Therapeutic use of fibroblast modulation of microglia

Abstract

Aspects of the disclosure include methods and compositions for medical conditions wherein an excess of microglia activity is directly or indirectly related. In specific aspects, the disclosure includes method of modulating activity of microglia cells by providing a therapeutically effective amount of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or other fibroblast-derived materials for a subject who has, or is at a risk for having, a medical condition associated with the central nervous system.

Description

THERAPEUTIC USE OF FIBROBLAST MODULATION OF MICROGLIACROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 512,930, filed July 11, 2023, which is incorporated by reference herein in its entirety.BACKGROUNDI. Technical Field

[0002] This disclosure relates at least the fields of cell biology, molecular biology, immunology, and medicine.II. Discussion of related art

[0003] Fibroblasts are no longer considered as mere structural components of organs but as dynamic participants in immune processes. Fibroblasts produce an environment that influences T regulatory cell migration, proliferation, ensure immunotolerance, and immune modulation through cytokine, chemokine, and growth factor excretion in multiple organs and tissues including the resident immune microglia cells of the brainfl].

[0004] Neuroinflammation, characterized by the activation of microglial cells, has been implicated in various neurological disorders, neurodegenerative diseases, traumatic brain injury, bacterial, viral, and fungal meningitis, and stroke[2]. As the only resident immune cells in the brain, they act as the first line of defense by phagocytosing harmful pathogens and cellular debris. Microglia, the resident immune cells of the central nervous system, play a crucial role in maintaining brain homeostasis. After central nervous system injury, microglia change their morphology and down-regulate genes which aid in homeostasis function and produce pro-inflammatory cytokines such as IL-1, IL-6, and TNF-a, chemokines from the CXCL and CCL family, and growth factors such as brain-derived neurotropic factor (BDNF)[3-8], Microglia operate as safeguards of the CNS, scanning the environment for danger cues and / or invading pathogens. Regularly distributed throughout the CNS, like watchmen, they undergo activation by local danger cues. Microglia actively adapt cell morphology in response to these signals, by increasing soma size and retracting their thin cytoplasmic processes[9],

[0005] Activation of microglia is protective for the brain. However, excessive, or chronic microglial activation contributes to neuronal damage and exacerbates neuroinflammatory processes. Thus, there is a need for innovative therapeutic strategies that can modulate microglia activity to mitigate the detrimental effects of neuroinflammationflO],

[0006] Using single cell fibroblasts, fibroblast spheroids, exosomes, or fibroblast-derived products which are natural immune modulators, one can achieve the same or better results in modulating microglia activity in the CNS, by controlling the expansion of the pathogenic immune cells, and their expression of pathogenic levels of cytokines, chemokines, and growth factors, without the adverse side effected noted in small molecule and antibody based immune modulators[ 11-13],SUMMARY OF THE INVENTION

[0007] Aspects of the present disclosure concern methods and compositions related to the treatment of medical conditions associated with the central nervous system (CNS). Aspects of the present disclosure concern methods and compositions related to the treatment of medical conditions associated with the brain. In specific aspects, the methods and compositions modulate activity and / or numbers of microglia cells, e.g., reduce activity (at least in part) and / or reduce numbers of microglia cells in the CNS. In specific aspects, the present disclosure concerns methods and compositions related to inflammation related to the expansion and / or activation of microglia to a level that that is deleterious to the subject, such as pathogenic levels. In particular aspects, there are methods and compositions that modulate microglial activity and / or numbers and, in certain aspects, subsequent attenuation of neuroinflammation.

[0008] In an aspect, the current disclosure encompasses a method of modulating activity of microglia cells in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of fibroblasts, one or more derivatives thereof, or combinations thereof, wherein the subject has, or is at risk for having, a medical condition associated with the central nervous system (CNS). The one or more derivatives of fibroblasts may comprise fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, other fibroblast-derived materials such as metabolites, or any combination thereof. In an aspect, the disclosed fibroblasts, or derivatives thereof may be administrated by intravenous injection or intrathecal injection. In an aspect, the medical condition of the CNS may be a neurological disorder, neurodegenerative disease, traumatic brain injury, bacterial meningitis, viral meningitis, fungal meningitis, or stroke.

[0009] In an aspect, the fibroblasts may be autologous, allogeneic, xenogeneic, or syngeneic with respect to the subject. The fibroblast may be genetically engineered to target one or more genes or gene products, to attenuate their immune modulation potential. Nonlimiting examples of the one or more genes or gene products that impact immune modulation potential include IL1RA, IL-4, IL-6, IL-10, IL-11, IL-13, TGFb, soluble TNF receptor p55, soluble TNF receptor p75, Soluble IL-1 receptor type 2, membrane-bound IL-1 receptor type 2, IL- 18 binding protein, prostaglandin E2, PD1, CTLA4, TIM3, LAG3, TIGIT, CD96, BTLA, VISTA, Arginase, prostaglandin E2, indoleamin 2,3 -dioxygenase, nitric oxide synthase. The fibroblasts may be chemically, physically or epigenetically activated. In particular aspects, the fibroblasts used in the composition and / or the fibroblasts from which the spheroids, exosomes, apoptotic bodies, conditioned media, and other derived materials were obtained, may be modified or unmodified. In specific aspects, the cells are modified genetically, such as with CRISPR / CAS9, novel Cas Nucleases (NCN), CasMINI, Cas-CLOVER, Prokaryotic Argonautes (NgAgo), Zinc Finger Nucleases (ZFNs), and or Transcription activator-Like Effector Nucleases (TALENs) systems. Such methods can be used to add, delete, and / or modify fibroblast cell surface markers, e.g., to attenuate their immune modulation potential. The fibroblasts may be activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof, prior to administration. The fibroblasts may be cultured fibroblasts. The fibroblasts may be cultured in a medium free of fetal bovine serum. The medium may optionally comprises one or more of human platelet rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, one or more cytokines, fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin like growth factor, angiopoietin, vascular endothelial growth factor, or any combination thereof.

[0010] The fibroblasts may be derived from skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, progenitor cells, placental fibroblasts, omental tissue derived fibroblasts, cord blood fibroblasts, or any combination thereof.

[0011] In an aspect, the current disclosure also encompasses a method of treating a medical condition associated with the central nervous system (CNS) of a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of fibroblasts, or derivatives thereof, for example fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic cell bodies, conditioned media from fibroblasts, fibroblast-derived materials such as metabolites, or any combination thereof, optionally wherein the fibroblastsare activated with one or more agents; and (2) one or more types of non-pathogenic immune cells and / or one or more derivative agents thereof.

