciliated cells, efferent tubule ciliated cells, ciliated ependymal cells, epidermal keratinocytes, epidermal basal cells, nail and toenail keratinocytes, nail bed basal cells, medullary hair stem cells, cortical hair stem cells, hair cuticle stem cells, hair cuticle root sheath cells, Huxley layer root sheath cells, Henle layer root sheath cells, outer root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, epithelial basal cells, urothelial cells, auditory inner hair cells of the organ of Coty, auditory outer hair cells of the organ of Coty, olfactory epithelial basal cells, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, epidermal Merkel cells, olfactory receptor neurons, pain-sensitive primary sensory neuronsPhotoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, tactile primary sensory neurons, type I carotid somatic cells, type II carotid somatic cells (blood pH sensor), type I hair cells of the vestibular organ (acceleration and gravity), type II hair cells of the vestibular organ, type I taste bud cells, cholinergic neurons, adrenergic neurons, peptidergic neurons, inner column cells of the organ of Coty, outer column cells of the organ of Coty, inner finger cells of the organ of Coty, outer finger cells of the organ of Coty, limbic cells of the organ of Coty, Henson cells of the organ of Coty, vestibular organ supporting cells, taste bud supporting cells. Cells, olfactory epithelial supporting cells, Schwann cells, satellite cells, enteric glial cells, astrocytes, neurons, oligodendrocytes, spindle neurons, anterior lens epithelial cells, lens fiber cells containing lens proteins, hepatocytes, adipocytes, white adipocytes, brown adipocytes, hepatic adipocytes, glomerular parietal cells, glomerular podocytes, proximal renal tubule brush border cells, thin segment cells of the loop of Henry, distal renal tubule cells, renal collecting duct cells, type I alveolar cells, pancreatic duct cells, styloid duct cells, duct cells, intestinal brush border cells, exocrine gland styloid duct cells, gallbladder epithelial cells, efferent duct nonciliated cells, epididymal chief cells, epididymal basal cells, and so on. Ameloblastic epithelial cells, semilunar epithelial cells, interdental epithelial cells of organ of Corti, loose connective tissue fibroblasts, corneal keratinocytes, tendon fibroblasts, bone marrow reticular tissue fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblasts / cementocytes, odontoblasts, dentin cells, hyaline cartilage chondrocytes, fibrochondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, vitreous cells, stellate cells (ear), hepatic stellate cells (Itor cells), pancreatic stellate cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nucleus bag cells, muscle spindle nucleus chain cells, satellite cells Cells, including: ordinary cardiomyocytes, connective cardiomyocytes, Purkinje fibroblasts, smooth muscle cells, iris myoepithelial cells, exocrine gland myoepithelial cells, reticulocytes, megakaryocytes, monocytes, connective tissue macrophages, epidermal Langerhans cells, dendritic cells, microglia, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, melanocytes, retinal pigment epithelial cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia, sperm, ovarian follicular cells, Sertoli cells, thymic epithelial cells, and / or renal interstitial cells.

[0336] In some embodiments, one or more culture methods or systems of the present invention utilize a bioreactor to generate or culture cells and to induce or maintain the generation of one or more soluble factors from cells at various stages of dedifferentiation or differentiation. In one embodiment, a culture system is provided comprising: a bioreactor in which tissue is cultured on a porous membrane located around the inner periphery of a cylindrical shell, a first space between the tissue and the cylindrical shell, and a second central space within the cylindrical shell defined by opposite sides of the tissue; a culture medium inlet port for providing a first culture medium flow to the first space and a second culture medium flow to the second space; and at least one culture medium outlet port. The first and second culture medium flows may have different concentrations of solutes, including nutrients, growth factors, and gases, as described above.

[0337] In some embodiments, methods for inducing tissue growth involve seeding structural support cells and parenchymal cells onto a porous substrate, such as a mesh, to form a co-culture that will generate 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 culture medium flow. A first culture medium flow having predetermined concentration levels of solutes such as various nutrients, gases, and growth factors flows in direct contact with cells on one side of the mesh portion. A second culture medium flow having different, typically varying, concentrations of solutes such as nutrients, gases, and growth factors flows in direct contact with the opposite side of the mesh portion. The difference in solute concentration in each culture medium flow creates a diffusion gradient or osmotic pressure gradient that drives the diffusion of various substances required for cell growth through the tissue layer. Simultaneously, metabolic waste diffuses into the culture medium flow and is carried away. In another use, a culture medium flow can be pressurized to create a hydrostatic pressure differential, thereby counteracting the osmotic pressure and providing further control over nutrient delivery. This invention offers advantages over previous tissue culture systems because nutrient delivery is guided inward from two opposing planes, allowing for the creation of a thicker tissue substrate and maintenance of its viability. Therefore, the diffusion gradient bioreactor of this invention mimics in vitro the mechanisms of nutrient delivery to and removal of waste from tissues in vivo, thereby enabling the culture of a wide variety of three-dimensional tissues.

