Means and methods of preventing or reversing aging

Abstract

To provide systems, methods, and compositions to provide one or more regenerative factors to one or more subjects.SOLUTION: The present disclosure provides means of regenerating / restoring aged and / or damaged tissue or treating a medical condition by providing a circuit extracorporeally containing cells, including fibroblasts or dedifferentiated fibroblasts, that are in contact with plasma but not with blood cells in a manner allowing exchange of factors between a subject and an extracorporeally residing mass of cells. The disclosure provides means of titrating factors produced by the extracorporeal regenerative cell mass, thus allowing a regenerative effect.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 758,240, filed on Nov. 9, 2018, which is incorporated herein by reference in its entirety.

[0002] Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, biology, immunology, and medicine.

Background Art

[0003] Previous studies have demonstrated that the circulatory connection between young and old animals produces a systemic anti-aging effect. For example, in one study, by establishing a shared circulatory system between young and old mice (heterochronic parabiosis), or by exposing old mice to factors present in young serum, the effect of systemic factors on aged progenitor cells from liver tissue was investigated. In particular, this pairing restored the activation of Notch signaling, as well as the proliferation and regenerative capacity of aged satellite cells. Furthermore, exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), enhanced Notch activation, and enhanced proliferation in vitro. Additionally, heterochronic parabiosis increased the proliferation of aged hepatocytes and restored the cEBP-α complex to levels seen in young animals. It was suggested that the decline in progenitor cell activity associated with aging may be regulated by systemic factors that change with aging [1]. In another study, it was revealed that young blood contains factors that induce vascular remodeling, and as a result, using a similar heterochronic parabiosis model, neurogenesis increased and olfactory discrimination improved in aged mice. As a result of identifying molecular factors related to improved cerebrovascular distribution and neurogenesis, the role of a molecule called GDF11 became clear [2].

[0004] Unfortunately, means of connecting human circulations are extremely difficult and unethical. Therefore, in the art, there is a need to supply regenerative factors to subjects in need of such factors in a practical manner.

SUMMARY OF THE INVENTION

[0005] The present disclosure is directed to systems, methods, and compositions for providing one or more regenerative factors to one or more subjects in need thereof. Aspects of the present disclosure provide systems, compositions, and methods in which the circulatory system of a subject having a medical condition and / or being aged is exposed to one or more beneficial regenerative factors produced from specific cells; wherein the regenerative factors are produced by exposure of the cells to blood or plasma from the subject. In other embodiments, the subject is exposed to the system to prevent or delay the onset of one or more symptoms of aging. In certain embodiments, when cells that are inline in the system with the subject are exposed to one or more degenerative factors arising from the subject's circulation, one or more regenerative factors are produced, and the one or more regenerative factors are provided to the subject that is inline in the system. Such regenerative factors then improve at least one symptom of the medical condition and / or reduce at least some of the effects of aging.

[0006] In certain embodiments, the present disclosure utilizes an extracorporeal circuit to provide one or more regenerative factors to one or more subjects in need thereof. In certain embodiments, the extracorporeal circuit includes tubing that couples at least one subject with at least one bioreactor. In certain embodiments, the present disclosure encompasses a circuit that couples a subject and a bioreactor, where one or more specific factors are shared between the subject and the bioreactor. In certain embodiments, the present disclosure separates one or more cellular components of the subject from one or more cellular components of one or more bioreactors. In certain embodiments, this separation occurs by using at least one semipermeable membrane at a location within the tubing (unless hollow fibers are utilized in certain embodiments), the subject, and / or the bioreactor. In some embodiments, the bioreactor includes cells that secrete one or more factors useful to the subject. In some embodiments, the bioreactor detects one or more factors from the subject. In certain embodiments, the bioreactor secretes factors at a rate that depends on the rate of factors detected from the subject.

[0007] In some embodiments, the subject is an individual having at least one medical condition in need of treatment and / or an age in need of replenishment of one or more factors and / or an age in need of reduction of at least one or more harmful factors. In at least some cases, the subject is an individual having an abnormal level of an aging-related factor. In other aspects, the subject is an individual having, for example, a chronic or acute abnormal level of a degenerative factor. In some embodiments, the subject is an organ. In at least some cases, the organ is derived from an individual having an abnormal level of one or more degenerative factors and / or one or more factors related to aging. In other cases, the organ is donor-derived, where the regenerative factors supplied by the bioreactor are useful for keeping the organ fully viable; this organ may need to be used, for example, for transplantation. In certain embodiments, the subject is one or more tissues.

[0008] Embodiments of the present invention provide means for generating and / or utilizing cells in a bioreactor. In some embodiments of the present disclosure, the cells to be generated and / or utilized are derived from a young subject or a subject lacking one or more signs of aging. In certain embodiments of the present disclosure, the cells to be generated and / or utilized have one or more cell surface molecules. In certain embodiments, the cells to be generated and / or utilized lack one or more specific cell surface molecules. In certain embodiments of the present disclosure, the cells to be generated and / or utilized are cultured using a method that secretes one or more regenerative factors and / or enhances the ability of the cells to detect or respond to one or more degenerative factors. In at least some cases, the cells to be generated and / or utilized are further manipulated, for example, by culturing using one or more viral and / or one or more non-viral methods.

[0009] Embodiments of the present disclosure include a system for treating a subject, the system comprising at least one bioreactor containing cells, At least one selectively permeable membrane, and tubing for connecting the at least one bioreactor, the at least one selectively permeable membrane, and the subject, and circulating fluid from the subject to the bioreactor and, in certain embodiments, the membrane is optional provided that the tubing includes hollow fibers. The system may further include blood or plasma from a subject, or may include an organ preservation solution. The cells may be regenerative cells that enable regeneration of cells or tissues, and in certain cases, the regenerative cells are fibroblasts, dedifferentiated fibroblasts, induced pluripotent cells, parthenogenic derived cells, mesenchymal stem cells, or hematopoietic stem cells, although the stem cells may be of any type. The regenerative cells can secrete one or more regenerative factors at a basal rate or an inducible rate. Examples of regenerative factors include AKT, BAMBI, BCL-2, BCL-2XL, BDNF, BIRC5 CDA, CXCR4, dominant negative CCL2, EGF, exosomes, FGF-2, GATA-4 GDF-11, GDNF, hCG, HGF, HIF-1α, HLA-G, HO-1, hTERT, IFN-b, IGF-1, IFT-1, LIGHT, miR-126, NK4, NUR77, OCT-4, PGE-1, SDF-1, STC-1, TERT, TRAIL, VEGF, WNT11, XIAP, and combinations thereof. The regenerative cells can produce one or more regenerative factors in response to one or more modifying factors from the subject's blood or plasma. In some cases, the regenerative cells secrete regenerative factors at a set rate upon detection of a modifying factor. The rate of regenerative factor secretion from the cells can be a ratio to the rate of modifying factor detection by the cells. In certain embodiments, the ratio of regenerative factor secretion rate to modifying factor detection rate is selected from the group consisting of 50:1, 25:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:25, 1:50, and any ratio therebetween.

[0010] In certain embodiments of the present disclosure, the selective permeability membrane is disposed within tubing, e.g., within tubing between a bioreactor and a subject; the tubing may be composed of the selective permeability membrane; and / or the bioreactor may be at least partially composed of the selective permeability membrane. In some cases, the selective permeability membrane inhibits or reduces the passage of cellular material (whole cells or fragments thereof) between the subject and the bioreactor. One or more selective permeability membranes may permit the passage of one or more regenerative factors and one or more modifying factors.

