Therapeutic use of fibroblasts for the treatment of systemic lupus erythematosus

Abstract

Embodiments of the disclosure include methods, systems, and compositions for the treatment of systemic lupus erythematosus (SLE), such as single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells and / or fibroblast-derived products. In certain embodiments, such treatment leads to reduction of the proliferation of SLE-associated pathogenic immune cells that may express pathogenic levels of IL-1, IL2, IL-6, IL-10, IL-21, IL-22, IL-17, IL-12, TNF, INF-γ, TGF-β1, or a combination thereof.

Description

THERAPEUTIC USE OF FIBROBEASTS FOR THE TREATMENT OF SYSTEMICEUPUS ERYTHEMATOSUSBACKGROUNDCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 569456, filed March 25, 2024, which is incorporated by reference herein in its entirety.I. Technical Field

[0002] Embodiments of the disclosure concern at least the fields of cell biology, molecular biology, immunology, and medicine.II. Background

[0003] Systemic lupus erythematosus (SLE), commonly referred to as lupus, is a chronic autoimmune disease with diverse clinical manifestation that impacts one or more organs, and there is a global prevalence rate of 3.4 million diagnosed, with 0.4 million newly diagnosed annually. According to data from the Lupus Foundation of American, in the U.S. there are currently 1.5 million diagnosed with lupus, and 16,000 new cases are reported each year. The majority of those impacted are women with a current prevalence of 3 million impacted globally.

[0004] SLE is characterized by the chronic production of autoantibodies and immune complexes, leading to chronic inflammation and often permanent tissue damage across a wide range of organs and tissues, such as the skin, joints, heat, lung, kidney, circulating blood cells, and brain. Factors such as genetics, environment, and hormonal imbalance have been implicated as major contributors to SLE, however the exact cause has not yet been determined [1]. The breakdown of self-tolerance has been implicated as a critical role in the development of SLE. Dysregulation of the innate and adaptive immune system lead to the activation of neutrophils, monocytes, and self-reactive lymphocytes [2].

[0005] Currently, physicians depend on the use of a wide range of non-specific immunosuppressants, such as antimalarial drugs, glucocorticosteroids steroids, and targeted monoclonal antibody-based therapies that directly or indirectly target B-cell inhibition or T- cell inhibition. All of these non-specific immunosuppressants carry major immediate side effects impacting biological functions, such as gastrointestinal, hematological, metabolic,cardiomyopathy, and nephrotoxicity functions. These same treatments also carry long-term use risks, such as malignancies, teratogenic effects, and susceptibility to pathogenic infections [2].

[0006] The present disclosure provides solutions to a long-felt need in the art of at least SLE treatment.SUMMARY

[0007] Embodiments of the disclosure encompass systems, methods, and compositions for treatment of SLE. In particular embodiments, one or more symptoms of SLE are treated, including one or more of chronic inflammation and / / or permanent tissue damage in tissues such as the skin, joints, heat, lung, kidney, circulating blood cells, and / or brain. In certain embodiments, the disclosure provides methods and compositions for reducing in part or in full chronic production of autoantibodies and immune complexes. In some embodiments, the systems, methods, and compositions reduce or eliminate the need for one or more types of nonspecific immunosuppressants for an individual with SLE.

[0008] Embodiments of the disclosure encompass compositions, methods, and systems for an individual with SLE or at risk for SLE or suspected of having SLE include introducing single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials by any suitable administration route, including topically, subcutaneously, or intravenously. In some embodiments, fibroblasts themselves or fibroblasts that produce spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials are modified. In some embodiments, existing fibroblast cells within damaged tissues of any kind in an individual with SLE are modified, such as for regeneration of its function. In specific embodiments, for an individual with SLE single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells and / or fibroblast-derived products that are natural immune modulators achieve the same or better results (as compared to bone marrow-derived stem cells or adipose-derived stem cells) in modulating the immune system, controlling the expansion of pathogenic immune cells, and / or reducing the expression of pathogenic levels of cytokines, chemokines, growth factors, and complement factors with little to no immediate or long-term side effects for the individual.