[0338] The bioreactor and the resulting cultures have a variety of applications, ranging from in vivo transplantation or implantation of tissues grown in the bioreactor, continuous in vitro production of biomaterials, and use as ex vivo organ assist devices for patients with organ failure. Furthermore, tissue growth in the reactor can be used for in vitro toxicology testing, either after removal to a static environment or by other means to assess, such as drug metabolism 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 to produce a wide variety of biomaterials and drugs, including but not limited to monoclonal antibodies, viral vaccines, hormones, lymphokines, enzymes, tumor-specific antigens, and viral pesticides. To grow tissues, structural support cells are seeded onto the mesh and provided with a culture medium stream to deliver the required nutrients while simultaneously removing waste products generated by the cells. Once the structural support cells have established, such as when they have grown to confluence, tissue-specific cells or parenchymal cells are added to the mesh. The contents of one or both of the culture medium streams can then be modified to meet the needs of the tissue-specific cells or the entire organism. Once the cells have grown into a three-dimensional tissue, the culture medium flow can be stopped, and the tissue can be used or preserved for future use. To preserve the tissue, a cryoprotectant is added, and the tissue is frozen, preferably in the same container holding the tissue in the bioreactor. The tissue can be thawed later and used as a bioreactor to produce biomaterials. To use the tissue, a first culture medium flow having a predetermined concentration of solutes such as nutrients, growth factors, and gases flows in direct contact with one side of the tissue. A second culture medium flow having a different concentration of solutes flows in direct contact with the opposite side of the tissue. Mass transfer within the bioreactor is achieved through a diffusion gradient. The concentrations of solutes and nutrients provided are sufficient to allow the three-dimensional tissue to produce the desired bioproducts. The products are removed from the bioreactor via the first culture medium flow or the second culture medium flow, or both. As described above, the three-dimensional tissue can be used or cryopreserved for future use. The tissue can be thawed later and used as an in vitro liver device. To use the tissue, blood is drawn from a patient with liver dysfunction. If necessary, plasma can be separated from the blood. The blood or plasma is then passed through the container, with liver tissue in contact with one side of the tissue, while culture medium flows through the opposite side of the tissue. The tissue will act as a functional liver for the blood or plasma, which can then be reinfused into the patient.

[0339] In some embodiments, the inventors disclose not only in vitro devices with regenerative cells and / or immunomodulatory cells, but also implantable devices. In some embodiments, the implantable device includes: a therapeutic cell population, a matrix capable of maintaining the viability and function of the therapeutic cell population, and a selectively permeable material layer surrounding the matrix capable of maintaining the viability and function of the therapeutic cell population. In some embodiments, the therapeutic cell population includes donor cells or recipient cells, and wherein the therapeutic cell population has the ability to induce self-reactive apoptosis or inactivation. In some embodiments, the therapeutic cell population is selected from the group consisting of: differentiated cells expressing high growth factor production activity, stem cells, and one or more cells expressing immunomodulatory factors. In some embodiments, the differentiated cells expressing high growth factor production activity are conferred the ability to produce high levels of growth factors through ex vivo manipulation. In some embodiments, the differentiated cells expressing high growth factor production activity naturally possess the ability to express high levels of growth factors. In some embodiments, the stem cells are selected from the group consisting of: embryonic stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neural stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue-derived stem cells, desquamated tooth-derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenetic stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the cell or multiple cells expressing immunomodulatory factors resemble a thymic cell population.

[0340] In some embodiments, the matrix capable of maintaining the viability and function of the therapeutic cell population is a macroporous polymer scaffold comprising a substantially non-membrane porous wall composed of microporous polymer struts defining macropores interconnected by macroporous channels. Each microporous polymer strut contains microporous channels extending through it, such that macropores on either side of a given microporous polymer strut are connected via the given microporous polymer strut. The macropores have an average diameter in the range of about 0.5 to about 3.5 mm, and the macroporous polymer scaffold has a porosity of at least 50%. In some embodiments, the matrix capable of maintaining the viability and function of the therapeutic cell population is selected from the group consisting of irradiated bone, decellularized bone, decellularized placental matrix, decellularized thymus, and decellularized organs. In some embodiments, the matrix capable of maintaining the viability and function of the therapeutic cell population is made partly or entirely of a material selected from the group consisting of: glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulfone, polyvinyl alcohol, acrylonitrile polymer, polyacrylamide, polycarbonate, polypentene, polypentane, nylon, magnetite, natural polysaccharides, modified polysaccharides, collagen, gelatin, and modified gelatin.

[0341] In some embodiments, the selectively permeable material layer is made using a biocompatible material that allows particles smaller than 1 micrometer to diffuse freely. In some embodiments, the selectively permeable material layer is made using a biocompatible material that does not allow for significant immune recognition of the cells within the material.