[0011] The system may be applied to any subject, such as a living animal, or the subject may be an organ (any part of an organ or any part of an organ system) or tissue derived from an animal, and the organ or tissue may be derived from a donor. Examples of organs include one or more selected from the group consisting of liver, pancreas, gallbladder, stomach, small intestine, large intestine, lung, kidney, heart, spleen, brain, eye, and combinations thereof.

[0012] When the subject includes an organ or tissue, the system may also include a container connected to at least one bioreactor and at least one selective permeability membrane for enclosing the subject. In certain embodiments the subject is an organ or tissue and the container is suitable for holding the subject such that transfer of an organ preservation solution or other fluid into tubing is possible.

[0013] Embodiments of the present disclosure include an ex vivo method of producing a regenerative factor, the method including subjecting a subject to a system encompassed by the present disclosure under conditions that enable secretion of one or more regenerative factors from cells in the system. The cells may be fibroblasts derived from tissues including skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue, placenta, and / or foreskin. The fibroblasts may have one or more surface markers selected from the group consisting of CD73, CD90, CD56, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, CD49b, CD105, aminopeptidase N, hyaluronic acid binding receptor, collagen / laminin binding integrin α2, OCT4, NANOG, SOX-2, and combinations thereof. In certain embodiments, the fibroblasts lack one or more surface markers selected from the group consisting of CD14, CD34, CD45, HLA class II, and combinations thereof.

[0014] In certain embodiments of the method, the method comprises culturing fibroblasts in an undifferentiated state for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days or more, culturing the fibroblasts of step (a) in the presence of one or more factors selected from the group consisting of nerve growth factor, bFGF, dibutyryl cAMP, IBMX, retinoic acid, exendin-4, and combinations thereof, and activating the fibroblasts. Activating the fibroblasts can include exposing the fibroblasts to one or more cytokines in the cell culture medium. Examples of cytokines are one or more selected from the group consisting of IL-1, IFNγ, and combinations thereof. The cytokine may be at a specific concentration. For example, the concentration of IL-1 may be 1-100 ng / mL, 5-100 ng / mL, 10-100 ng / mL, or 20-40 ng / mL, and the concentration of IL-1 may be 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL. The concentration of IFNγ may be 1-1000 IU, 5-1000 IU, 10-1000 IU, 1-500 IU, 5-500 IU, 10-500 IU, 100-500 IU, or 250 IU, and the concentration of IFNγ may be 1 IU, 5 IU, 10 IU, 50 IU, 100 IU, 200 IU, 250 IU, 300 IU, 400 IU, 500 IU, 600 IU, 700 IU, 800 IU, 900 IU, or 1000 IU. Such concentrations are merely exemplary.

[0015] In certain embodiments of the present disclosure, exposing fibroblasts to one or more cytokines induces an increase in the expression of one or more complement inhibitory molecules from the fibroblasts. Examples of complement inhibitory molecules are selected from the group consisting of CD35, CD46, C4BP, CD55, factor H, and combinations thereof. Activating the fibroblasts can include transfecting the fibroblasts with one or more viral and / or non-viral expression systems to induce the expression of one or more regenerative factors. In a specific embodiment, the regenerative factors are selected from the group consisting of AKT, BAMBI, BCL-2, BCL-2XL, BDNF, BIRC5 CDA, CXCR4, dominant negative CCL2, EGF, exosomes, FGF-2, GATA-4 GDF-11, GDNF, hCG, HGF, HIF-1α, HLA-G, HO-1, hTERT, IFN-b, IFT-1, LIGHT, miR-126, NK4, NUR77, OCT-4, PGE-1, SDF-1, STC-1, TERT, TRAIL, VEGF, WNT11, XIAP, and combinations thereof.

[0016] Embodiments of the present disclosure include kits that include some or all of any of the systems included herein.

[0017] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure so that the detailed description that follows may be better understood. Additional features and advantages that form the subject of the claims of this specification will be described below. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. Also, it should be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed to be characteristic of the design disclosed herein, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. However, it should be clearly understood that each drawing is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

Brief Description of the Drawings

[0018] To more fully understand the present disclosure, reference is made to the following description in conjunction with the accompanying drawings.

[0019]

Figure 1

[0020]

Figure 2

[0021]

Figure 3

Modes for Carrying Out the Invention

[0022] [I. Examples of Definitions] In accordance with long-standing patent law convention, when the words "a" and "an" are used in cooperation with the words "comprising" and "comprise", including in the claims, they mean "one or more", and some embodiments of the disclosure can consist of, or consist essentially of, one or more elements, method steps, and / or methods of the disclosure. It is intended that any method or composition described herein can be used with respect to any other method or composition described herein. As used herein, the terms "about" or "approximately" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length. In certain embodiments, the term "about" or "approximately" appears before a numerical value and represents a plus or minus value within the range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within 5-fold, more preferably within 2-fold. Unless otherwise specified, the term "about" means within an acceptable error range for a particular numerical value.

[0023] As used herein, the term "aging" refers to a subject in whom signs of age-related degeneration have begun to appear. Examples of age-related degeneration include a decrease in telomere length, an increase in oxidative stress, the formation of cellular tangles, the development of tauopathy in the central nervous system, and / or an increase in the number of misfolded proteins.

[0024] As used herein, the term "bioreactor" refers to a vessel or container that contains, or is capable of containing, for example, cells, cell components, and / or other biological materials. A bioreactor can be composed of any suitable material. In some embodiments, the bioreactor comprises at least one semipermeable membrane described herein, at least in part. The bioreactors of the present disclosure can include an environment that allows cells to survive while perfusing the tissue culture medium through living cells in a manner that allows the removal of growth factors and therapeutic agents from the tissue culture medium in which the cells are growing in the environment. In one embodiment, the cells are perfused with plasma from the blood of an individual in need of treatment.

[0025] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted as specifying the presence of stated steps or groups of elements but not as precluding the presence of others. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the expression "consisting of" indicates that the listed elements are required or essential and that no other elements may be present. The expression "consisting essentially of" means including any elements listed after the phrase and limited to other elements that do not prevent or contribute to the activity or action specified in the disclosure of the recited elements. The phrase "consisting essentially of" indicates that the listed elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0026] As used herein, the terms "modifying" or "modifying agent" refer to one or more factors that are harmful to the subject. Such agents can be one or more age-related factors that include inflammatory molecules selected from the group consisting of at least one or more of IL-1, TNF-α, IL-6, IL-17, IL33 and combinations thereof, or other factors known in the art.

[0027] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "a particular embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0028] As used herein, the term "agent" refers to a molecule such as a protein, lipid, nucleic acid, metabolite, hormone, biosynthetic product, or other molecule produced by a subject and / or a bioreactor.

[0029] The terms "decrease", "inhibit", "reduce", "suppress", "lower", "prevent" and grammatical equivalents (including "lower", "less", etc.) when referring to the manifestation of any symptom in a treated subject as compared to an untreated subject, mean that the amount and / or magnitude of the symptom in the treated subject is lower by any amount clinically recognized as relevant by any person trained in medicine as compared to the untreated subject. In one embodiment, the amount and / or magnitude of the symptom in the treated subject is at least 10% lower, at least 25% lower, at least 50% lower, at least 75% lower, and / or at least 90% lower than the amount and / or magnitude of the symptom in the untreated subject.

[0030] As used herein, the term "regenerate" or "regenerative agent" refers to one or more agents that act to fully or partially restore a subject to a state prior to exposure to a denaturing stimulus. Such regenerative stimuli can be said to act, for example, in a manner opposite to that of the associated denaturing agent.