[0009] In particular embodiments, Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR- associated protein 9 (Cas9) CRISPR / Cas9 systems may be used to add, delete, or modify fibroblast cell surface markers to allow targeting of the cells to a specific organ or tissue of interest. In particular embodiments the disclosure concerns interaction of afirst type of cells with a second type of cells and / or certain agent(s) and includes modification(s) to the first and / or second type of cells as a result of the interaction. In specific embodiments, the disclosure includes compositions, methods, and systems in which fibroblasts are modified upon exposure to certain cells and / or one or a combination of certain agent(s) including nucleic acids, cytokines, chemokines, or growth factors. Introduction of these modified or unmodified fibroblasts (e.g., intravenously) can modulate the immune system, including by bringing the immune system back to homeostasis. In particular embodiments, the modulation of the immune system may be achieved through one or more of the following steps, as examples only:1. Administration of single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials (e.g., intravenously, subcutaneously, or topically) to an individual with SLE or at risk for SLE or suspected of having SLE that in specific embodiments reduces the imbalance of Thl / Th2 cells that have been implicated in SLE (e.g. through downregulation of Thl)[3].2. Administration of single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials (e.g., intravenously, subcutaneously, or topically) to an individual with SLE or at risk for SLE or suspected of having SLE that in specific embodiments reduces the imbalance of Thl7 / Treg cells that have been implicated in SLE (e.g., through downregulation of Thl7 and upregulation of Treg)[3-5].3. Modulation of the immune system of an individual with SLE or at risk for SLE or suspected of having SLE through single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials to control the expansion and localization of pathogenic immune cells and the expression of pathogenic levels from those cells of cytokine, chemokines and growth factors associated with inflammation. These cells include T cells, dendritic cells macrophages, monocytes, NK cells, and neutrophils, expressing pathogenic levels of IL-1, IL2, IL-6, IL-10, IL-21, IL-22, IL- 17, IL-12, TNE, INE- y, TGE- pi, in specific embodiments.

[0010] Levels of T cells, dendritic cells macrophages, and / or neutrophils may be monitored in an individual with SLE or at risk for SLE or suspected of having SLE, including before, during, and / or after treatment with systems, methods, and compositions encompassed herein. Such monitoring may occur by measuring Complete Blood Count, White Blood Count, and / or tissue levels of T cells, dendritic cells, and / or macrophages. In some embodiments, IL-2, IL- 1, IL-10, IL-21, IL-22, IL-17, IL-12, TNE, INE- y, and / or TGE-pi levels may be monitoredbefore, during, and / or after treatment with systems, methods, and compositions encompassed herein, e.g., using ELISA and / or bead-based immunoassays. The monitoring may be done before therapy for baseline and / or after therapy for determining impact of treatment. The monitoring may be done before therapy for baseline and / or after 1, 2, 3, 4, 5., 6, 7, 8, 9, 10, or more administrations of systems, methods, and compositions of the disclosure.

[0011] In specific embodiments, the disclosure concerns compositions, methods, and systems in which certain cells are modified upon exposure to fibroblasts and / or certain agent(s) excreted from fibroblasts. In particular embodiments, the interaction of 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 embodiments, the other types of cells include at least immune cells.

[0012] In specific embodiments, methods of the disclosure occur ex vivo, such as in a culture and can also occur in vivo. In particular cases, the methods occur by the hand of 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 embodiments, the concentrations of cells used in a method of exposing one type of cells to another type of cells does not occur in nature and does not happen randomly in nature. In specific embodiments, the concentration of one or more agents used in a method of exposing the one or more agents 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 embodiment, tissue biopsy from the 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.

[0013] 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 single cell fibroblasts, fibroblast spheroids, or fibroblast-derived materials have been modified e.g., by exposure or treatment ex vivo with growth factors, chemokine or cytokines) prior to their exposure to the immune cells and other cells found in the blood and skin tissue, such as chemically, physically, or epigenetic ally activated, or exposed to 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 fibroblasts.

[0014] Embodiments of the disclosure provide means of utilizing fibroblasts as allogeneic, autologous (or xenogeneic or syngeneic) therapeutic cells through modification of culture conditions. In one embodiment of the disclosure, fibroblasts are extracted from sources with lower immunogenicity (e.g., placental fibroblasts, omental tissue derived fibroblasts, cord blood-derived fibroblasts, etc.).

[0015] In one embodiment 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 of fibroblasts by the recipient immune system. In one embodiment, fibroblasts and / or fibroblast spheroids may be cultured in conditions that lack xenogeneic components, such as xenogeneic-free medium; in some cases, for example, the media may be free of fetal calf serum. In specific embodiments, 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 / or vascular endothelial growth factor.