[0342] In some embodiments, the implantable device may include a porous base structure to which therapeutic cells are attached, the porous base structure being implantable into and removable from a patient. In another embodiment, the medical device includes a porous base seeded with therapeutic cells, the device including an additional layer that allows for free molecular exchange without cell escape. The medical device is seeded with autologous or allogeneic pluripotent stem cells. Where tolerance to allogeneic responsive donor cells is required, the thymic medullary epithelial cells or their progenitor cells are derived from recipient cells. Where tolerance to allogeneic responsive recipient cells is required, the thymic medullary epithelial cells or their progenitor cells are derived from donor cells.

[0343] Some embodiments disclosed herein relate to a method for treating inflammatory conditions. In some embodiments, the method includes selecting a cell population that produces soluble anti-inflammatory mediators; culturing the cells producing soluble anti-inflammatory mediators in vitro with a matrix to enable the cells to adhere to the matrix; coating the cells and the matrix with a selectively permeable material to allow soluble factors to diffuse through the material without cell escape; and implanting the encapsulated 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-α, interferon-γ, and TRANCE. In some embodiments, the inflammatory condition is selected from the group consisting of autoimmunity, transplant rejection, and aging. In some embodiments, the cell population generating the soluble anti-inflammatory mediator is selected from the group consisting of: immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow monocytes, mesenchymal stem cells, regulatory T cells, NKT cells, hematopoietic stem cells, umbilical cord stromal monocytes, adipose tissue monocytes, placental stromal monocytes, umbilical cord blood monocytes, and CD5-positive B cells. In some embodiments, the cell population is autologous, allogeneic, or xenogeneic. In some embodiments, the cells are derived from one or more cell lines. In some embodiments, the encapsulated matrix containing therapeutic cells is administered subcutaneously in a manner that allows for subsequent removal.

[0344] Some embodiments disclosed herein relate to a method for altering an immune response. In some embodiments, the method includes selecting a cell population that produces soluble immunomodulators, culturing the cells producing the soluble immunomodulators in vitro with a matrix to enable the cells to adhere to the matrix, coating the cells and the matrix with a selectively permeable material to allow soluble factors to diffuse through the material without cell escape, and implanting the encapsulated 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.

[0345] Some embodiments disclosed herein relate to implantable devices comprising a therapeutic cell population, a matrix capable of maintaining the viability and function of the therapeutic cell population, and a selectively permeable material layer surrounding the matrix capable of maintaining the viability and function of the therapeutic cell population. The implanted cells include regenerative cells and regenerative cells pre-stimulated for a specific function. For example, if a patient to be treated is suspected of having sepsis, the regenerative cells in the device can be pre-stimulated with sepsis-related molecular factors such as TLR4 agonists. In some embodiments, the therapeutic cell population is selected from the group consisting of differentiated cells exhibiting high growth factor producing activity, stem cells, and one or more cells expressing immunomodulatory factors. In some embodiments, the differentiated cells exhibiting high growth factor producing activity are conferred the ability to produce high levels of growth factors through ex vivo manipulation. In some embodiments, the differentiated cells exhibiting high growth factor producing activity naturally possess the ability to express high levels of growth factors. In some embodiments, the stem cells are selected from the following stem cell group: embryonic stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neural stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue-derived stem cells, exfoliated tooth-derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenetic stem cells, reprogrammed stem cells, and lateral population stem cells. In some embodiments, the cell or multiple cells expressing immunomodulatory factors are similar to a thymic cell population. In some embodiments, the matrix capable of maintaining the viability and function of the therapeutic cell population is a macroporous polymer scaffold comprising a substantially non-membrane porous wall comprising microporous polymer struts defining macropores interconnected by macroporous channels. In some embodiments, the microporous polymer struts contain microporous channels extending through the microporous polymer struts, such that macropores on either side of a given microporous polymer strut are connected through that given microporous polymer strut. In some embodiments, the average diameter of the macropores is in the range of 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 the viability and function of the therapeutic cell population is selected from the group consisting of: irradiated bone, decellularized bone, decellularized placental matrix, decellularized thymus, and decellularized organs. In some embodiments, the source of the matrix is ​​selected from the group consisting of: autologous, allogeneic, and xenogeneic.In some implementations, the matrix capable of maintaining the viability and function of the therapeutic cell population is made, in whole or in part, of a material selected from the group consisting of: glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulfone, polyvinyl alcohol, acrylonitrile polymer, polyacrylamide, polycarbonate, polypentene, polypentane, nylon, magnetite, natural polysaccharides, modified polysaccharides, collagen, gelatin, and modified gelatin.