[0031] As used herein, the term "selectively permeable membrane" refers to one or more devices or materials that can selectively allow the passage of agents while restricting the passage of cells. In some embodiments, the selectively permeable membrane utilized is a hollow fiber membrane. A hollow fiber membrane is a semi-permeable membrane consisting of long porous filaments having an inner and an outer side, capable of selectively allowing a particular composition to pass from the inside to the outside or from the outside to the inside, while restricting such passage of other compositions. In some embodiments, the hollow fiber membrane allows the passage of agents while restricting the passage of cells. In certain embodiments, the membrane achieves two things:

[0032] In certain embodiments, the membrane does not permit blood cells from the individual to contact the cells in the bioreactor: and only plasma from the individual's blood contacts the regenerative cells in the bioreactor. In certain cases, the membrane does not permit cells from the bioreactor to leak into the individual's blood.

[0033] The term "cellular senescence" (or simply "senescence") is a stress-induced persistent cell cycle arrest of cells that were previously replicable.

[0034] "Treatment", "treating", or "treatment" means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing not only the symptoms but also the disease or condition itself. Treatment can be any decrease from the pre-treatment level and can include, but is not limited to, complete elimination of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the severity of an established disease or disease progression, including a decrease in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered a treatment if there is a detectable decrease in the immunogenicity of the cells compared to the pre-treatment level in the same subject or control subject. Thus, the decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between compared to the native or control level. It is understood and contemplated herein that "treatment" does not necessarily refer to a cure of the disease or condition, but rather means an improvement in the outlook of the disease or condition. In certain embodiments, treatment can refer to a decrease in the severity or degree of at least one symptom and, alternatively or additionally, can refer to a delay in the onset of at least one symptom.

[0035] In some embodiments of the present disclosure, the cells generated and / or utilized are derived from a "young subject", which can be a subject of an age of 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 years old or less than 1 year old.

[0036] In certain embodiments, a subject lacking one or more signs of aging may not be of a specific age, but can be 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 years old or less than 1 year old.

[0037] [II. In Vitro System] In certain embodiments, the present disclosure describes a system having the ability to induce one or more regenerative changes in a subject, e.g., a system having the ability to induce one or more regenerative changes systemically or locally in a subject. In some embodiments, the system generates regenerative feedback proportional to the degree of degenerative stimulus and, in certain aspects, can counteract one or more degenerative stimulus effects. In certain embodiments, the device generates, releases, or secretes one or more units of regenerative factor per one or more units of degenerative agent detected by the system. In some cases, the degenerative stimulus includes aging-related stimuli and / or aging-related inflammation. More precisely, one or more of some inflammatory mediators are associated with various aspects of aging or degeneration. Without being bound by theory, certain embodiments of the systems encompassed herein are exogenous bioreactors in which regenerative cells in the system contact circulating factors from a subject linked to the bioreactor and the cells in the system produce one or more regenerative factors in response to the circulating factors in the circulation in the system. In some embodiments of the present disclosure, the circulating factors from the subject are aging-related factors such as inflammatory mediators. The object of the present invention is to create an artificial environment in which inflammation or other degeneration can induce the production of regenerative factors based on the needs of the body. In some embodiments of the present disclosure, the regenerative factors include agents such as GDF-11, exosomes, or other agents related to regeneration such as BDNF, EGF, hCG, VEGF, and IGF-1.

[0038] In some embodiments, the subject may be aging. Aging may be natural aging, accelerated aging, or aging may be a time-dependent decline in physiological function. In some embodiments, aging is associated with enhanced fibrosis, enhanced inflammation, and / or decreased telomere length, all compared to normal. In some embodiments, the subject may have abnormal levels of degenerative agents associated with a disease state. In some embodiments, the subject may have a particular trauma or injury or medical condition that changes the levels of the subject's degenerative and / or regenerative factors.

[0039] In certain embodiments, the subject can be an organ from an individual that is, for example, aging, has a disease, or has an injury or trauma. In other embodiments, the subject can be an organ used for transplantation. In the case of transplantation, the system is used to keep the organ in a viable state. In further embodiments, the subject can be a tissue or set of tissues that maintains a viable state. In embodiments of the present disclosure where the subject is an organ, the subject can be the liver, pancreas, gallbladder, stomach, small intestine, large intestine, lung, kidney, heart, spleen, brain, eye, omentum, small intestinal mucosa, or other organ, or any part of an organ, or any part of an organ system.

[0040] Referring to FIG. 1, system 100 shows an example of an extracorporeal system for preventing or reversing the aging of subject 101 and / or treating the medical condition of subject 101. In one example of the system, the circulatory system of subject 101 is attached inline to tubing 102 such that tubing 102 can transfer blood or plasma out of subject 101 and extracorporeally. Tubing 102 transfers blood or plasma from subject 101 to a bioreactor 103 containing cells that can detect the presence of one or more denaturing factors in the blood or plasma of subject 101. When the cells in bioreactor 103 are exposed to the blood or plasma from subject 101 transferred through tubing 102, the cells in bioreactor 103 produce one or more regenerative factors. Next, one or more regenerative factors from the cells in bioreactor 103 are transferred into the blood or plasma through tubing 104 and returned to subject 101 because tubing 104 is inline with the system and attached to the circulatory system of subject 101. A semipermeable membrane may be incorporated at any location in the system, such as in and / or throughout tubing 102 and / or 104, or may at least partially enclose bioreactor 103.

[0041] Referring to FIG. 2, system 200 shows an example of an extracorporeal system for preventing or reversing the aging of an organ or tissue 202 and / or treating the medical condition of an organ or tissue 201. In an example of the system, the circulatory system of the organ or tissue 201 is attached in-line to tubing 202 so that the tubing 202 can transfer blood or plasma from the organ or tissue 201 outside of the organ or tissue. The organ or tissue may be housed within container 205. The tubing 202 transfers organ preservation fluid (commercially available) from the organ or tissue 201 to a bioreactor 203 that houses cells capable of detecting the presence of one or more denaturing factors in the blood or plasma from the organ or tissue 201. When the cells in the bioreactor 203 are exposed to the blood or plasma from the organ or tissue 201 transferred through the tubing 202, the cells in the bioreactor 203 produce one or more regenerative factors. Next, one or more regenerative factors from the cells in the bioreactor 203 are transferred into the blood or plasma through tubing 204 and returned to the organ or tissue 201 because the tubing 204 is in-line with the system and attached to the circulatory system of the organ or tissue 201. A semi-permeable membrane may be incorporated at any location in the system, such as within and / or throughout the tubing 202 and / or 204, or may at least partially enclose the bioreactor 203 and / or the container 205.

[0042] In certain embodiments of the present disclosure, a bioreactor is provided that includes a compartment suitable for containing a regenerative biological material that includes cells. In one aspect, the bioreactor includes at least one selectively permeable membrane that can contact the cells. In certain embodiments, the bioreactor has at least one selectively permeable wall. In certain embodiments, the selectively permeable membrane can be a selectively permeable hollow fiber. The bioreactor can include a plurality of selectively permeable hollow fibers passing through the compartment, through which one or both of a gas and a fluid containing nutrients for the cells can pass. In certain embodiments, the bioreactor includes a plurality of hollow fibers having selective permeability passing through the bioreactor. In other embodiments, the selectively permeable membrane or membranes are separated from the bioreactor. In certain embodiments, the membrane(s) are disposed within tubing connecting at least one of the bioreactor and the subject. The bioreactor and the selectively permeable membrane can be connected to the subject by tubing. The tubing can be made of any material that is flexible or rigid and biocompatible. In some embodiments, the tubing is connected to the subject's circulatory system at at least one point in the subject's circulatory system. In certain embodiments, the tubing is connected to a container that houses the subject (e.g., if the subject is a tissue or organ). In such embodiments, the tubing allows for circulation of fluid from the subject and / or around the subject. In certain embodiments, a method and / or device for circulating fluid within the tubing connecting the subject, the bioreactor, and the selectively permeable membrane is used.