[0012] Also disclosed herein is a method of treating a medical condition associated with the CNS of a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of (1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic cell bodies, conditioned media from fibroblasts, fibroblast-derived materials such as metabolites, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents; and (2) one or more type of immune cells and / or one or more derivative agents thereof.

[0013] In an aspect, the administering is into the blood stream or intrathecally. In an aspect, administering of the fibroblasts results in a decrease in the expression of pathogenic levels of IL-23, IL-1, IL- 10, IL-6, IL-22, IL- 17, IL- 12, TNF, INF- , TGF-01, or a combination thereof from the microglia, T cells, dendritic cells, macrophages, or a combination thereof in the subject.

[0014] Also disclosed herein is a method of epigenetic reprogramming of localized microglia, or oligodendrocyte, keratinocyte cells in a subject with a medical condition associated with the brain or CNS, comprising the step of administering an effective amount of: (1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, fibroblast-derived materials such as metabolites, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or conditions, and (2) one or more type of immune cells and / or one or more derivative agents thereof. In an aspect, the administering steps is into the blood stream of the subject or intrathecally to the subject.

[0015] In an aspect, disclosed herein is a method of enabling the activation and migration of localized stem cell niches to replace damaged oligodendrocytes and / or damaged myelin sheaths in a subject in need thereof, comprising the step of administering to the subject an effective amount of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, fibroblast-derived materials or products, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or one or more conditions; and optionally one or more type of immune cells and / or one or more derivative agents thereof. The administering may be in the blood stream of the subject or intrathecally. In fibroblasts, derivatives thereof, and / or the immune cells may be administered with one or more adjuvants, non-limiting examples include chemical-based, viral-based, or bacterial product-based.

[0016] Disclosed herein is a composition comprising fibroblasts, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, fibroblast-derived materials or products, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or one or more conditions, for use in the treatment of medical condition associated with the CNS. The composition may further comprise one or more type of immune cells and / or one or more derivative agents thereof. The composition may be formulated for intravenous or intrathecal administration.

[0017] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific aspect disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

[0018] It is contemplated that any aspect encompassed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0019] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the subject matter of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0021] FIG. 1 EAE severity of the treated animals over time with administration of fibroblasts, compared to adipose-derived MSCs followed over a period of 60 days. This demonstrates that treatment with fibroblasts significantly reduced neuroinflammation and neuronal damage as compared to control and MSC treated animals.

[0022] FIG. 2. Immunohistochemistry (IHC) Fluorescent staining of treated and controls animal cerebral cortex with antibodies specific to myelin and neuron fibers. This demonstrates that the animals treated with fibroblasts contained significantly less myelin and neuron fiber degradation.

[0023] FIG. 3. Quantitative measurement of myelin in the cerebral cortex of control and treated animals. This data demonstrates that fibroblast treated animals had significantly higher density of myelin.

[0024] FIG. 4. Immunohistochemistry (IHC) Fluorescent staining of treated and controls animal Spinal cord with antibodies specific for microglia (CD68 and IBA-1). This data clearly indicates that while the control animals and adipose derived MSC treated animals had regions of hyperactive microglia activity, the fibroblast treated animals did not show any overactive microglia.

[0025] FIG. 5. Immunohistochemistry (IHC) Fluorescent staining of treated and controls animal cerebral cortex with antibodies specific for microglia (CD68 and IBA-1). This data clearly indicates that while the control animals and adipose derived MSC treated animals had regions of hyperactive microglia activity, the fibroblast treated animals did not show any overactive microglia.

[0026] FIG. 6 In vivo assay for IL-10 which is a biomarker for T-regulatory cells (T-reg). This data demonstrates that fibroblasts significantly enhance stimulation of T-reg cells as compared to the control animals and bone marrow-derived mesenchymal stem cells (BM- MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs).

[0027] FIG. 7. In vivo assay for Th-17 cells expressing and secreting IL-17 inflammatory cytokine. The data demonstrates that as compared to control and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells(Adipose-MSCs), fibroblast treated animals have significantly suppressed levels of IL- 17 inflammatory cytokine.

[0028] FIGS. 8A-8C. Assays for key biomarkers responsible for inflammation. This data demonstrates that as compared to control and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs), fibroblast treated animals have significantly downregulated inflammatory biomarkers CD-40 (FIG. 8A), CD-80 (FIG. 8B), and CD-86 (FIG. 8C).

[0029] FIGS. 9A-9C. Assays for key biomarkers of anti-inflammatory cytokines IL- 10, IL- IRA and programmed cell death ligand (PDL). This data demonstrates that as compared to control and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs), fibroblast treated animals have significantly upregulated anti-inflammatory cytokines IL- 10 (FIG. 9A), IL- IRA (FIG. 9B), and programmed cell death ligand (PDL) (FIG. 9C).DETAILED DESCRIPTIONI. Examples of Definitions

[0030] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some aspects of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0031] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0032] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular aspects, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value.

[0033] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0034] Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” or “a further aspect” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0035] As used herein, the term “activated immune cells” refers to immune cells treated with one or more stimuli capable of inducing one or more alterations in the cell: metabolic, immunological, epigenetic, growth factor secreting, surface marker expression, and production and excretion of microvesicles.

[0036] The term "administered" or "administering", as used herein, refers to any method of providing a composition to a subject such that the composition has its intended effect on the subject. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, applicator gun, syringe etc. A second exemplary method of administering is by a direct mechanism such as, local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository etc.

[0037] As used herein, “allogeneic” refers to tissues or cells from another body that in a natural setting are immunologically incompatible or capable of being immunologically incompatible, although from one or more subjects of the same species.

[0038] As used herein, “autologous” refers to tissues or cells that are derived or transferred from the same subject's body (z.e., autologous blood donation; an autologous bone marrow transplant).

[0039] As used herein, “agent” refers to nucleic acids, cytokines, chemokines, transcription factors, epigenetics factors, growth factors, or hormones.

[0040] As used herein, “xenogeneic” refers to tissues or cells from a species different from the patient.

[0041] Cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent or (actively) dividing. In a cell culture, cells are grown and maintained at an appropriate temperature, typically a temperature of 37°C and under an atmosphere typically containing oxygen and CO2. Culture conditions may vary widely for each cell type though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly varied factor in culture systems is the growth medium. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0042] The term "subject", or “individual” as used herein, refers to a human or animal that may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The subject may be receiving one or more medical compositions via the internet. A subject may comprise any age of a human or non-human animal and therefore includes both adults and juveniles (z.e., children) and infants. It is not intended that the term "subject" connotes a need for medical treatment, therefore, a subject may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. The term “subject” or “subject” may be used interchangeably and refers to any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0043] The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent" and grammatical equivalents (including "lower," "smaller," etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one aspect, the quantity and / or magnitude of the symptoms in the treated subjectis at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject.