[0346] In some embodiments, the selectively permeable material layer is made using a biocompatible material that allows particles smaller than 1 micrometer to diffuse freely. In some embodiments, the selectively permeable material layer is made using a biocompatible material that does not allow significant immune recognition of the cells within the material. Some embodiments disclosed herein relate to a method for treating an inflammatory condition. In some embodiments, the method includes selecting a cell population that produces soluble anti-inflammatory mediators, culturing the cells producing soluble anti-inflammatory mediators in vitro with a matrix to enable the cells to adhere to the matrix, coating the cells and the matrix with a selectively permeable material to allow soluble factors to diffuse through the material without cell escape, and implanting the encapsulated 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-α, interferon-γ, and TRANCE. In some embodiments, the inflammatory condition is selected from the group consisting of: autoimmunity, transplant rejection, and aging. In some embodiments, the cell population producing soluble anti-inflammatory mediators is selected from the group consisting of: immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow monocytes, mesenchymal stem cells, regulatory T cells, NKT cells, hematopoietic stem cells, umbilical cord stromal mononuclear cells, adipose tissue mononuclear cells, placental stromal mononuclear cells, umbilical cord blood mononuclear cells, and CD5-positive B cells. In some embodiments, the cell population is selected from the group consisting of autologous, allogeneic, and xenogeneic sources. In some embodiments, the cells are derived from one or more cell lines. In some embodiments, the encapsulated matrix containing therapeutic cells is administered subcutaneously in a manner that allows for subsequent removal.

[0347] Some embodiments disclosed herein relate to a method for modifying antibody-mediated immune responses. In some embodiments, the method includes selecting a cell population that produces soluble immunomodulators; culturing the cells producing the soluble immunomodulators in vitro with a matrix to enable the cells to adhere to the matrix; coating the cells and the matrix with a selectively permeable material to allow soluble factors to diffuse through the material but prevent cell escape; and implanting the sealed matrix containing therapeutic cells into a patient requiring this method. In some embodiments, an immunosuppressant is administered before, and / or simultaneously with, and / or after implantation of the sealed matrix containing therapeutic cells. In some embodiments, the immunosuppressant selectively inhibits B cell responses. In some embodiments, the immunosuppressant selectively inhibits T cell responses. In some embodiments, the cell population generating the soluble anti-inflammatory mediator is selected from the following cell groups: immature dendritic cells, lymphoid dendritic cells, alternative activated macrophages, bone marrow monocytes, mesenchymal stem cells, regulatory T cells, NKT cells, hematopoietic stem cells, umbilical cord stromal monocytes, adipose tissue monocytes, placental stromal monocytes, umbilical cord blood monocytes, and CD5-positive B cells. In some embodiments, the cell population is selected from sources consisting of autologous, allogeneic, and xenogeneic sources. In some embodiments, the sealed matrix containing therapeutic cells is administered subcutaneously in a manner that allows for subsequent removal. Some embodiments disclosed herein relate to a method for altering cell-mediated immune responses. In some embodiments, the method includes selecting a cell population that produces soluble immunomodulators, culturing the cells producing soluble immunomodulators in vitro with a matrix to enable the cells to adhere to the matrix, coating the cells and the matrix with a selectively permeable material to allow the soluble factor to diffuse through the material but prevent cell escape, and implanting the sealed matrix containing therapeutic cells into a patient in need. In some embodiments, an immunosuppressant is administered before, and / or simultaneously with, and / or after implantation of the sealed matrix containing therapeutic cells. In some embodiments, the immunosuppressant selectively inhibits B cell responses. In some embodiments, the immunosuppressant selectively inhibits T cell responses. In some embodiments, the cell population producing the soluble immunomodulator is selected from the following cell groups: immature dendritic cells, lymphoid dendritic cells, alternatively activated macrophages, bone marrow monocytes, mesenchymal stem cells, regulatory T cells, NKT cells, hematopoietic stem cells, umbilical cord stromal monocytes, adipose tissue monocytes, placental stromal monocytes, umbilical cord blood monocytes, and CD5-positive B cells. In some embodiments, the cell population is selected from sources consisting of autologous, allogeneic, and xenogeneic cells.In some implementations, the enclosed matrix containing therapeutic cells is administered subcutaneously in a manner that allows for subsequent removal.

[0348] Some embodiments disclosed herein relate to a method for stimulating endogenous cellular repair processes. In some embodiments, the method includes selecting a cell population that produces soluble factors capable of enhancing the endogenous cellular repair process; culturing the cells producing the soluble factors in vitro with a matrix, thereby enabling the cells to adhere to the matrix; covering the cells and the matrix with a selectively permeable material, thereby allowing the soluble factors to diffuse through the material but preventing cell escape; and implanting the encapsulated matrix containing therapeutic cells into a patient in need. In some embodiments, the cells producing the soluble factors capable of enhancing the endogenous cellular repair process are manipulated to enhance the production of the soluble factors. In some embodiments, the cells producing the soluble factors capable of enhancing the endogenous cellular repair process are selected from a group of cells comprising: differentiated cells expressing high growth factor production activity, stem cells, and cells expressing one or more immunomodulatory factors. In some embodiments, the stem cells are selected from the group consisting of: embryonic stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neural stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germline stem cells, adipose tissue-derived stem cells, degenerated tooth-derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenetic stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the cell population is selected from the group consisting of autologous, allogeneic, and xenogeneic sources. In some embodiments, the sealed matrix containing therapeutic cells is administered subcutaneously in a manner that allows for subsequent removal.