[0016] In one embodiment of the disclosure, effective amounts of single cell fibroblasts, fibroblast spheroids, and / or fibroblast-derived materials as prepared in methods encompassed by the disclosure are administered to an individual for a therapy or prevention of one or more medical conditions, including SLE, or they are administered to reduce the severity of and / or delay onset of one or more symptoms of SLE. In specific embodiments, the fibroblasts are administered to improve keratinocyte, epithelial, fibroblast, and / or resident immune cell responsiveness, modulation of immune cell activity, and / or cell differentiation.

[0017] Embodiments of the disclosure provide methods for co-administration of universal donor single cell fibroblasts, fibroblast spheroids, and / or fibroblast-derived materials with one or more agents that modulate the immune cell activity in the site of inflammation. In a specific embodiment of the disclosure, methods are provided for co-administration of universal donor single cell fibroblasts, fibroblast spheroids, and / or fibroblast-derived materials with one or a combination of growth factors, chemokines, and / or cytokines. In one embodiment of the disclosure, universal donor fibroblasts derived from fibroblasts that have been treated under conditions (e.g., culturing conditions such as using media and media components that do not over stimulate or stimulate the expression of markers that will illicit an immune response upon administration) to reduce immunogenicity are utilized to stimulate vascular endothelial growthfactor (VEGF) production by the introduced modified / unmodified fibroblasts or from endogenous cells under the regulatory control of nerve growth factor (NGF) of the individual thereby leading to tissue regeneration and revascularization of the damaged tissue (such as skin).

[0018] Embodiments of the disclosure provide methods of reducing immunogenicity of particular types of fibroblasts. Single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / 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) and / or upon autopsy. In some aspects, the cells comprise fibroblasts, which can be from a fetal, neonatal, adult origin, or a combination thereof.

[0019] Single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells and / or fibroblast-derived materials or products for use in any methods of the disclosure may be exposed to certain one or more medium component(s), in specific embodiments.

[0020] Embodiments of the disclosure encompass an in vivo method of treating SLE using an intravenous or subcutaneous injection (as optional routes only) of single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials. In specific embodiments, this modulates the immune system and to reduce the expansion proliferation of SLE-associated pathogenic T cells, pathogenic dendritic cells, pathogenic macrophages, and / or pathogenic neutrophils, including any cells expressing pathogenic levels of IL-1, IL2, IL-6, IL-10, IL-21, IL- 22, IL-17, IL-12, TNE, INE- y, and / or TGE- pi, as examples. In specific embodiments, the method occurs in vivo. The fibroblast cells, or the products derived therefrom may be autologous, allogeneic, xenogeneic, or syngeneic with respect to a recipient individual.

[0021] Introduction of single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials into an individual with SLE modulates the expansion and proliferation of pathogenic immune cells in the individual, in specific embodiments. Introduction of single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials or products that are or have been activated with one or more agents (e.g., pre-treatment of the products with a growth factor, chemokine, or cytokine to increase the immune modulation capacity, for example pre-treatment with TNE alpha) modulates the expansion and proliferation of pathogenic immune cells in an individual with SLE or at risk thereof or suspected of having SLE, in specific embodiments. Introductionof single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials plus one or more type of immune cells or a derivative agent thereof modulates the expansion and proliferation of pathogenic immune cells in an individual with SLE or at risk for SLE or suspected of having SLE, in specific embodiments. In some embodiments, the introduction is, or is not. into the blood stream and / or skin tissue of an individual in need thereof. In some embodiments, the modulation of the pathogenic immune cells in the individual modulates the expression of pathogenic levels of IL-1, IL2, IL-6, IL-10, IL-21, IL-22, IL-17, IL-12, TNE, INE- y, and / or TGE- pi, for example. In some embodiments, the introduction of single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials, or any of these activated with one or more agents, and / or any of these plus one or more type of immune cells or derivative agent enable the epigenetic reprogramming of damaged tissue-localized fibroblasts and / or epithelial cells. In some embodiments, any of these may be provided to enable the activation and / or migration of localized stem cell niches to replace the damaged localized fibroblasts and / or epithelial cells.