[0043] Biological materials contained in the bioreactor: 1) sense a predetermined level of a denaturing factor, 2) generate an appropriate level of one or more appropriate reaction regeneration factors, and 3) in some embodiments, also release a diagnostic marker that clarifies the degree of the denaturing factor generated by the subject. The biological material for use in the present apparatus can be any cell or cell material or plurality of cells effective for use in a bioreactor, and can be heterologous cells, syngeneic, allogeneic, or autologous cells with respect to the individual being treated by the use of the bioreactor. In certain embodiments where the biological material is a cell, the cell can detect the denaturing factor and secrete the regeneration factor at a rate commensurate with the rate of denaturing factor detection. The corresponding rate may be the ratio of regeneration secretion to denaturing detection. This ratio can be, for example, 50:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:50, and any ratio therebetween.

[0044] In certain embodiments, the biological material comprises cells. In certain embodiments, the cells are cultured in a bioreactor. In other embodiments, the cells are cultured outside of the bioreactor in a suitable cell culture vessel and then harvested for use in the bioreactor. In certain embodiments, the cells utilized are fibroblasts. The cells used, including where they can be fibroblasts, can have one or more surface markers selected from the group consisting of CD73, CD90, CD56, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, CD49b, CD105, aminopeptidase N, hyaluronic acid binding receptor, collagen / laminin binding integrin α2, and combinations thereof. At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% of the cell population used in the present disclosure can express one or more specific surface markers. The cells used may also lack one or more surface markers (e.g., one or more selected from the group consisting of CD14, CD34, CD45, HLA class II, and combinations thereof). In certain embodiments of the present disclosure, the cells can self-renew in tissue culture, maintain euploidy for over one year in culture, share markers with human ES cells, and / or differentiate into all three germ layers of the developing embryo. In certain embodiments, the cells are fibroblasts obtainable from amniotic membranes taken during the second trimester of human pregnancy. Fibroblasts include a plurality of morphologically distinguishable cell types, and most of the cells are prone to senescence and are known to be lost from the culture. In one embodiment, cells are grown from fibroblast harvests from normal 16- to 18-week pregnancies using fibronectin-coated plates and culture conditions as described in U.S. Patent No. 7,569,385, which is incorporated herein by reference. In one embodiment, the cells of the present disclosure are of fetal origin and can have a normal diploid karyotype. The regenerative cells can be isolated from any mammal, including any primate (including humans). However, the regenerative cells of fibroblasts may be isolated in a similar manner from other species.Examples of species that can be used to induce regenerative cells of fibroblasts include, but are not limited to, mammals, humans, primates, dogs, cats, goats, elephants, sheep, endangered species, cows, horses, pigs, mice, rabbits, and the like.

[0045] Cells used in the present disclosure can be recognized by specific cell surface proteins or by the presence and / or absence of specific cellular proteins. Typically, a specific cell type has specific cell surface proteins. These surface proteins can be used as markers to determine or confirm a specific cell type. Typically, these surface markers can be visualized using antibody-based techniques or other detection methods. In certain embodiments, the markers are selected from the group consisting of CD73, CD90, CD56, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, CD49b, CD105, aminopeptidase N, hyaluronic acid binding receptor, collagen / laminin binding integrin α2, and combinations thereof.

[0046] In certain embodiments of the present disclosure, the cells are human stem cells that can be continuously cultured in an undifferentiated state and grown for an indefinite period. The term "undifferentiated" refers to cells that have not become a specialized cell type. The cells are cultured in a nutrient medium. The nutrient medium can contain any one or more of isotonic saline, buffers, amino acids, antibiotics, serum or serum substitutes, and exogenously added factors in appropriate combinations. The cells can grow in an undifferentiated state for a desired period and then be cultured under specific conditions to progress to a differentiated or activated state. In some embodiments, the cells are cultured in an undifferentiated state for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days or more before the cells are activated. In certain embodiments, the cells are cultured in the presence of factor(s) before the cells are activated. In further embodiments, the cells are cultured in the presence of nerve growth factor, bFGF, dibutyryl cAMP, IBMX, retinoic acid, exendin-4, or other factors that can be included alone or in combination and are useful for producing the desired activated cells. By being activated, the term means the process by which non-specialized cells acquire the characteristics of specialized cells such as cells of the heart, liver, muscle, pancreas or other organs or tissues. The cells in the present disclosure can be activated into any cell type that can be used for the described system.

[0047] General methods related to cell differentiation techniques that may be useful for differentiating the cells of the present disclosure are described in general texts such as (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998); and in Stem cell biology (L. M. Reid, Curr. Opinion Cell Biol. 2:121, 1990), each of which is hereby incorporated by reference in its entirety.

[0048] In some embodiments of the present disclosure, cells are cultured in the presence of one or more cytokines to activate the cells for use in a system. In certain embodiments, the cytokines used are IL-1, IFNγ, or both, either simultaneously or sequentially. In some embodiments, the concentration of IL-1 supplied to the cell culture can be, for example, 1 - 100 ng / mL, 5 - 100 ng / mL, 10 - 100 ng / mL, or 20 - 40 ng / mL. In other embodiments, the concentration of IL-1 can be, for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL. In one embodiment, the concentration of IFNγ can be, for example, 1 - 1000 IU, 5 - 1000 IU, 10 - 1000 IU, 1 - 500 IU, 5 - 500 IU, 10 - 500 IU, 100 - 500 IU, or 250 IU. In further embodiments, the concentration of IFNγ can be, for example, 1 IU, 5 IU, 10 IU, 50 IU, 100 IU, 200 IU, 250 IU, 300 IU, 400 IU, 500 IU, 600 IU, 700 IU, 800 IU, 900 IU, or 1000 IU.

[0049] In certain embodiments, the cells can be cultured prior to use in the system. In certain cases, culturing the cells induces the expression of complement inhibitory molecules. These molecules can include CD35, CD46, C4BP, CD55, factor H, or other factors that reduce the activation of the complement system.

[0050] In some embodiments of the present disclosure, cells are transfected to have enhanced regenerative properties. Transfection can be achieved by the use of one or more viral vectors (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus) or one or more non-viral vectors (e.g., plasmid). Means for performing transfection are well known in the art and are discussed in the following references [3-9]. Specific examples of genes include SDF-1

[10] for promoting the homing of stem cells, particularly hematopoietic stem cells, GDNF

[11] for treating Parkinson's disease in animal models, HGF

[12] for promoting remyelination in brain injury models, akt

[13] for protecting against pathological circulatory remodeling and necrotic cell progeny, TRAIL [14-17] for inducing apoptosis of tumor cells, PGE-1 synthase

[18] for cardioprotection, NUR77

[19] for promoting migration, BDNF

[20] for reducing optic neuropathy associated with hypertension, HIF-1α

[21] for stimulating bone formation, dominant negative CCL2

[22] for suppressing pulmonary fibrosis, interferon-β

[23] for suppressing tumor progression, HLA-G

[24] for enhancing immunosuppressive activity, hTERT

[25] for inducing differentiation along the hepatocyte lineage, cytosine deaminase

[26] , OCT-4 [27,28] for reducing aging, BAMBI

[29] for reducing TGF expression and tumor-inducing effects, HO-1

[30] for radiation protection, LIGHT

[31] for inducing antitumor activity, miR-126 [32, 33] for promoting angiogenesis, bcl-2

[34] for inducing the generation of nucleus pulposus cells, telomerase

[35] for inducing neurogenesis, CXCR4

[36] for promoting hematopoietic recovery and

[37] reducing unwanted immunity, wnt11

[38] for promoting regenerative cytokine production, HGF antagonist NK4

[39] for reducing cancer, etc. Other factors known in the art for suppressing degeneration or inducing regeneration can also be used.