[0349] Some embodiments disclosed herein relate to a method for accelerating hematopoietic recovery following injury to the hematopoietic system. In some embodiments, the method includes selecting a cell population that produces soluble factors capable of enhancing hematopoietic recovery; culturing the cells producing the soluble factors in vitro with a matrix, thereby enabling the cells to adhere to the matrix; covering the cells and the matrix with a selectively permeable material, thereby allowing the soluble factors to diffuse through the material but preventing cell escape; and implanting the encapsulated matrix containing therapeutic cells into a patient in need. In some embodiments, the cell population producing the factors capable of enhancing hematopoietic recovery is selected from the following cell groups: embryonic stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, amniotic membrane stem cells, neural stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germline stem cells, adipose tissue-derived stem cells, desquamated tooth-derived stem cells, supporting cells, hair follicle stem cells, dermal stem cells, parthenogenetic stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the matrix is ​​a macroporous polymer scaffold comprising a substantially non-membrane porous wall. In some embodiments, the porous wall comprises microporous polymer struts defining macropores interconnected by macropore channels. In some embodiments, the microporous polymer struts contain micropore channels extending through the microporous polymer struts, such that macropores on either side of a given microporous polymer strut are connected through the given microporous polymer struts. In some embodiments, the macropores have an average diameter in the range of 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 the matrix group consisting of irradiated bone, decellularized bone, decellularized placental matrix, decellularized thymus, and decellularized organs. In some embodiments, the cells are derived from a source group consisting of allogeneic, autologous, and xenogeneic sources. In some embodiments, one or more doses of factors capable of enhancing hematopoietic recovery are also administered. In some embodiments, the factors enhancing hematopoietic recovery are selected from the group consisting of: G-CSF, GM-CSF, human chorionic gonadotropin, GSK-3β inhibitors, histone deacetylase inhibitors, DNA methyltransferase inhibitors, hyaluronic acid fragments, IL-1, IL-3, IL-7, thrombopoietin, and parathyroid hormone. In some embodiments, the permeable materials disclosed herein are not used to allow implanted cells to migrate out of the matrix. In some embodiments, in cases requiring hematopoietic stem cell transplantation that prevents donor-receptor reactivity to recipient tissue, the therapeutic cell population is derived from recipient cells. In some embodiments, the therapeutic cell population is a cell population capable of inducing apoptosis or inactivation of self-reactive cells.In some embodiments, the autoreactive cells are lymphocytes that recognize recipient tissue in the case of hematopoietic stem cell transplantation. In some embodiments, the autoreactive cells are lymphocytes that recognize donor tissue in the case of solid organ transplantation.

[0350] Some embodiments disclosed herein relate to an implantable device comprising: a therapeutic cell population, a matrix capable of maintaining the viability and function of the therapeutic cell population, and a selectively permeable material layer surrounding the matrix capable of maintaining the viability and function of the therapeutic cell population. In some embodiments, the therapeutic cell population is derived by the following steps: extracting adult tissue, dedifferentiating the adult tissue to impart lineage plasticity, and differentiating the adult tissue into thymic medullary epithelial cells or their progenitor cells. 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 stem cells. In some embodiments, the stem cells are selected from the following cell groups: embryonic stem cells, induced pluripotent stem cells, nuclear transfer-derived stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neural stem cells, circulating peripheral blood stem cells, mesenchymal stem cells, germline stem cells, adipose tissue-derived stem cells, desquamated tooth-derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenetic stem cells, reprogrammed stem cells, and side population stem cells. In some embodiments, the embryonic stem cells are pluripotent and express one or more antigens selected from the following: stage-specific embryonic antigen (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 umbilical cord blood stem cells are pluripotent and capable of differentiating into endothelial cells, smooth muscle cells, and neurons. In some embodiments, the umbilical cord blood stem cells are identified based on the expression of one or more antigens selected from the following: SSEA-3, SSEA-4, CD9, CD34, c-kit, OCT-4, Nanog, and CXCR-4. In some embodiments, the umbilical cord blood stem cells do not express one or more markers selected from the following: CD3, CD34, CD45, and CD1 lb. In some embodiments, the placental stem cells are isolated from placental structures. In some embodiments, the placental stem cells are identified based on the expression of one or more antigens selected from the following: 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 comprise bone marrow mononuclear cells.In some embodiments, the bone marrow stem cells are selected based on their ability to differentiate into one or more of endothelial cells, smooth muscle cells, and neurons. In some embodiments, the bone marrow stem cells are selected based on the 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 Wharton's gum. In some embodiments, the placental stem cells are morphologically mesenchymal. In some embodiments, the placental cells express one or more cytokines associated with regenerative activity. In some embodiments, the lineage-plastic cells differentiate into thymic medullary epithelial cells by culturing in a medium lacking leukemia inhibitory factors and by exposure to human fibroblast growth factor 7, human fibroblast growth factor 10, human bone morphogenetic protein 4, and human 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 epidermal growth factor is 50 ng / mL.