[0022] Adjuvants may be used in combination with single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials to activate the immune system of an individual with SLE or at risk for SLE or suspected of having SLE. These adjuvants could be chemical, viral, or bacterial product-based and a mixture thereof may be used in some cases.

[0023] In some embodiments, the combination of encapsulated RNA, microRNA, and / or RNAi with single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, and / or fibroblast-derived materials, may be used to locally activate damaged tissue regeneration or reverse tissue atrophy in an individual with SLE, suspected of having SLE, or at risk for SLE.

[0024] Individuals at risk for SLE include at least those with a family history of lupus and / or having genes associated with autoimmune disorders; people of African American, Hispanic, Asian American, Native American, and Pacific Islander descent; women; and individuals between the ages of 15 and 44.

[0025] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated bythose skilled in the art that the conception and specific embodiment 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 subject matter of the disclosure as set forth in the appended claims. The novel features that are believed to be characteristic of the subject matter of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description. It is to be expressly understood, however, that the subject matter of the disclosure is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present embodiments and aspects.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 certain embodiments presented herein.

[0027] FIGS. 1A-1I. Immunomodulatory effects of fibroblasts on monocytes and dendritic cells. Monocytes were isolated from human blood and co-cultured with fibroblasts. Fibroblasts suppressed monocyte activation in a dose-dependent manner, reducing the production of TNF- a (1A) and IL-17 (IB) in response to LPS and IL-6 stimulation, respectively. Fibroblasts cocultured with dendritic cells inhibited LPS-induced dendritic cell maturation, as indicated by the downregulation of CD40 (1C), CD80 (ID), CD86 (IE), and IL- 12 (IF), along with the upregulation of inhibitory molecules IL- 10 (1G), IL- IRA (1H), and PD-L1 (II). Mean fluorescence intensity is MFI.

[0028] FIGS. 2A-2F. Fibroblast therapy restored hematological balance and mitigated systemic inflammation. Fibroblasts demonstrated systemic immunomodulatory effects by improving red blood cell count (RBC; 2A), hemoglobin levels (HGB; 2B), and platelet numbers (PLT; 2C) in mice with a skin inflammatory disease (Imiquimod; IMQ (a TLR7 agonist)+Control group). The treatment significantly suppressed immune cell activation, as evidenced by a reduction in circulating neutrophils (NEUT; 2D) and monocytes (MONO; 2E) (IMQ+HDF group). The white blood cell (WBC) differential count is shown in FIG. 2F. For both IMQ+Control and IMQ+HDF, the bottom of the bars represents neutrophils, the middleof the bars represents lymphocytes, and towards the top of the bars represents monocytes. For IMQ+HDF, the very top of the bar represents eosinophils.

[0029] FIGS. 3A-3E. Fibroblasts demonstrated inhibitory effects on immune cell populations within the spleen in a mouse model of skin inflammatory disease (IMQ+Control group). Treatment reduced the populations of neutrophils (3A), CD3+T cells (3B), CD4+T cells (3C), CD8+T cells (3D), and CD25+T cells (3E) (IMQ+HDF group), suggesting a broad immunomodulatory impact on innate and adaptive immune responses.

[0030] FIGS. 4A-4D. Fibroblast treatment exerted immunomodulatory effects in the inguinal lymph nodes in a mouse model of skin inflammatory disease (IMQ+Control group), leading to a reduction in CD3+T cells (4A), CD4+T cells (4B), CD8+T cells (4C), and CD25+T cells (4D) (IMQ+HDF group).

[0031] FIGS. 5A-5E. Fibroblasts modulate both systemic and tissue-specific inflammation. In a mouse model of acute skin inflammatory disease, fibroblast treatment reduced circulating IL-17F (5A) while enhancing IL- 10 production in the skin (5B), indicating a shift toward an anti-inflammatory environment. In a chronic skin inflammatory disease model induced by cyclic immune activation, systemic cytokine levels remained unchanged; however, fibroblasts significantly decreased local expression of pro-inflammatory cytokines IL-23 (5C), TNF-a (5D), and IL-12p70 (5E).

[0032] FIGS. 6A-6E. Fibroblasts reduce tissue inflammation and immune cell infiltration. Histological analysis showed that fibroblasts mitigated skin inflammation by reducing both epidermal thickening (6A and 6B and 6E) H&E and Ki67+ keratinocytes) and immune cell infiltration (CD3+ T lymphocytes (6C) and F4 / 80+ macrophages (6D)), contributing to the restoration of normal epidermal and dermal architecture.DETAILED DESCRIPTIONI. Examples of Definitions

[0033] 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 embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0034] 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 embodiments, 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 more preferably within 2-fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value.