[0051] [III. Method for Treating a Medical Condition, or Method for Reversing or Retarding Aging] Embodiments of the present disclosure include methods of treating a medical condition or reducing the effects of the natural process of aging. The medical condition can be an aging-related disorder or an age-related disorder or process. An individual may have a cellular accumulation of damage and other detrimental changes, including the loss of at least some cells and structures, compared to a younger individual. In certain cases, the individual has lost the ability of adult somatic cells to be converted into cells that have been partially reprogrammed for the individual or induced pluripotent stem cells.

[0052] In some embodiments of the present disclosure, the method and system include extending the lifespan of cells and / or organisms compared to individuals not subjected to the method and system of the present disclosure. In certain embodiments, the method and system of the present disclosure enable the reversal or delay of cellular aging and the irreversible loss of replicative capacity in somatic cells. In certain cases, the method and system prevent or reduce changes in mitochondrial homeostasis associated with telomere dysfunction and / or cellular aging.

[0053] In certain embodiments, the medical condition is an aging-related disease, for example, a disease that becomes more frequent as aging progresses and is most frequently seen. In at least some cases, age-related diseases are complications resulting from aging. With the exception of a few rare cases, all adult animals age, but not all adult animals experience all age-related diseases, so in certain embodiments, age-related diseases can be distinguished from the aging process itself. In certain cases, age-related diseases include neurodegenerative diseases, atherosclerosis, cardiovascular diseases, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, Parkinson's disease, amyotrophic lateral sclerosis, and / or Alzheimer's disease.

Examples

[0054] [IV. Examples] The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. However, they should in no way be construed as limiting the broad scope of the present disclosure.

[0055] [Example 1. Collection of Cells for Use in the System] The surface markers of isolated pluripotent amniotic fluid stem cells (MAFSC) derived from independently collected fibroblast samples were tested for the range of cell surface and other markers using monoclonal antibodies and FACS analysis. These cells can be characterized by the following cell surface markers: SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54. MAFSC cells can be distinguished from mouse ES cells in that they do not express the cell surface marker SSEA1. Furthermore, MAFSC expresses Oct-4, a stem cell transcription factor. MAFSC cells can be recognized by the presence of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or all of the following cell markers: SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, and Oct-4.

[0056] MAFSC cultures express little or no SSEA-1 marker. In addition to the embryonic stem cell markers SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4, fibroblast regenerative cells expressed high levels of cell surface antigens that are normally found on human mesenchymal stem cells but not on human embryonic stem cells. This set of markers includes CD13 (99.6%) aminopeptidase N, CD44 (99.7%) hyaluronic acid binding receptor, CD49b (99.8%) collagen / laminin binding integrin α2, and CD105 (97%) endoglin. Since both embryonic stem cell markers and hMSC markers are present in MAFSC cell cultures, the fibroblast-derived MAFSC cells grown and expanded as described herein are shown to be a new class of human stem cells with characteristics of both hES cells and hMSC cells.

[0057] To determine the quality of the MAFSC cultures, flow cytometry was performed on all cultures to examine the surface expression of SH-2, SH-3, SH-4 MSC markers and the absence of contamination with CD14-positive and CD-45-positive cells. Cells were detached with 0.05% trypsin-EDTA, washed with DPBS + 2% bovine albumin, fixed in 1% paraformaldehyde, blocked with 10% serum, and incubated separately with primary antibodies for SH-2, SH-3, and SH-4, followed by a PE-conjugated anti-mouse IgG(H+L) antibody. Confluent MSC in 175 cm 2 flasks were washed with Tyrode's salt solution, incubated with Medium 199 (M199) for 60 minutes, and detached with 0.05% trypsin-EDTA (Gibco). Cells from 10 flasks were detached at once, and the MSC were resuspended in 40 ml of M199 + 1% human serum albumin (HSA; American Red Cross, Washington, DC, USA). MSC collected from each set of 10 flasks were stored at 4°C for up to 4 hours and pooled at the end of the collection. A total of 2 - 10'10 6 / kg of MSC were resuspended in M199 + 1% HSA and centrifuged at 460 g for 10 minutes at 20°C. The cell pellet was resuspended in fresh M199 + 1% HSA medium and centrifuged at 460 g for 10 minutes at 20°C three more times. The total recovery time was 2 - 4 hours based on the MSC yield per flask and the desired dose. The recovered MSC were cryopreserved in Cryocyte (Baxter, Deerfield, IL, USA) freezer bags using a rate-controlled freezer at a final concentration of 10% DMSO (Research Industries, Salt Lake City, UT, USA) and 5% HSA.

[0058] In one embodiment of the present disclosure, after the MAFSC are allowed to adhere for 72 hours, the medium is changed every 3 - 4 days. The adherent cells are removed with 0.05% trypsin-EDTA, and 1'10 per 175 cm 2 6 ​Re-plating is performed at the concentration of. MAFSC can be administered intravenously or, in a preferred embodiment, intrathecally in patients suffering from neurodegenerative symptoms associated with radiation. The dosage can be determined by those skilled in the art and depends on various patient characteristics, but for intravenous administration, it can be carried out at a concentration in the range of 1 million to 10 million MSCs / kg, and the preferred dosage is about 2 million to 5 million cells / kg.

[0059] [Example 2. Examples of Transfected Cells for the System] In one embodiment of the present invention, MAFSCs are transfected with anti-apoptotic proteins to extend their in vivo lifespan. The present disclosure includes methods of using MAFSCs cultured under conditions that express increased amounts of at least one anti-apoptotic protein as a treatment for inhibiting or preventing apoptosis. In one embodiment, the MAFSCs used as a treatment for inhibiting or preventing apoptosis are contacted with apoptotic cells. The present invention is based on the discovery that MAFSCs in contact with apoptotic cells express high levels of anti-apoptotic molecules. In some examples, MAFSCs in contact with apoptotic cells secrete high levels of at least one anti-apoptotic protein, including but not limited to STC-1, BCL-2, XIAP, survivin, and Bcl-2XL. Methods of transfecting MAFSCs with anti-apoptotic genes applicable to the present invention have been previously described, and non-limiting examples of such anti-apoptotic genes that can be utilized in the practice of the present invention include GATA-4

[40] , FGF-2

[41] , bcl-2 [34, 42], and HO-1

[43] . Based on the disclosure provided herein, MAFSCs can be obtained from any source. MAFSCs may be autologous (obtained from the same host) with respect to the subject and / or recipient, or may be allogeneic with respect to the subject and / or recipient. Additionally, MAFSCs can be xenogeneic (obtained from animals of a different species) with respect to the subject and / or recipient. In one embodiment of the present invention, MAFSCs are pretreated with an agent for inducing the expression of anti-apoptotic genes, and in one example, this is pretreatment with exendin-4 as previously described

[44] . In further non-limiting embodiments, the MAFSCs used in the present invention can be isolated from the bone marrow of any species of mammal, including but not limited to humans, mice, rats, primates, rhesus monkeys, and cows. In non-limiting embodiments, MAFSCs are isolated from humans, mice, or rats. In another non-limiting embodiment, MAFSCs are isolated from humans.