[0351] In some embodiments, mesenchymal stem cells are dedifferentiated by methods selected from: somatic cell nuclear transfer, cytoplasmic transfer, treatment with an epigenetic agent, treatment with a known factor inducing induced pluripotent stem cell formation, and treatment with acidic conditions. In some embodiments, the somatic cell nuclear transfer is performed by introducing the nucleus of the donor mesenchymal stem cell into an enucleated recipient oocyte. In some embodiments, a method is performed to induce activation of the enucleated recipient oocyte. In some embodiments, the pluripotent stem cells are derived from the activated recipient oocyte. In some embodiments, the pluripotent stem cells have the ability to self-renew for more than one year. In some embodiments, the pluripotent stem cells express hTERT. In some embodiments, inhibition of spontaneous differentiation of the pluripotent stem cells is achieved by growth in a culture medium conditioned with feeder cells. In some embodiments, inhibition of spontaneous differentiation of the 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 the pluripotent stem cells to mesenchymal stem cells derived from an allogeneic graft donor. In some embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, parthenogenetic stem cells, stem cells generated from somatic cell nuclear transfer, and induced pluripotent stem cells. In some embodiments, the cytoplasmic transfer is induced by permeabilizing the mesenchymal stem cell membrane using an agent that induces pore formation in the membrane. In some embodiments, the agent capable of inducing pore formation in the membrane is streptococcal hemolysin O. In some embodiments, membrane permeability is induced using an electric current. In some embodiments, the epigenetic agent is a DNA methyltransferase inhibitor. In some embodiments, the epigenetic agent is a histone deacetylase inhibitor. 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 chlorate. In some embodiments, the induced pluripotent stem cells are generated by introducing genes selected from the group consisting of SOX-2, Oct4, c-Myc, Klf4, nanog, LIN28, and Glis1.

[0352] Example 1: Reducing aging by culturing pluripotent cells in conditioned medium Purification and Senescence Induction of Fibroblasts Skin punch biopsy tissue was obtained using standard procedures (e.g., with a 4mm circular Visipunch instrument) and preserved on ice in complete DMEM 20% FBS medium.

[0353] The skin biopsy tissue was then placed at the bottom of a 10 cm culture dish and dissected under a dissecting microscope in a laminar flow hood. The dissection began by fixing the biopsy tissue in place with a scalpel and then using a second scalpel to cut the tissue block into equal halves with a unidirectional rolling motion, thereby cutting the 4 mm circular skin biopsy tissue into 12-15 uniformly sized and sharply edged tissue blocks.

[0354] Using pointed forceps, place 2-3 biopsy tissue blocks into each well of a prepared 6-well plate containing 800 μl of DMEM containing 20% ​​FCS, instead of placing them on dry wells. Use tapping or sliding motions to attach the tissue blocks to the bottom of the wells. Sometimes, a scalpel is used to remove any biopsy tissue blocks from the forceps. The 6-well plate is then placed in a 37 °C incubator. Monitor daily during the first week to ensure a layer of culture medium covering the bottom of the wells; add approximately 200 μl every 2 days to replace any evaporated medium.

[0355] One week later, increase the culture medium volume to 2 ml of complete DMEM / 20% FBS and change it every 2-3 days. Once the fibroblasts in each well have merged to the point where they reach the edge of the well, trypsinize the cells and passage them from the 6-well plate to 2X T75 culture flasks (passage 1). The tissue blocks can also be transferred. They will not adhere and will be washed out during the next medium change. Once the fibroblasts have merged, transfer them to 3X T175 culture flasks (passage 2) at 1×10⁶ cells per flask. 6 Cells / ml were frozen in complete DMEM medium with 10% DMSO.

[0356] To induce senescence, cells were treated with 50 nm doxorubicin for 48 h 24 h after seeding in 6-well plates, and then cultured in fresh complete medium for three days. Controls were represented by cells incubated in complete medium containing 0.0005% DMSO (used as a diluent for doxorubicin) for 48 h, followed by three days in fresh complete medium. The appearance of the senescence phenotype was confirmed by p16 expression.

[0357] Inhibition of aging Pluripotent iPSC conditioned medium was extracted from serum-free medium and added to fibroblasts exposed to doxorubicin at 5% v / v, 10% v / v, and 15% v / v. As shown in the following figures, based on p16 expression, the conditioned medium derived from pluripotent stem cells reduced the generation of senescent cells. The conditioned medium was generated 48 hours after culturing the pluripotent stem cells. Results are shown in… Figure 1 .