[0035] 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.

[0036] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0037] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0038] 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.

[0039] The term "administered" or "administering", as used herein, refers to any method of providing a composition to an individual such that the composition has its intended effect on the patient. 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.

[0040] 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 individuals of the same species.

[0041] As used herein, “autologous” refers to tissues or cells that are derived or transferred from the same individual's body (z.e., autologous blood donation; an autologous bone marrow transplant).

[0042] As used herein, “agent” refers to nucleic acids, cytokines, chemokines, transcription factors, epigenetics factors, growth factors, or hormones.

[0043] As used herein, “xenogeneic” refers to tissues or cells from a species different from the patient.

[0044] ‘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.

[0045] The term "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 individual may be receiving one or more medical compositions via the internet. An individual 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 "individual" connotes a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. The term “subject” or “individual” 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.

[0046] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particularfeature, structure or characteristic described in connection with the embodiment is included in at least one embodiment 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 embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0047] 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 embodiment, the quantity and / or magnitude of the symptoms in the treated subject is 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.

[0048] As used herein, the term “transplantation” refers to the process of taking living tissue or cells and implanting it in another part of the body or into another body.

[0049] ‘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 embodiments, 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.

[0050] It is contemplated that any embodiment discussed 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.

[0051] 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 embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0052] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.I. Administration of Therapeutic Compositions

[0053] The fibroblast-related therapy (single cell fibroblasts, fibroblast spheroids, exosomes, lysates, apoptotic cells, and / or fibroblast-derived materials) provided herein may comprise administration of one or a combination of therapeutic agents, such as those therapeutic for SLE. The therapy or therapies may be administered in any suitable manner known in the art and may be administered once or multiple times. In some embodiments, when multiple therapeutic agents are administered, they may or may not be in a separate composition. In some embodiments, they are in the same composition.

[0054] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition orin more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0055] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the SLE therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, and / 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 individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0056] 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 injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0057] In some embodiments, a dose of between 1 mg / kg and 5000 mg / kg. In some embodiments, the dose is administered 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, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526,527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564,565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0058] In specific embodiments, a dose is based on the number of cells and / or number of spheroids per kg body weight. Cell doses may include about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, or greater, and any range derivable therein.

[0059] 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 certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg may be used. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, g / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0060] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to50 pM; or about 50 pM to 150 pM; or about 50 |aM to 100 |aM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most 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 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0061] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. 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.

[0062] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. 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.

[0063] In certain instances, it will be desirable to have multiple administrations of the composition or compositions, 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 (or day or month) intervals, including all ranges there between.

[0064] 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. Theuse 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 aboutby the use in the compositions of agents delaying absorption, for example, aluminum mono stearate and gelatin.

[0069] 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-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0070] 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.

[0071] 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.II. Cellular Therapies

[0072] In particular embodiments, fibroblasts are cultured prior to use or subject to one or more particular agents and / or conditions. In particular embodiments, fibroblast spheroids are cultured prior to use and / or subject to one or more particular agents and / or conditions. In particular embodiments, prior to obtaining exosomes, lysates, conditioned media, apoptotic cells, etc, the fibroblasts from which they are derived are cultured and / or subject to one or more particular agents and / or conditions, although in other embodiments they are not.

[0073] In some embodiments, fibroblasts and / fibroblast spheroids are subject to one or more cytokines and / or growth factors, such as VEGF, HGF, IGF-1, FGF-2, PDGF, SDF1, CXCL-1, RANTES, MCP-1, PGE2, TGF- , IL-10, IL-6, G-CSF and / or M-CSF. In some embodiments, fibroblasts and / fibroblast spheroids are subject to one or more proteins, such asTIMP-2, SPARC, and / or angiopoietin-1. In some embodiments, fibroblasts and / fibroblast spheroids are subject to Lipids, DNA, miRNAs, and / or IncRNA. In some embodiments, fibroblasts and / fibroblast spheroids are subject to one or more types of Extracellular Vesicles (EVs). In some embodiments, fibroblasts and / fibroblast spheroids are subject to hypoxic conditions, such as less than or equal to about 5% oxygen.A. Cell Culture

[0074] In some embodiments, 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 embodiments, 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 culturing may or may not be continuous. The culturing may utilize media changes and / or splitting to reach a desired number of cells for treatment.