[0060] [Example 3. Fibroblast conditioned medium reduces senescence] Foreskin fibroblasts were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer's instructions. To prepare fibroblast conditioned medium, fibroblasts were isolated based on CD73 expression and cultured for 24 hours at a concentration of 1 million cells per 10 ml of RPMI medium containing 10% fetal bovine serum, and the medium was used as the conditioned medium.

[0061] To assess senescence, foreskin fibroblasts were exposed to an accelerated senescence protocol induced by exposure to the indicated concentration of H 2 O 2 for 48 hours. Cells were cultured in control medium (RPMI) (left bar of the three-bar group), 5% conditioned medium (middle bar of the three-bar group), or 10% conditioned medium (right bar of the three-bar group). Senescence was detected by fixing the cells with 4% paraformaldehyde and staining for SA-β-Gal using a senescent cells histochemical staining kit (Sigma Aldrich, St Louis, MO, USA). Three images were collected per well and SA-β-Gal stained cells were counted.

[0062] A decrease in senescence-associated β-galactosidase was observed in the conditioned medium from fibroblasts selected by CD73.

[0063] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the design defined by the appended claims. Further, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As will be readily understood by those skilled in the art, processes, machines, manufactures, compositions of matter, means, methods, or steps that presently exist or will later be developed and that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, means, methods, or steps.