[0044] Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pre-treatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered to be a treatment if there is a detectable reduction in the immunogenicity of cells when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition. In specific aspects, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.II. Overview

[0045] The current disclosure is based in part on the unexpected discovery that compositions comprising fibroblasts, derivatives thereof, or equivalent thereof, for example fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites, are effective in alleviating, treating or reducing symptoms of autoimmune encephalomyelitis (EAE) in Lewis rats. Further analysis revealed that treated rats showed significantly lower neuroinflammation and neuronal damage, less myelin and neuron fiber damage, reduced microglial hyperactivity, greater stimulation of non-pathogenic immune cells, and significantly suppressed levels of inflammatory biomarkers. The disclosed compositions can be administered intravenously and can effectively overcome the blood brain barrier, or intrathecally. The disclosed compositions can be used, not only for EAE but also for other microglia associated diseases of the central nervous system (CNS).

[0046] Aspects of the present disclosure concern methods and compositions related to the treatment of medical conditions associated with the CNS. Aspects of the present disclosure concern methods and compositions related to the treatment of medical conditions associated with the brain. In specific aspects, the methods and compositions modulate activity and / or numbers of microglia cells, e.g., reduce activity (at least in part) and / or reduce numbers of microglia cells in the CNS. In specific aspects, the present disclosure concerns methods and compositions related to inflammation related to the expansion and / or activation of microglia to a level that that is deleterious to the subject, such as pathogenic levels. In particular aspects, there are methods and compositions that modulate microglial activity and / or numbers and, in certain aspects, subsequent attenuation of neuroinflammation.

[0047] The present disclosure encompasses methods and compositions for directly and / or indirectly modulating microglia activity and / or numbers in the brain. In specific aspects, a subject in need thereof is a subject that has or is at risk for having a neurological disorder, neurodegenerative disease, traumatic brain injury, bacterial meningitis, viral meningitis, fungal meningitis, or stroke. Aspects of the disclosure include providing a therapeutically effective amount of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites. In particular aspects, any of these may be administered intravenously or intrathecally. In particular cases, administration allows the fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites, to be effective inside the blood-brain barrier. Fibroblasts may be isolated and characterized to maximize their immune modulation potential. In specific aspects, one may directly or indirectly provide to a subject a therapeutically effective amount of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites.

[0048] In particular aspects, the fibroblasts used in the composition and / or the fibroblasts from which the spheroids, exosomes, apoptotic bodies, conditioned media, and other derived materials were obtained, may be modified or unmodified. In specific aspects, the cells are modified genetically, such as with CRISPR / CAS9, novel Cas Nucleases (NCN), CasMINI, Cas-CLOVER, Prokaryotic Argonautes (NgAgo), Zinc Finger Nucleases (ZFNs), and or Transcription activator-Like Effector Nucleases (TALENs) systems. Such methods can be used to add, delete, and / or modify fibroblast cell surface markers, e.g., to attenuate their immune modulation potential.

[0049] In particular aspects, the disclosure concerns interaction of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites, with a second type of cells (including immune cells) and / or certain agent(s) and includes modification(s) to the fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblast, and / or fibroblast-derived materials, such as metabolites and / or the second type of cells as a result of the interaction. In specific aspects, the disclosure includes compositions, methods, and systems in which fibroblasts are modified upon exposure to certain cells, including certain immune cells, and / or one or a combination of certain agent(s) including nucleic acids, cytokines, chemokines, growth factors, and / or metabolites. Introduction of these modified or unmodified fibroblasts (and spheroids, exosomes, apoptotic bodies, conditioned media, and other derived materials therefrom) intravenously or intrathecally can modulate and reprogram overactive microglia and other pro-inflammatory white blood cells (e.g., T cells, basophils, mast cells, neutrophils, macrophages, eosinophils, and combinations thereof), and oligodendrocytes, and initiate the foundation for remyelination. In particular aspects, the regeneration of these cells can be through one or more of the following steps, such as:(1) The differentiation of the modified or unmodified single cell fibroblasts, fibroblast spheroids, exosomes, or fibroblast-derived materials such as metabolites, and such as when provided to the subject intravenously or intrathecally.(2) Epigenetic reprogramming of overactive microglia and / or excess numbers of microglia, such as through direct contact with the modified or unmodified fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials, and / or through indirect contact with one or more secreted cytokines, one or more secreted chemokines, one or more secreted growth factors, or one or more secreted metabolites, through modulation and / or return to homeostasis and / or desired activity.(3) Epigenetic reprogramming of oligodendrocytes, such as through direct contact with the administered modified or unmodified fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials, or indirect contact with one or more secreted cytokines, one or more secreted chemokines, one or more secreted growth factors, or one or more secreted metabolites for proliferation and / or regeneration of the myelin sheath.(4) Reprogramming or activating microglia by one or more products excreted from the modified or unmodified fibroblasts, fibroblast spheroids, fibroblast exosomes,fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast- derived materials.(5) Recruitment, activation, and differentiation of local stem cells into active microglia and or oligodendrocyte through the direct / indirection action of the injected modified / unmodified single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast- derived materials such as from the modified / unmodified fibroblasts.(6) Modulation of the immune system through fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials to control the expansion and / or localization of pathogenic immune cells of any kind, including their expression of pathogenic levels of one or more cytokines, pathogenic levels of one or more chemokines, and / or pathogenic levels of one or more growth factors, including those associated any pro- inflammatory response of microglia. These cells include microglia, T cells, dendritic cells, macrophages, B cells, and neutrophils, expressing pathogenic levels of one or more of IL-23, IL-1, IL-6, IL- 10, IL-22, IL- 17, IL- 12, TNF, INF-y, TGF- 1.

[0050] In some aspects, the regeneration of overactive microglia, other pro-inflammatory white blood cells, and / or oligodendrocytes may occur by alternative mechanisms other than these.

[0051] Levels of microglia, T cells, dendritic cells, B cells, macrophages, and / or neutrophils may be monitored by one or more of a variety of ways, including complete blood count, white blood Count, and / or tissue levels of microglia, T cells, dendritic cells, B cells, macrophages. The level of one or more of IL-23, IL-1, IL-6, IL- 10, IL-22, IL- 17, IL- 12, TNF, INF-y, and TGF-pi may be monitored using ELISA and / or bead-based immunoassays, as examples.

[0052] In specific aspects, the disclosure concerns compositions, methods, and systems in which certain immune cells, including microglia, are modified upon exposure to fibroblasts and / or certain agent(s) excreted from fibroblasts, listed elsewhere herein. In particular aspects, the interaction of fibroblasts and / or certain agent(s) excreted from fibroblasts with one or more other types of cells (and optionally that interaction also includes one or more certain agent(s)) results in modification of the fibroblasts and / or the other type of cells. In specific aspects, the other types of cells include at least immune cells, such as microglia.