[0358] Example 2: Reducing aging by conditioned medium for mesenchymal stem cells derived from pluripotent cells Mesenchymal stem cells derived from pluripotent stem cells were created using the "MSC Switch" technique. In short, confluent 6-cm iPSCs were trypsin-digested at 37°C for 3 minutes and placed in gelatinized 10-cm culture dishes containing knockout Dulbecco modified Eagle medium (DMEM GIBCO) supplemented with 10% serum-replaced 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) to enrich MSC growth. After one week, differentiating iPSCs were harvested and incubated with CD24-phycoerythrin (PE) and CD105-FITC (BDPharMingen, San Diego, Calif). CD24-CD105+ was sorted using a fluorescence-activated cell sorting (FACS) system. CD24-CD105+ cells were seeded in 6-well plates at an initial density of 10,000 cells per well, under knockout DMEM supplementation with 10% FCS (GIBCO), bFGF (5 ng / mL), platelet-derived growth factor AB (10 ng / mL), and epidermal growth factor (10 ng / mL). When the CD24-CD105+ cultures reached confluence, one-quarter of the cells were passaged for pLL3.7-green fluorescent protein (GFP+) labeling, followed by limiting dilutions (0.5 cells per well in a 96-well plate). Wells containing a single cell as observed under a fluorescence microscope were selected, and wells containing more cells were excluded. When clones derived from single cells reached 60%–70% confluence, cells from each well were reseeded into one well of a 6-well plate, subsequently at a rate of 1 × 10⁶ cells per well. 4 Re-inoculate at densities of / mL to 25-, 75-, and 175-cm³ sequentially. 2 In tissue culture flasks. When cells are at 175-cm... 2 When the cells are combined in tissue culture flasks, they are designated as generation 1 and cryopreserved as cell reserves.

[0359] After thawing, MSC senescence was reduced by culturing in 5 nm / ml valproic acid. Cells were cultured in valproic acid-containing medium (10% FBS DMEM). Conditioned medium was obtained after culturing in serum-free DMEM without FBS for 48 hours. The conditioned medium was used to inhibit senescence induction, which was performed as described in Example 1.

[0360] Pluripotent MSCs were conditioned in serum-free medium and added to fibroblasts exposed to doxorubicin at 5% v / v, 10% v / v, and 15% v / v. Figure 1 As shown, based on p16 expression, conditioned medium derived from pluripotent stem cells was observed to reduce the generation of senescent cells. The conditioned medium was prepared 48 hours after culturing the pluripotent stem cells. Results are shown in... Figure 2 .

[0361] Example 3: Reduction of aging in vivo after administration of pluripotent stem cell conditioned medium Ten mice were divided into groups: a) control (cell-free culture medium concentrate); b) pluripotent stem cell conditioned medium concentrate (1 μg per mouse); c) pluripotent stem cell conditioned medium concentrate (10 μg per mouse); and d) pluripotent stem cell conditioned medium concentrate (100 μg per mouse). The concentrate was administered three times a week for three weeks.

[0362] The conditioned medium was prepared by culturing 1 million iPSCs per ml in serum-free DMEM medium for 48 hours. The medium was collected and filtered through a 0.2-μm filter to remove cell debris. The medium was then concentrated approximately 25-fold using an ultrafiltration device with a molecular weight cutoff of 3-kDa (Amicon Ultra-PL 3, Millipore, Billerica, MA).

[0363] Control or viable conditioned medium was administered to 20-month-old BALB / c mice. Age-related IL-11 levels were assessed before intervention, at the end of the 3-week treatment period, and 3 weeks after treatment. Results are shown in... Figure 3 .

[0364] Example 4: Reduction of aging in vivo after administration of conditioned medium for mesenchymal stem cells derived from pluripotent stem cells. Ten mice were divided into groups: a) control (cell-free culture medium concentrate); b) MSC conditioned medium concentrate derived from pluripotent stem cells (1 μg per mouse); c) MSC conditioned medium concentrate derived from pluripotent stem cells (10 μg per mouse); and d) MSC conditioned medium concentrate derived from pluripotent stem cells (100 μg per mouse). The mice were administered three times a week for three weeks.

[0365] The conditioned medium was prepared by culturing 1 million iPSCs per ml in serum-free DMEM medium for 48 hours. The medium was collected and filtered through a 0.2-μm filter to remove cell debris. The medium was then concentrated approximately 25-fold using an ultrafiltration device with a molecular weight cutoff of 3-kDa (Amicon Ultra-PL 3, Millipore, Billerica, MA).

[0366] Control or viable conditioned medium was administered to 20-month-old BALB / c mice. Age-related IL-11 levels were assessed before intervention, at the end of the 3-week treatment period, and 3 weeks after treatment. Results are shown in... Figure 4 .