[0064] [References] All patents and publications referred to herein are indicative of the level of skill of those in the art to which the present invention pertains. All patents and publications in this specification are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0065] 1.Conboy, I.M., et al., Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature, 2005. 433(7027): p. 760-4. 2.Katsimpardi, L., et al., Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science, 2014. 344(6184): p. 630-4. 3. Zhang, X.Y., et al., Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells. Mol Ther, 2002. 5(5 Pt 1): p. 555-65. 4. Kyriakou, C.A., et al., Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model. J Gene Med, 2006. 8(3): p. 253-64. 5. Worsham, D.N., et al., In vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector. Mol Ther, 2006. 14(4): p. 514-24. 6. Rabin, N., et al., A new xenograft model of myeloma bone disease demonstrating the efficacy of human mesenchymal stem cells expressing osteoprotegerin by lentiviral gene transfer. Leukemia, 2007. 21(10): p. 2181-91. 7. Kallifatidis, G., et al., Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer. Cancer Gene Ther, 2008. 15(4): p. 231-40. 8. Meyerrose, T.E., et al., Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease. Stem Cells, 2008. 26(7): p. 1713-22. 9. McGinley, L., et al., Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia. Stem Cell Res Ther, 2011. 2(2): p. 12. 10. Liang, X., et al., Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro. Int J Radiat Biol, 2010. 86(3): p. 230-7. 11. Glavaski-Joksimovic, A., et al., Glial cell line-derived neurotrophic factor-secreting genetically modified human bone marrow-derived mesenchymal stem cells promote recovery in a rat model of Parkinson's disease. J Neurosci Res, 2010. 88(12): p. 2669-81. 12. Liu, A.M., et al., Umbilical cord-derived mesenchymal stem cells with forced expression of hepatocyte growth factor enhance remyelination and functional recovery in a rat intracerebral hemorrhage model. Neurosurgery, 2010. 67(2): p. 357-65; discussion 365-6. 13. Yu, Y.S., et al., AKT-modified autologous intracoronary mesenchymal stem cells prevent remodeling and repair in swine infarcted myocardium. Chin Med J (Engl), 2010. 123(13): p. 1702-8. 14. Mueller, L.P., et al., TRAIL-transduced multipotent mesenchymal stromal cells (TRAIL-MSC) overcome TRAIL resistance in selected CRC cell lines in vitro and in vivo. Cancer Gene Ther, 2011. 18(4): p. 229-39. 15. Yan, C., et al., Suppression of orthotopically implanted hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with sTRAIL gene expression driven by AFP promoter. Biomaterials, 2014. 35(9): p. 3035-43. 16. Deng, Q., et al., TRAIL-secreting mesenchymal stem cells promote apoptosis in heat-shock-treated liver cancer cells and inhibit tumor growth in nude mice. Gene Ther, 2014. 21(3): p. 317-27. 17. Sage, E.K., et al., Systemic but not topical TRAIL-expressing mesenchymal stem cells reduce tumour growth in malignant mesothelioma. Thorax, 2014. 69(7): p. 638-47. 18. Lian, W.S., et al., In vivo therapy of myocardial infarction with mesenchymal stem cells modified with prostaglandin I synthase gene improves cardiac performance in mice. Life Sci, 2011. 88(9-10): p. 455-64. 19. Maijenburg, M.W., et al., Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration. Stem Cells Dev, 2012. 21(2): p. 228-38. 20. Harper, M.M., et al., Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes. Invest Ophthalmol Vis Sci, 2011. 52(7): p. 4506-15. 21. Zou, D., et al., In vitro study of enhanced osteogenesis induced by HIF-1alpha-transduced bone marrow stem cells. Cell Prolif, 2011. 44(3): p. 234-43. 22. Saito, S., et al., Mesenchymal stem cells stably transduced with a dominant-negative inhibitor of CCL2 greatly attenuate bleomycin-induced lung damage. Am J Pathol, 2011. 179(3): p. 1088-94. 23. Seo, K.W., et al., Anti-tumor effects of canine adipose tissue-derived mesenchymal stromal cell-based interferon-beta gene therapy and cisplatin in a mouse melanoma model. Cytotherapy, 2011. 13(8): p. 944-55. 24. Yang, H.M., et al., Enhancement of the immunosuppressive effect of human adipose tissue-derived mesenchymal stromal cells through HLA-G1 expression. Cytotherapy, 2012. 14(1): p. 70-9. 25. Liang, X.J., et al., Differentiation of human umbilical cord mesenchymal stem cells into hepatocyte-like cells by hTERT gene transfection in vitro. Cell Biol Int, 2012. 36(2): p. 215-21. 26. Fei, S., et al., The antitumor effect of mesenchymal stem cells transduced with a lentiviral vector expressing cytosine deaminase in a rat glioma model. J Cancer Res Clin Oncol, 2012. 138(2): p. 347-57. 27. Jaganathan, B.G. and D. Bonnet, Human mesenchymal stromal cells senesce with exogenous OCT4. Cytotherapy, 2012. 14(9): p. 1054-63. 28. Han, S.H., et al., Effect of ectopic OCT4 expression on canine adipose tissue-derived mesenchymal stem cell proliferation. Cell Biol Int, 2014. 38(10): p. 1163-73. 29. Shangguan, L., et al., Inhibition of TGF-beta / Smad signaling by BAMBI blocks differentiation of human mesenchymal stem cells to carcinoma-associated fibroblasts and abolishes their protumor effects. Stem Cells, 2012. 30(12): p. 2810-9. 30. Kearns-Jonker, M., et al., Genetically Engineered Mesenchymal Stem Cells Influence Gene Expression in Donor Cardiomyocytes and the Recipient Heart. J Stem Cell Res Ther, 2012. S1. 31. Ma, G.L., et al., [Study of inhibiting and killing effects of transgenic LIGHT human umbilical cord blood mesenchymal stem cells on stomach cancer]. Zhonghua Wei Chang Wai Ke Za Zhi, 2012. 15(11): p. 1178-81. 32. Huang, F., et al., Mesenchymal stem cells modified with miR-126 release angiogenic factors and activate Notch ligand Delta-like-4, enhancing ischemic angiogenesis and cell survival. Int J Mol Med, 2013. 31(2): p. 484-92. 33. Huang, F., et al., Overexpression of miR-126 promotes the differentiation of mesenchymal stem cells toward endothelial cells via activation of PI3K / Akt and MAPK / ERK pathways and release of paracrine factors. Biol Chem, 2013. 394(9): p. 1223-33. 34. Fang, Z., et al., Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro. Biochem Biophys Res Commun, 2013. 432(3): p. 444-50. 35. Madonna, R., et al., Transplantation of mesenchymal cells rejuvenated by the overexpression of telomerase and myocardin promotes revascularization and tissue repair in a murine model of hindlimb ischemia. Circ Res, 2013. 113(7): p. 902-14. 36. Zang, Y., et al., [Influence of CXCR4 overexpressed mesenchymal stem cells on hematopoietic recovery of irradiated mice]. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2013. 21(5): p. 1261-5. 37. Cao, Z., et al., Protective effects of mesenchymal stem cells with CXCR4 up-regulation in a rat renal transplantation model. PLoS One, 2013. 8(12): p. e82949. 38. Liu, S., et al., Overexpression of Wnt11 promotes chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in synergism with TGF-beta. Mol Cell Biochem, 2014. 390(1-2): p. 123-31. 39. Zhu, Y., et al., Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts. Drug Des Devel Ther, 2014. 8: p. 2449-62. 40. Yu, B., et al., Enhanced mesenchymal stem cell survival induced by GATA-4 overexpression is partially mediated by regulation of the miR-15 family. Int J Biochem Cell Biol, 2013. 45(12): p. 2724-35. 41. Xu, W., et al., Basic fibroblast growth factor expression is implicated in mesenchymal stem cells response to light-induced retinal injury. Cell Mol Neurobiol, 2013. 33(8): p. 1171-9. 42. Li, W., et al., Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells, 2007. 25(8): p. 2118-27. 43. Tsubokawa, T., et al., Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia. Am J Physiol Heart Circ Physiol, 2010. 298(5): p. H1320-9. 44. Zhou, H., et al., Exendin-4 protects adipose-derived mesenchymal stem cells from apoptosis induced by hydrogen peroxide through the PI3K / Akt-Sfrp2 pathways. Free Radic Biol Med, 2014. 77: p. 363-75. 45. Le Blanc, K., et al., Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004. 363(9419): p. 1439-41. 46. Lazarus, H.M., et al., Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant, 2005. 11(5): p. 389-98. 47. Bernardo, M.E., et al., Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J Cell Physiol, 2007. 211(1): p. 121-30. 48. Reinisch, A., et al., Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application. Regen Med, 2007. 2(4): p. 371-82. 49. Capelli, C., et al., Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant, 2007. 40(8): p. 785-91. 50. Lataillade, J.J., et al., New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy. Regen Med, 2007. 2(5): p. 785-94. 51. Seshareddy, K., D. Troyer, and M.L. Weiss, Method to isolate mesenchymal-like cells from Wharton's Jelly of umbilical cord. Methods Cell Biol, 2008. 86: p. 101-19. 52. Sensebe, L., Clinical grade production of mesenchymal stem cells. Biomed Mater Eng, 2008. 18(1 Suppl): p. S3-10. 53. otiropoulou, P.A., S.A. Perez, and M. Papamichail, Clinical grade expansion of human bone marrow mesenchymal stem cells. Methods Mol Biol, 2007. 407: p. 245-63. 54. Shetty, P., et al., Clinical grade mesenchymal stem cells transdifferentiated under xenofree conditions alleviates motor deficiencies in a rat model of Parkinson's disease. Cell Biol Int, 2009. 33(8): p. 830-8. 55. Zhang, X., et al., Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases. Int J Lab Hematol, 2010. 32(2): p. 256-64. 56. Arrigoni, E., et al., Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models. Cell Tissue Res, 2009. 338(3): p. 401-11. 57. Grisendi, G., et al., GMP-manufactured density gradient media for optimized mesenchymal stromal / stem cell isolation and expansion. Cytotherapy, 2010. 12(4): p. 466-77. 58. Prasad, V.K., et al., Efficacy and safety of ex vivo cultured adult human mesenchymal stem cells (Prochymal) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study. Biol Blood Marrow Transplant, 2011. 17(4): p. 534-41. 59. Sensebe, L., P. Bourin, and K. Tarte, Good manufacturing practices production of mesenchymal stem / stromal cells. Hum Gene Ther, 2011. 22(1): p. 19 - 26. 60. Capelli, C., et al., Minimally manipulated whole human umbilical cord is a rich source of clinical - grade human mesenchymal stromal cells expanded in human platelet lysate. Cytotherapy, 2011. 13(7): p. 786 - 801. 61. Ilic, N., et al., Manufacture of clinical grade human placenta - derived multipotent mesenchymal stromal cells. Methods Mol Biol, 2011. 698: p. 89 - 106. 62. Santos, F., et al., Toward a clinical - grade expansion of mesenchymal stem cells from human sources: a microcarrier - based culture system under xeno - free conditions. Tissue Eng Part C Methods, 2011. 17(12): p. 1201 - 10. 63. Timmins, N.E., et al., Closed system isolation and scalable expansion of human placental mesenchymal stem cells. Biotechnol Bioeng, 2012. 109(7): p. 1817 - 26. 64. Warnke, P.H., et al., A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies. J Craniomaxillofac Surg, 2013. 41(2): p. 153-61. 65. Fekete, N., et al., GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC. PLoS One, 2012. 7(8): p. e43255. 66. Hanley, P.J., et al., Manufacturing mesenchymal stromal cells for phase I clinical trials. Cytotherapy, 2013. 15(4): p. 416-22. 67. Trojahn Kolle, S.F., et al., Pooled human platelet lysate versus fetal bovine serum-investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use. Cytotherapy, 2013. 15(9): p. 1086-97. 68.Veronesi, E., et al., Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal / stem cells for bone regeneration. Tissue Eng Part C Methods, 2014. 20(3): p. 239-51. 69.Dolley-Sonneville, P.J., L.E. Romeo, and Z.K. Melkoumian, Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions. PLoS One, 2013. 8(8): p. e70263. 70.Siciliano, C., et al., Optimization of the isolation and expansion method of human mediastinal-adipose tissue derived mesenchymal stem cells with virally inactivated GMP-grade platelet lysate. Cytotechnology, 2015. 67(1): p. 165-74. 71.Martins, J.P., et al., Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data. Stem Cell Res Ther, 2014. 5(1): p. 9. 72. Iudicone, P., et al., Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells. J Transl Med, 2014. 12: p. 28. 73. Skrahin, A., et al., Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial. Lancet Respir Med, 2014. 2(2): p. 108-22. 74. Ikebe, C. and K. Suzuki, Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols. Biomed Res Int, 2014. 2014: p. 951512. 75. Chatzistamatiou, T.K., et al., Optimizing isolation culture and freezing methods to preserve Wharton's jelly's mesenchymal stem cell (MSC) properties: an MSC banking protocol validation for the Hellenic Cord Blood Bank. Transfusion, 2014. 54(12): p. 3108-20. 76. Swamynathan, P., et al., Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton's jelly derived mesenchymal stem cells? A comparative study. Stem Cell Res Ther, 2014. 5(4): p. 88. 77. Vaes, B., et al., Culturing protocols for human multipotent adult stem cells. Methods Mol Biol, 2015. 1235: p. 49-58. 78. Devito, L., et al., Wharton's jelly mesenchymal stromal / stem cells derived under chemically defined animal product-free low oxygen conditions are rich in MSCA-1(+) subpopulation. Regen Med, 2014. 9(6): p. 723-32.