[0053] In specific aspects, at least parts of methods of the disclosure occur ex vivo, such as in a culture, and this can also occur in vivo. In particular cases, the methods occur by the handof man and do not encompass ordinary or random occurrences in a body. The methods of the disclosure are non-natural, in particular aspects. In specific aspects, the concentrations of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials used in a method of exposing one type of cells or cell products to another type of cells (including microglia) does not occur in nature and does not happen randomly in nature. In specific aspects, the concentration of one or more fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials used in a method of exposing the fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials to one or more types of cells does not occur in nature and does not happen randomly in nature. The modification of any types of cells encompassed by the disclosure that occurs ex vivo or in vitro does not occur in vivo naturally in the same manner. In such aspect, tissue biopsy from a donor is used to isolate, characterize, if necessary, activate, expand, and reintroduce back into the donor one or a combination of the cells for the purpose of modulating the immune system and reprogramming resident microglia and / or oligodendrocytes.

[0054] The disclosure encompasses therapeutic uses of cells, including epithelial cells, keratinocytes, epithelial cells, dendritic cells, macrophages, B lymphocytes, myeloid cells, endothelial cells, fibroblasts, immune cells, and mixtures thereof. In at least some cases, the fibroblasts, fibroblast spheroids, or fibroblast-derived materials such as fibroblast exosomes, and fibroblast metabolites have been modified prior to their delivery in vivo to the immune cells (e.g., microglia) and other cells, including those found in the CNS. The modification may be chemically activated, physically activated, and / or epigenetically activated, and / or exposed to one or more conditions that are not normally found in the body, and in other cases immune cells, or their derivatives, have been modified, such as activated, prior to their exposure to the modified or unmodified single cell fibroblasts, or fibroblast spheroids.

[0055] Aspects of the disclosure provide means of utilizing fibroblasts as allogeneic, autologous, xenogeneic, or syngeneic therapeutic cells through modification of culture conditions. In one aspect of the disclosure, fibroblasts are extracted from sources with inherently lower immunogenicity (e.g. placental fibroblasts, omental tissue derived fibroblasts, cord blood derived fibroblasts, etc. .

[0056] In one aspect of the disclosure, single cell fibroblasts and / or fibroblast spheroids are cultured in vitro for preserving viability and proliferative ability of fibroblasts. The disclosure provides for the modification of known culture techniques to decrease recognition offibroblasts and their derived materials listed elsewhere herein by the recipient immune system. In one aspect, fibroblasts and / or fibroblast spheroids are cultured in conditions that lack xenogeneic components, such as xenogeneic-free medium; in some cases, for example, the media is free of fetal calf serum. In specific aspects, the disclosure encompasses the substitution of fetal calf serum with one or more other agents, such as those that facilitate reduction of immunogenicity of fibroblasts, for example, human platelet rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, and / or one or more defined cytokines, such as one or a combination of fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin like growth factor, angiopoietin, and vascular endothelial growth factor.

[0057] In one aspect of the disclosure, effective amounts of single cell fibroblasts, fibroblast spheroids, or fibroblast-derived materials as prepared in methods encompassed by the disclosure are administered to a subject for a therapy or prevention of one or more medical conditions. In specific aspects, the fibroblasts are administered to improve resident immune cell responsiveness, modulation of immune cell activity, and cell differentiation.

[0058] Aspects of the disclosure provide methods for co-administration of universal donor single cell fibroblasts, fibroblast spheroids, or fibroblast-derived materials with one or more agents that modulate the immune cell activity in the site of inflammation, such as in the CNS. In a specific aspect of the disclosure, methods are provided for co-administration of universal donor single cell fibroblasts, fibroblast spheroids, and / or fibroblast-derived materials, such as exosomes and / or metabolites with one or a combination of one or more growth factors, one or more chemokines, one or more cytokines, and / or one or more metabolites. In one aspect of the disclosure, universal donor fibroblasts derived from fibroblasts that have been treated under conditions to reduce immunogenicity are utilized to stimulate chemokine production and release leading to myelin regeneration and expansion of oligodendrocytes.

[0059] Aspects of the disclosure provide methods of reducing immunogenicity of particular types of fibroblasts. Single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, or fibroblast-derived materials may be derived from various tissues or organs, including, but not limited to skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin, which can be obtained by biopsy (where appropriate) or upon autopsy. In some aspects, the cells comprise fibroblasts, which can be from a fetal, neonatal, adult origin, or a combination thereof.

[0060] Single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials or products for use in any methods of the disclosure may be exposed to certain medium component s), in specific aspects.

[0061] Aspects of the disclosure include an in vivo method of modulating microglia activity through, e.g., intravenous or intrathecal injection of single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysate, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials, such as metabolites, to modulate the immune system and to reduce the uncontrolled activity of microglia-associated various neurological disorders, neurodegenerative diseases, traumatic brain injury, bacterial, viral, and fungal meningitis, and stroke, as examples.

[0062] Aspects of the disclosure include introduction into the blood stream or the spinal cord of single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysate, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast-derived materials such as metabolites, or products activated with one or more agents, or single cell fibroblasts, fibroblast spheroids, and / or fibroblast-derived materials, in addition to one or more type of immune cells and / or one or more derivative agents thereof, to modulate the expansion and proliferation of pathogenic immune cells, such as microglia.

[0063] Aspects of the disclosure include introduction into the blood stream or intrathecally (as examples) of single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysate, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast- derived materials such as metabolites, activated with one or more agents, including in some cases to one or more type of immune cells and / or one or more derivative agents thereof, to modulate the expression of pathogenic levels of one or more of IL-23, IL-1, IL-10, IL-6, IL- 22, IL-17, IL-12, TNF, INF- y, TGF- pi. In some aspects, the one or more of IL-23, IL-1, IL- 10, IL-6, IL-22, IL-17, IL-12, TNF, INF- y, TGF- pi are secreted by microglia, T cells, dendritic cells, B cells, macrophages, and / or neutrophils.

[0064] Aspects of the disclosure include introduction of single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, or fibroblast-derived materials, such as metabolites, or products activated with an agent, including in some cases with one or more type of immune cells and / or derivative agents thereof into the blood stream of a subject or intrathecally to the CNS to enable the epigenetic reprogramming of localized microglia, oligodendrocytes, and / or keratinocyte cells in the CNS.

[0065] Aspects of the disclosure include introduction into the blood stream or intrathecally to the CNS of single cell fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or fibroblast- derived materials or products, in some cases activated with one or more agents, in addition to one or more type of immune cells and / or one or more derivative agents thereof, to enable theactivation and / or migration of localized stem cell niches to replace the damaged oligodendrocytes and myelin sheath.