Claims

1. A method for treating a subject suffering from a disease, condition, or symptom of biological aging, the method comprising: a) Identify subjects with age-related diseases, conditions, or symptoms; b) Isolate primary somatic cells from the subject's biological fluid or tissue; c) Provide a first cell culture system in which the primary cells are provided with culture conditions and treated to induce their dedifferentiation into induced pluripotent stem cells; d) Provide a second cell culture system, wherein culture conditions are provided for the induced pluripotent stem cells to induce their differentiation into somatic cells; e) Provide a third cell culture system in which culture conditions are provided for the differentiated somatic cells to induce or maintain the production of one or more soluble factors, wherein the one or more soluble factors are secreted into the culture medium, and wherein the one or more soluble factors have regenerative, immunomodulatory or both functions. f) Harvesting or isolating one or more soluble factors from the culture medium of the third culture system; and g) Applying one or more soluble factors to the subject, wherein the one or more soluble factors have regenerative, immunomodulatory, or both functions.

2. The method according to claim 1, wherein the differentiated somatic cells are selected from the group consisting of: mature dendritic cells, immature dendritic cells, lymphoid dendritic cells, myeloid dendritic cells, monocytes, macrophages, alternatively activated macrophages, mesenchymal stem cells, T cells, B cells, CD5-positive B cells, regulatory T cells, neutrophils, eosinophils, basophils, mast cells, myeloid cells, common myeloid progenitor cells, common lymphoid progenitor cells, hematopoietic stem cells, natural killer (NK) cells, NKT cells, fibroblasts, epithelial cells, epithelial progenitor cells, endothelial cells, endothelial progenitor cells, hepatocyte-like cells, hepatic stellate cells, cardiomyocytes, keratinocytes, kidney progenitor cells, neural stem cells, neural progenitor cells, neurons, or any tissue-specific stem cells or progenitor cells.

3. The method of claim 1, wherein the age-related disease comprises at least one disease selected from the group consisting of: autoimmune diseases, arthritis, cardiovascular diseases, hypertension, stroke, heart failure, diabetes, 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 a decrease in endothelial function in an organ or tissue.

5. The method of claim 1, wherein the disease, disorder, or symptom of biological aging is related to 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 decrease in the cytotoxic activity of T cells in the subject.

7. The method of claim 6, wherein the cytotoxic activity of the T cells is measured based on perforin production, granzyme B production, Fas ligand expression, 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 more cytokines by cells isolated from the subject's tissue.

9. The method of claim 8, wherein the cells are selected from the group consisting of 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 more cytokines include C-reactive protein (CRP), interferon-γ, TNF-α, IL-1α, IL-1β, IL-6, IL-12, IL-15, IL-17, IL-18, IL-27, G-CSF, GM-CSF, and MCP-1.

11. The method according to claim 1, wherein the primary somatic cells are selected from the group consisting of: fibroblasts, T cells, B cells, myeloid cells, neutrophils, eosinophils, basophils, myeloblasts, monocytes, macrophages, mesenchymal stem cells, keratinocytes, epithelial cells, exfoliated renal epithelial cells, epithelial progenitor cells, hematopoietic stem cells, common myeloid progenitor cells, natural killer (NK) cells, NK T cells, common lymphoid progenitor cells, endothelial cells, endothelial progenitor cells, synovial cells, pancreatic cells, mobilized CD34+ peripheral blood mononuclear cells, isolated blood mononuclear cells, or tissue-specific stem cells or progenitor cells.

12. The method of claim 1, wherein the soluble factor is selected from the group consisting of: proteins, glycoproteins, lipids, glycolipids, carbohydrates, antigens, epitopes, antibodies, extracellular vesicles, exosomes, apoptotic bodies, DNA molecules, or RNA molecules.

13. The method of claim 1, wherein the regenerative function comprises one or more of the following effects on cells, organs and / or tissues in the subject: reversing or preventing epigenetic alterations, stopping or reducing telomere depletion, inducing telomere elongation, improving genome stability by promoting the detoxification of DNA damaging agents, reducing or improving protein homeostasis, regulating nutrient signaling pathways, preventing or reversing mitochondrial dysfunction and damage, preventing cellular senescence, counteracting stem cell depletion and loss of function, and producing anti-inflammatory effects.

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

15. The method of claim 2, wherein the second culture system generates regulatory T cells, and wherein the regulatory T cells include effector regulatory T cells, memory regulatory T cells, or chimeric antigen receptor (CAR) regulatory T cells.

16. The method of claim 1, wherein the induced pluripotent stem cells are generated by transfecting somatic cells with pluripotency inducing factors to induce stem cell reprogramming, and wherein the pluripotency inducing factors include one or more 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 more agents comprising cellular stressors.

18. The method of claim 17, wherein the one or more agents comprising cellular stressors initiate or maintain the one or more soluble factors in the third cell culture system.

19. The method of claim 17, wherein the one or more cellular stressors comprise molecules or agents that induce inflammation.

20. The method of claim 19, wherein the inflammatory molecule or agent activates one or more Toll-like receptors, and wherein the molecule or agent is selected from the group consisting of: β-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 cells exposed to hyperthermia.

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