Claims

1. 1. A system for preventing or reversing aging in a subject, comprising: (a) at least one bioreactor containing cells; (b) at least one selectively permeable membrane; and (c) tubing connecting at least one of the bioreactors, at least one of the selectively permeable membranes, and the subject, for circulating fluid from the subject to the bioreactors (provided that if the tubing includes hollow fibers, the membrane is optional); wherein the cell is a fibroblast and the fibroblast expresses CD73.

2. The system of claim 1 , further comprising blood or plasma from the subject, or comprising an organ preservation fluid.

3. The system of claim 1 , wherein the cells secrete one or more regenerative factors at a basal or inducible rate.

4. The system of claim 3, wherein the regenerative factor is selected from the group consisting of AKT, BAMBI, BCL-2, BCL-2XL, BDNF, BIRC5 CDA, CXCR4, dominant negative CCL2, EGF, exosomes, FGF-2, GATA-4 GDF-11, GDNF, hCG, HGF, HIF-1α, HLA-G, HO-1, hTERT, IFN-b, IGF-1, IFT-1, LIGHT, miR-126, NK4, NUR77, OCT-4, PGE-1, SDF-1, STC-1, TERT, TRAIL, VEGF, WNT11, XIAP, and combinations thereof.

5. The system of any one of claims 1 to 4, wherein the cells produce one or more of the regenerative factors in response to a degenerative factor from the blood or plasma of the subject.

6. The system according to any one of claims 1 to 5, wherein the cells secrete the regenerative factor at a rate determined by the detection of a degenerative factor.

7. The system of claim 6, wherein the ratio of the secretion rate of the regeneration factor to the detection rate of the denaturation factor is selected from the group consisting of 50:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:50, and any ratio therebetween.

8. The method of claim 1, wherein at least one of the selectively permeable membranes is disposed within the tubing between the bioreactor and the subject. the tubing is constructed from a selectively permeable membrane; and / or The bioreactor is composed, at least in part, of a selectively permeable membrane. A system according to any one of claims 1 to 7.

9. The system described in claim 8, wherein at least one of the selectively permeable membranes inhibits or reduces the passage of cellular material between the subject and the bioreactor.

10. The system of claim 1, wherein at least one of the selectively permeable membranes allows the passage of regenerative and denaturing factors.

11. The system according to any one of claims 1 to 10, wherein the subject is an animal.

12. The system according to any one of claims 1 to 10, wherein the target is an organ or tissue derived from an animal.

13. The system according to any one of claims 1 to 12, wherein the subject is an organ or tissue derived from a donor.

14. 14. The system of claim 12 or 13, wherein the organ is selected from the group consisting of liver, pancreas, gallbladder, stomach, small intestine, large intestine, lung, kidney, heart, spleen, brain, eyeball, and combinations thereof.

15. The system according to any one of claims 12 to 14, wherein the object is any part of an organ or any part of an organ system.

16. The object is an organ or tissue, the system further comprising a vessel connected to at least one of the bioreactors and to at least one selectively permeable membrane for containing the subject; A system according to any one of claims 1 to 10 or 12 to 15.

17. The object is an organ or tissue, the container is adapted to hold the object so as to allow transfer of organ preservation fluid or other fluid to the tubing; 17. The system of claim 16.

18. 1. An ex vivo method for producing a regenerative factor, comprising:

20. A method comprising the step of subjecting the subject to a system according to any one of claims 1 to 17 under conditions that permit secretion of one or more regenerative factors from cells in the system, unless the subject is a human or is taken from a human and returned to the human from whom it was taken for therapeutic purposes.

19. 20. The method of claim 18, wherein the fibroblasts are derived from tissues including skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue, placenta, and / or foreskin.

20. 20. The method of claim 19, wherein the fibroblasts have one or more surface markers selected from the group consisting of CD73, CD90, CD56, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, CD49b, CD105, aminopeptidase N, hyaluronan binding receptor, collagen / laminin binding integrin alpha 2, OCT4, NANOG, SOX-2, and combinations thereof.

21. The method of any one of claims 18 to 20, wherein the fibroblasts lack one or more surface markers selected from the group consisting of CD14, CD34, CD45, HLA class II, and combinations thereof.

22. (a) culturing fibroblasts in an undifferentiated state for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days; (b) culturing the fibroblasts from step (a) in the presence of one or more factors selected from the group consisting of nerve growth factor, bFGF, dibutryl cAMP, IBMX, retinoic acid, exendin-4, and combinations thereof; and (c) activating the fibroblasts from step (b); The method according to any one of claims 19 to 21, comprising:

23. The method of claim 22, wherein step (c) of activating the fibroblasts comprises exposing the fibroblasts from step (b) to one or more cytokines in cell culture medium.

24. 24. The method of claim 23, wherein the cytokine is selected from the group consisting of IL-1, IFNγ, and combinations thereof.

25. 25. The method of claim 24, wherein the concentration of IL-1 is 1 to 100 ng / mL, 5 to 100 ng / mL, 10 to 100 ng / mL, or 20 to 40 ng / mL.

26. The method of claim 24 or 25, wherein the concentration of IL-1 is 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL.

27. 25. The method of claim 24, wherein the concentration of IFNγ is 1 to 1000 IU, 5 to 1000 IU, 10 to 1000 IU, 1 to 500 IU, 5 to 500 IU, 10 to 500 IU, 100 to 500 IU, or 250 IU.

28. 28. The method of claim 24 or 27, wherein the concentration of IFNγ is 1 IU, 5 IU, 10 IU, 50 IU, 100 IU, 200 IU, 250 IU, 300 IU, 400 IU, 500 IU, 600 IU, 700 IU, 800 IU, 900 IU, or 1000 IU.

29. 29. The method of any one of claims 23 to 28, wherein exposing the fibroblasts to one or more cytokines induces increased expression of one or more complement inhibitory molecules from the fibroblasts.

30. 30. The method of claim 29, wherein the complement inhibitory molecule is selected from the group consisting of CD35, CD46, C4BP, CD55, Factor H, and combinations thereof.

31. The method of claim 22, wherein activating the fibroblasts in step (c) comprises transfecting the fibroblasts from step (b) with one or more viral and / or non-viral expression systems to induce expression of one or more regenerative factors.

32. 32. The method of claim 31, wherein the regenerative factor is selected from the group consisting of AKT, BAMBI, BCL-2, BCL-2XL, BDN, BIRC5 CDA, CXCR4, dominant negative CCL2, EGF, exosomes, FGF-2, GATA-4 GDF-11, GDNF, hCG, HGF, HIF-1α, HLA-G, HO-1, hTERT, IFN-b, IFT-1, LIGHT, miR-126, NK4, NUR77, OCT-4, PGE-1, SDF-1, STC-1, TERT, TRAIL, VEGF, WNT11, XIAP, and combinations thereof.

33. A kit comprising the system according to any one of claims 1 to 17.

34. A system described in any one of claims 1 to 17 for treating an aging-related disease.

35. The system described in claim 34, wherein the aging-related disease is selected from the group consisting of neurodegenerative diseases, arteriosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease.

Images