[0066] In method encompassed herein may include the use of one or more adjuvants in combination with single cell fibroblasts, fibroblast spheroids, or fibroblast-derived materials such as metabolites to activate the immune system. These adjuvants may be chemical, viral, or bacterial product-based.III. Administration of Therapeutic Compositions

[0067] Aspects of the disclosure encompass methods and compositions for treating a medical condition associated with the CNS. Aspects of the disclosure encompass methods and compositions for treating medical conditions in which excess in microglia activity and / or numbers is associated with the medical condition. In specific aspects, the medical condition is a neurological disorder, neurodegenerative disease, traumatic brain injury, bacterial meningitis, viral meningitis, fungal meningitis, or stroke, as examples.

[0068] Examples of neurological disorders (e.g., diseases of the brain, spine, and the nerves that connect them) include at least Alzheimer's Disease, Multiple Sclerosis, frontotemporal dementia, psychiatric disorders, Amyotrophic Lateral Sclerosis (ALS), Ataxia, Bell's Palsy, Brain Tumors, Cerebral Aneurysm, Encephalitis, Epilepsy, Seizures, Guillain-Barre Syndrome, Dementia, Migraine, or Parkinson's Disease. A subject with any neurological disorder may be subject to methods and compositions of the disclosure. A subject with symptoms such as headache, seizure, loss of feeling or tingling, weakness or loss of muscle strength, loss of sight or double vision, memory loss, impaired mental ability, lack of coordination, and / or stroke may be subject to methods and compositions of the disclosure. A subject who is at risk for a neurological disorder, such as having an associated genetic mutation and / or a family and / or personal history, may be subject to methods and compositions of the disorder.

[0069] Examples of neurodegenerative diseases (e.g., when cells of the central nervous system stop working or die) include at least Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, Spinal muscular atrophy. In particular aspects, the subject with the neurodegenerative disorder gets worse over time. The neurodegenerative disease may be caused by any reason, such as an associated genetic mutation, from a tumor, from a stroke, be an alcoholic, exposure to toxins (mercury, aluminum, copper, lead, manganese, P-N-methylamino-L-alanine), exposure tochemicals (pesticides, metal-based nanoparticles), exposure to certain viruses (Herpes simplex virus, varicella zoster virus, some strains of influenza virus, and viruses that cause encephalitis or meningitis), or have an unknown cause. A subject may have one or more of the following symptoms prior to onset of treatment with the methods and compositions of the disclosure: a loss of inhibition, anxiety, agitation, apathy, difficulty with movement, forgetfulness, memory loss, and mood changes. In particular aspects, the methods and compositions prevent the onset of one or more symptoms, delay the onset of one or more symptoms, or reduce the severity of one or more symptoms.

[0070] For some medical conditions, the condition may be considered a neurological disorder and a neurodegenerative disease.

[0071] Traumatic brain injury (TBI) including brain dysfunction may be caused by an outside force, such as a violent blow to the head. It may occur as a result of a severe sports injury, a car accident, etc. In some aspects, it is followed by immediate or delayed symptoms such as confusion, blurry vision, concentration difficulty, headache, convulsions or seizures, blurred or double vision, unequal eye pupil size or dilation, etc. The TBI may be a penetrating TBI or a non-penetrating TBI.

[0072] Administration of one or more therapeutic compositions of the disclosure include administration of a therapeutically effective amount of one or more of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or other fibroblast-derived materials, such as metabolites. Without being bound by theory, in specific aspects the therapeutic composition modulates the activity and / or numbers of microglia cells in the receiving subject. Without being bound by theory, in specific aspects the therapeutic composition decreases the activity and / or numbers of microglia cells in the receiving subject. Without being bound by theory, in specific aspects the therapeutic composition reduces inflammation of the CNS in the receiving subject. In particular aspects, the subject has a medical condition associated with the central nervous system (CNS) or is at risk thereof.

[0073] In an aspect, the administration of the one or more of fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and / or other fibroblast-derived materials, such as metabolites, may be coupled with administration of one or more non-pathogenic immune cells.

[0074] In particular cases, the subject being treated with methods and compositions of the disclosure also receives one or more other therapies. In specific cases, the one or more other therapies may or may not treat one or more symptoms of the medical condition rather than theunderlying cause. Examples include one or more of surgery; rehabilitation, including physical therapy, occupational therapy, and / or speech therapy; dopaminergic treatment; antiinflammatory; analgesic; cholinesterase; antipsychotic, etc. The one or more other therapies may be provided to the subject before, during, and / or after the methods and compositions of the disclosure. The one or more other therapies and the methods and compositions of the disclosure may or may not be administered via the same route. The one or more other therapies and the methods and compositions of the disclosure may or may not be in the same composition.

[0075] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapeutic composition(s) is administered intravenously, intrathecally, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the subject, the subject's clinical history and response to the treatment, and the discretion of the attending physician.

[0076] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single delivery but may comprise continuous delivery over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0077] In some aspects, the therapy is administered at a dose of between 100 cells and 10 million cells. In some aspects, the therapy is administered at a dose of at least, at most, or about 100-1 million, 100-100,000, 100-10,000, 100-1000, 1000-10 million, 1000-1 million, 1000- 100,000, 1000-10,000, 10,000-10 million, 10,000-1 million, 10,000-100,000, 100,000-10 million, 100,000-1 million, or 1 million -10 million cells, or products therefrom, or any range derivable therein.

[0078] In some aspects, a single dose of the therapy is administered. In some aspects, multiple doses of the therapy are administered. In some aspects, the therapy is administered at a dose of between 100 cells-10 million cells, or products therefrom. In some aspects, the therapy is administered at a dose of at least, at most, or about 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg / kg.

[0079] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 100 cells to 10 million cells can affect the protective capability of these agents. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0080] Precise amounts of the therapeutic composition may also depend on the judgment of the practitioner and may be peculiar to each subject. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0081] It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0082] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0083] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0084] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions;solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0085] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0086] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0087] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0088] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0089] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0090] In some aspects, between about 105and about 1013cells per 100 kg are administered to a subject per infusion. In some aspects, between about 1.5 x 106and about 1.5 x 1012cells are infused per 100 kg. In some aspects, between about 1 x 109and about 5 x 1011cells are infused per 100 kg. In some aspects, between about 4 x 109and about 2 x 1011cells are infused per 100 kg. In some aspects, between about 5 x 108cells and about 1 x 1011cells are infused per 100 kg. In some aspects, a single administration of cells is provided. In some aspects, multiple administrations are provided. In some aspects, multiple administrations are provided over a period of time. In some aspects, 3-7 administrations are provided over the course of 3-7 consecutive days. In some aspects, 5 administrations are provided over the course of 5 consecutive days. In some aspects, a single administration of between about 105and about 1013cells per 100 kg is provided. In some aspects, a single administration of between about 1.5 x 108and about 1.5 x 1012cells per 100 kg is provided. In some aspects, a single administration of between about I x 109and about 5 x 1011cells per 100 kg is provided. In some aspects, a single administration of about 5 x 1010cells per 100 kg is provided. In some aspects, a single administration of 1 x 1010cells per 100 kg is provided. In some aspects, multiple administrations of between about 105and about 1013cells per 100 kg are provided

[0091] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.IV. Cellular TherapiesA. Cells and Cell Culture

[0092] In certain aspects, the fibroblasts may be derived from tissues comprising skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue,foreskin, placental, and / or umbilical cord. In specific aspects, the fibroblasts are placental, fetal, neonatal, or adult or mixtures thereof. In an aspect, the fibroblasts may be differentiated from stem cells, for example embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, or progenitor cells. In an aspect, the fibroblasts may be modified or unmodified. The term modified fibroblasts includes, but is not limited to, genetically modified fibroblasts, chemically modified fibroblasts, physically modified fibroblast, or epigenetically modified fibroblasts. Fibroblasts may be modified in vitro, ex vivo or in vivo. The fibroblasts may be autogenic, allogeneic, xenogeneic, or syngenetic to the subject. Thus in some aspects, the fibroblasts may be isolated from an autogenic, allogeneic, xenogeneic, or syngenetic source, and manipulated prior to administration to the subject. Manipulation may include culturing the cells, modifying the cells, for example by chemical, physical, epigenetic or genetic manipulations.

[0093] In an aspect, the cells may be genetically manipulated. In an aspect, the cells may be genetically engineered to impact their immune modulation potential. In an aspect, the cells may be genetically engineered to target one or more genes or gene products to attenuate their immune modulation potential. For example, the one or more genes or gene products may comprise: cytokine genes, non-limiting examples of which include IL1RA, IL-4, IL-6, IL-10, IL-11, IL-13, TGFb; genes that impact levels or function of soluble receptors, non-limiting examples of which include soluble TNF receptor p55, soluble TNF receptor p75, soluble IL-1 receptor type 2, membrane-bound IL-1 receptor type 2, IL- 18 binding protein and corresponding; genes that impact levels or function of mediators like prostaglandin E2; genes that impact levels or function of cell surface receptors, non-limiting examples of which include PD1, CTLA4, TIM3, LAG3, TIGIT, CD96, BTLA, VISTA; and other genes that impact levels or function of arginase, prostaglandin E2, indoleamin 2,3 -dioxygenase, nitric oxide synthase etc. The one or more cells, including fibroblasts, may be engineered as disclosed herein above. Genetic modification methods or compositions may be used to introduce nucleic acids into cells, edit genomic DNA or transcripts using gene editing, homologous and non-homologous recombination, TALENS, CRISPR, zinc finger nucleases or any combination thereof. These genetic modifications may introduce exogenous genes, delete, or mutate endogenous genes, introduce selection or identification markers or any combination thereof. These methods are known in the art and may be used in isolation or a combination of these methods may be used to obtain cells with desired properties.

[0094] In an aspect, the cells are chemically, physically or epigenetically activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors,growth factors, hormones, or any combination thereof, prior to administration. In an exemplary aspect, isolated cells may be cultured in the presence of other immune cells. Non-limiting examples include macrophages, microglia, dendritic cells, Treg cells etc.

[0095] In some aspects, fibroblast cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some aspects, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0096] Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum may be added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. In an aspect, the media may be free of fetal bovine serum. In an aspect, the medium may comprise one or more of human platelet rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, one or more cytokines, fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin like growth factor, angiopoietin, vascular endothelial growth factor, or any combination thereof. Media useful in the methods of the disclosure may further comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells.

[0097] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated. One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein.

[0098] In particular aspects, the cells of the disclosure may be specifically formulated and / or they may be cultured in a particular medium. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

[0099] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0100] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, caninclude a medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0101] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolglycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0102] In certain aspects, the medium may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and / or T3 (triodo-I-thyronine). . In specific aspects, one or more of these may be explicitly excluded.

[0103] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In some aspects, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine,linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific aspects, one or more of these may be explicitly excluded.

[0104] The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts. . In specific aspects, one or more of these may be explicitly excluded.

[0105] In specific aspects, the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular aspects the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing. The cells may be grown at temperaturesbetween 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The culturing conditions can vary depending on the specific method and components of the culture. In some aspect, the fibroblasts may be cultures at a fixed or variable temperature ranging from about 20° to about 40 °C, or about 25 °C to about 38 °C, or about 37 °C. In an aspect, the organoid may be cultured at a fixed, or variable carbon dioxide concentration ranging from 1-10%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% carbon dioxide concentration. In an aspect, the oxygen tension can range from 1-20%, for example about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% and any combination of ranges derivable therein.

[0106] The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure. Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular aspects, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.B. Cell Generation

[0107] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In particular aspects of the present disclosure, cells are plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cellsand the substrate surfaces may ensue. Accordingly, in certain aspects cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such aspects encompass plating onto a substrate, which allows adherence of cells thereto.

[0108] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation of these cells can be achieved, for example, through flow cytometry, ELISA, and / or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and / or to monitor changes in gene expression in response to differentiation. In certain aspects, the marker proteins used to identify and characterize the cells are selected from the list consisting of c-Kit, Nanog, Sox2, Heyl, SMA, Vimentin, Cyclin D2, Snail, E- cadherin, Nkx2.5, GATA4, CD105, CD90, CD29, CD73, Wtl, CD34, CD45, and a combination thereof.C. Pharmaceutical Compositions

[0109] In certain aspects, the compositions, or agents for use in the methods, such as fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast microvesicles, fibroblast lysate, fibroblast apoptotic bodies, fibroblast conditioned media, or a combination thereof, are suitably contained in a pharmaceutically acceptable carrier. In an aspect, the pharmaceutical composition may further comprise one or more non-pathogenic immune cells and / or one or more derivative agents thereof. Non-pathogenic cells, as used herein, comprise any immune cell that does not directly cause disease or harm to the body. Non-limiting examples include regulatory T-cells (Tregs, non-pathogenic Thl7 cells, M2 macrophages, B-cells, dendritic cells, antigen presenting cells (APC) and eosinophils. Thus in an aspect, the disclosed pharmaceutical compositions may comprise fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast microvesicles, fibroblast lysate, fibroblast apoptotic bodies, fibroblast conditioned media, or a combination thereof and non-pathogenic immune cells, and a pharmaceutically acceptable carrier.

[0110] The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (z.e., to a specific location of the body, such as on or in the skin or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalantsand injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like. In an exemplary aspect, the composition is formulated for intravenous or intrathecal delivery.[OHl] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0112] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay, or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0113] In an aspect, the pharmaceutical composition may further comprise one or more adjuvants. In an aspect, the adjuvants may be chemical-based, viral-based, or bacterial productbased.

[0114] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the subject.

[0115] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0116] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions;solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0117] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

[0118] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0119] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other excipients.

[0120] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0121] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each subject. Factors affecting the dose include the physicaland clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability, and toxicity of the particular therapeutic substance.V. Kits of the Disclosure

[0122] Any of the cellular and / or non-cellular compositions described herein or similar thereto may be comprised in a kit. In a non-limiting example, one or more reagents for use in methods for preparing cellular therapy may be comprised in a kit. Such reagents may include cells, IFN-gamma, platelet rich plasma, platelet lysate, one or more angiogenic factors, one or more growth factors, vector(s) one or more costimulatory factors, media, enzymes, buffers, nucleotides, salts, primers, and so forth. The kit may comprise any protein listed in the disclosure. The kit components are provided in suitable container means.

[0123] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0124] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and / or other such like apparatus, or may be a substrate with multiple compartments for a desired reaction.

[0125] Some components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent.

[0126] In specific aspects, reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include apparatus or reagents for isolation of a particular desired cell(s).

[0127] In particular aspects, there are one or more apparatuses in the kit suitable for extracting one or more samples from A subject. The apparatus may be a syringe, fine needles, scalpel, and so forth.EXAMPLES

[0128] The following examples are included to demonstrate particular aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1THERAPEUTIC USE OF FIBROBLAST MODULATION OF MICROGLIAMETHODS

[0129] Animals and neuroinflammation disease model. Adult male Lewis rats (9-10 weeks of age, 200-250 g) where assigned to four groups: normal control group (n = 10), BM- MSC group (n = 10), Adipose MSC group (n = 10), and Fibroblast group (n=10). Experimental autoimmune encephalomyelitis (EAE) was induced by subcutaneous injection of guinea pig spinal cord homogenate (GPSCH) emulsified at a 1 : 1 ratio with complete Freund adjuvant (CFA) containing heat killed Mycobacterium tuberculosis. Each rat received an intraperitoneal injection of 300 ng Pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 ml distilled water immediately after the subcutaneous injection and again 48 hours later. All experimental procedures followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

[0130] Clinical assessment of neuroinflammation. The clinical manifestations of EAE were assessed daily until the time of sacrifice. Disease severity was scored on a 5-point scale:0 = no signs, 1 = partial loss of tail tonicity, 2 = loss of tail tonicity, 3 = unsteady gait and mild paralysis, 4 = hind limb paralysis and incontinence, and 5 = moribund or death. Disease scoring was performed by pathologists blinded to treatment conditions.

[0131] Cell culture aud treatment. Fibroblast cells were used at passage 3-5 and maintained in fibroblast growth medium at 37°C with 5% CO2 until 70-80% confluence. For preparing HDF (Human Dermal Fibroblast) spheroids, HDF were trypsinized using 0.25% trypsin for 5 min. Cells were then placed in hanging drop culture on the inner surface of a Petri dish lid and incubated until they form the true 3D spheroids. After 3 days, spheroids (150uM spheroids containing 10000 cell each) were collected using a Pl 000 pipette and washed twice with PBS for further cell-based treatment in rats. HDF spheroids (100 of 150 uM) were then suspended in saline and administered intravenously. Control animals were treated with

[0132] Statistical analysis. Statistical analysis was performed using GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA). Results are expressed as the mean ± SEM. Normality tests were performed using D’Agostino-Pearson. The statistical difference between groups was analyzed using one-way ANOVA tests followed by post-hoc comparisons. P value of less than 0.05 was considered statistically significant.Example 2RESULTS

[0133] EAE severity of the animals treated with administration of fibroblasts was compared to animals treated with adipose derived mesenchymal stem cells (MSC) as shown in FIG. 1. Results show that treatment with fibroblasts significantly reduced neuroinflammation and neuronal damage as compared to control and MSC treated animals. This was further confirmed using immunohistochemistry (IHC). Sections of rat cerebral cortex were subjected to immunohistochemistry using antibodies specific to myelin and neuron fibers. Animals treated with fibroblasts contained significantly less myelin and neuron fiber degradation compared to the control (see FIG. 2). Quantitation of the data is provided in FIG. 3, which clearly demonstrates that fibroblast treated animals had significantly higher density of myelin.

[0134] Next, spinal cord sections of treated and control animals were subjected to IHC staining using antibodies specific for microglia (CD68 and IBA-1). Representative images are shown in FIG. 4. The data clearly indicates that while the control animals and adipose derived MSC treated animals had regions of hyperactive microglia activity, the fibroblast treated animals did not show any overactive microglia. These experiments were repeated with corticalsections. As with the spinal cord, the data indicates that while the control animals and adipose derived MSC treated animals had regions of hyperactive microglia, the fibroblast treated animals did not show any overactive microglia (see FIG. 5).

[0135] IL-10 is a biomarker for T-regulatory cells (T-regs). Levels of IL-10 was assayed from isolated samples from control and treated animals. A quantitation of the data is provided in FIG. 6. These data demonstrates that fibroblasts significantly enhance stimulation of T-reg cells as compared to the control animals and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs).

[0136] Th-17 cell samples were also tested for secretion of IL-17 inflammatory cytokine.As shown in FIG. 7, compared to control and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs), fibroblast treated animals have significantly suppressed levels of IL- 17 inflammatory cytokine.

[0137] Samples from control and treated animals were also assayed for key biomarkers linked to inflammation, including CD-40, CD-80, and CD-86. Data from these experiments show that compared to control and bone marrow-derived mesenchymal stem cells (BM-MSCs) and Adipose derived Mesenchymal Stem Cells (Adipose-MSCs), fibroblast treated animals have significantly downregulated inflammatory biomarkers CD-40, CD-80, and CD-86 (see FIG. 8A, FIG. 8B and FIG. 8C respectively). Samples were also tested for key antiinflammatory cytokines IL- 10, IL- IRA and programmed cell death ligand (PDL) (See FIG. 9A, FIG. 9B and FIG. 9C respectively). Consistent with the biomarker assays, these data also demonstrate that compared to the controls, fibroblast treated animals had significantly upregulated anti-inflammatory cytokines IL- 10, IL- IRA and programmed cell death ligand (PDL).

[0138] Data presented herein clearly shows the efficacy of using fibroblasts and derived products modulate microglial activities in the central nervous system.* * *

[0139] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Clark, R.A. and T.S. Kupper, IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin. Blood, 2007. 109(1): p. 194-202.2. Uriarte Huarte, O., et al., Microglia in Health and Disease: The Strength to Be Diverse and Reactive. Front Cell Neurosci, 2021. 15: p. 660523.3. Araki, T., Y. Ikegaya, and R. Koyama, The effects of microglia- and astrocyte-derived factors on neurogenesis in health and disease. Eur J Neurosci, 2021. 54(5): p. 5880- 5901.4. Wang, W.Y., et al., Role of pro-inflammatory cytokines released from microglia in Alzheimer's disease. Ann Transl Med, 2015. 3(10): p. 136.5. Guedes, J.R., et al., Roles of Microglial andMonocyte Chemokines and Their Receptors in Regulating Alzheimer's Disease-Associated Amyloid- fl and Tau Pathologies. Front Neurol, 2018. 9: p. 549.6. Chauhan, P., et al., Differential Cytokine-Induced Responses of Polarized Microglia. Brain Sci, 2021. 11(11).7. Smith, J. A., et al., Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull, 2012. 87(1): p. 10-20.8. Wojcieszak, J., K. Kuczynska, and J.B. Zawilska, Role of Chemokines in the Development and Progression of Alzheimer's Disease. J Mol Neurosci, 2022. 72(9): p. 1929-1951.9. Szalay, G., et al., Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Nat Commun, 2016. 7: p. 11499.10. Muzio, L., A. Viotti, and G. Martino, Microglia in Neuroinflammation and Neurodegeneration: From Understanding to Therapy. Front Neurosci, 2021. 15: p. 742065.11. Lowes, M. A., M. Suarez-Farinas, and J.G. Krueger, Immunology of psoriasis. Annu Rev Immunol, 2014. 32: p. 227-55.Rendon, A. and K. Schakel, Psoriasis Pathogenesis and Treatment. Int J Mol Sci, 2019. 20(6). Samotij, D., et al., Pathogenesis of psoriasis in the "omic" era. Part I. Epidemiology, clinical manifestation, immunological and neuroendocrine disturbances. Postepy Dermatol Alergol, 2020. 37(2): p. 135-153.

Claims

WHAT IS CLAIMED IS:

1. A method of modulating activity of microglia cells in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of fibroblasts, one or more derivatives thereof, or any combination thereof, wherein the subject has, or is at risk for having, a medical condition associated with the central nervous system (CNS).

2. The method of claim 1, wherein the one or more derivatives of fibroblasts comprise fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, and other fibroblast-derived materials such as metabolites.

3. The method of claims 1 or 2, wherein the administration is by intravenous injection or intrathecal injection.

4. The method of claim 1, wherein the medical condition is a neurological disorder, neurodegenerative disease, traumatic brain injury, bacterial meningitis, viral meningitis, fungal meningitis, or stroke.

5. The method of claims 1 or 2, wherein the fibroblasts are autologous, allogeneic, xenogeneic, or syngeneic with respect to the subject.

6. The method of any one of claims 1-5, wherein the fibroblast are genetically engineered to target one or more genes or gene products to attenuate their immune modulation potential.

7. The method of claim 6, wherein the one of more genes or gene products is selected from a group consisting of IL1RA, IL-4, IL-6, IL-10, IL-11, IL-13, TGFb, soluble TNF receptor p55, soluble TNF receptor p75, Soluble IL-1 receptor type 2, membrane-bound IL-1 receptor type 2, IL- 18 binding protein, prostaglandin E2, PD1, CTLA4, TIM3, LAG3, TIGIT, CD96, BTLA, VISTA, Arginase, prostaglandin E2, indoleamin 2,3- dioxygenase, nitric oxide synthase.

8. The method of any one of claims 1-7, wherein the fibroblasts are chemically, physically or epigenetically activated.

9. The method of claim 8, wherein the fibroblasts are activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof.

10. The method of any one of claims 1-9, wherein the fibroblasts are cultured fibroblasts.

11. The method of claim 10, wherein the fibroblasts are cultured in a medium free of fetal bovine serum, and optionally wherein the medium comprises one or more of human platelet rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, one or more cytokines, fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin like growth factor, angiopoietin, vascular endothelial growth factor, or any combination thereof.

12. The method of any one of claims 1-11, wherein the fibroblasts are derived from embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, progenitor cells, placenta, omental tissue, cord blood, skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin or any combination thereof.

13. A method of treating a medical condition associated with the central nervous system (CNS) of a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic cell bodies, conditioned media from fibroblasts, fibroblast-derived materials such as metabolites, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents; and(2) one or more types of immune cells and / or one or more derivative agents thereof.

14. The method of claim 13, wherein the immune cell is a non-pathogenic immune cell.

15. The method of claim 14, wherein the administering is into the blood stream or intrathecally.

16. The method of claim 14 or 15, wherein following the administering step, there is a decrease in the expression of pathogenic levels of IL-23, IL-1, IL-10, IL-6, IL-22, IL-17, IL-12, TNF, INF-P, TGF-pi, or a combination thereof from the microglia, T cells, dendritic cells, macrophages, or a combination thereof in the subject.

17. A method of epigenetic reprogramming of localized microglia, or oligodendrocyte, keratinocyte cells in a subject with a medical condition associated with the brain or CNS, comprising the step of administering an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast apoptotic bodies, conditioned media from fibroblasts, fibroblast-derived materials such as metabolites, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or conditions, and(2) one or more type of immune cells and / or one or more derivative agents thereof.

18. A method of enabling the activation and migration of localized stem cell niches to replace damaged oligodendrocytes and / or damaged myelin sheaths in a subject in need thereof, comprising the step of administering to the subject an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, fibroblast-derived materials or products, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or one or more conditions; and(2) one or more type of immune cells and / or one or more derivative agents thereof.

19. The method of any one of claims 17 or 18, wherein the administering is into the blood stream of the subject or intrathecally.

20. The method of any one of claims 1-19, wherein one or more adjuvants are administered in combination with single cell fibroblasts, fibroblast spheroids, or fibroblast-derived materials such as metabolites.

21. The method of claim 20, wherein the adjuvants are chemical -based, viral -based, or bacterial product-based.

22. A pharmaceutical composition for use in the treatment of medical condition associated with the CNS, wherein the pharmaceutical composition comprises fibroblasts,fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, fibroblast-derived materials or products, or any combination thereof, optionally wherein the fibroblasts are activated with one or more agents and / or one or more conditions; and at least one carrier or excipient.

23. The composition of claim 22, wherein the pharmaceutical composition is formulated for intravenous or intrathecal administration.

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