[0075] 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), RPML1640, 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 embodiments, 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 embodiments may employ further basal media formulations, such as chosen from the ones above.

[0076] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium(aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is 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. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 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 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.

[0077] 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., E-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.

[0078] 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 butknown 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 embodiments, 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

[0079] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In particular embodiments 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 cells and the substrate surfaces may ensue. Accordingly, in certain embodiments 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 embodiments encompass plating onto a substrate, which allows adherence of cells thereto.

[0080] 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 embodiments, 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, CD 105, CD90, CD29, CD73, Wtl, CD34, CD45, KGF, VEGF, IL-6TNF, HGF, CD44, CD49b, CD87, Ly-6C, and a combination thereof.C. Pharmaceutical Compositions

[0081] In certain aspects, the compositions or agents for use in the methods, such as single cell fibroblasts, fibroblast spheroids, exosomes from fibroblasts, lysates from fibroblasts, apoptotic cells from fibroblasts, conditioned media from fibroblast culture, and / or fibroblast- derived materials, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biologicalactivity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as 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, inhalants and 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.

[0082] 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 diglycerides. 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.

[0083] 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.

[0084] 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 individual subject.

[0085] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. 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.

[0086] 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 purposecomprises 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.

[0087] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. 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, antgifungal agents, anti-oxidants, 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.

[0088] 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.

[0089] 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. This may include oral, nasal, buccal, rectal, vaginal, or topical. 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. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0090] 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.

[0091] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include thephysical and 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.III. Formulations and Culture of the Cells

[0092] In particular embodiments, 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.

[0093] 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.

[0094] 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, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0095] 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'- thiolgiycerol, 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).

[0096] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitaminssuch 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 embodiments, one or more of these may be explicitly excluded.

[0097] In some embodiments, the medium further comprises vitamins. In some embodiments, 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 embodiments, 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 B 12. In some embodiments, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In some embodiments, the medium comprises or futher comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, 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-camitine, 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 embodiments, one or more of these may be explicitly excluded.

[0098] 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 embodiments, one or more of these may be explicitly excluded.

[0099] 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.

[0100] In specific embodiments, 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 embodiments 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.

[0101] In particular embodiments, the cells of the disclosure comprise an exogenous TCR, which may be of a defined antigen specificity. In some embodiments, the TCR can be selected based on absent or reduced alloreactivity to the intended recipient. In the example where the exogenous TCR is non-alloreactive, during T cell differentiation the exogenous TCR suppresses rearrangement and / or expression of endogenous TCR loci through a developmental process called allelic exclusion, resulting in T cells that express only the non-alloreactive exogenous TCR and are thus non-alloreactive. In some embodiments, the choice of exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some embodiments, the endogenous TCR genes have been modified by genome editing so that they do not expressa protein. Methods of gene editing such as methods using the CRISPR / Cas9 system are known in the art and described herein.

[0102] In some embodiments, the cells of the disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which a CAR may be directed include at least 5T4, 8H9, avP6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD 171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRD, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), Her2, IL-13RD2, Eambda, Eewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-l / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmell7, TRP-1 / -2, P. polypeptide, MC1R, Prostate-specific antigen, P-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-PRII, or VEGF receptors (e.g., VEGFR2), for example. The CAR may be a first, second, third, or more generation CAR. The CAR may be bispecific for any two nonidentical antigens, or it may be specific for more than two nonidentical antigens.IV. Examples

[0103] The following examples are included to demonstrate certain aspects of the subject matter of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practice of the invention, and thus can be considered to constitute particular 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 that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the subject matter of the disclosure.EXAMPLE 1THERAPEUTIC USE OF FIBROBEASTS FOR THE TREATMENT OF SYSTEMIC EUPUS ERYTHEMATOSUS

[0104] Immunomodulatory effects of fibroblasts on monocytes and dendritic cells were characterized (FIG. 1). Monocytes were isolated from human blood and co-cultured with fibroblasts. Fibroblasts suppressed monocyte activation in a dose-dependent manner, reducing the production of TNF-a (FIG. 1A) and IL- 17 (FIG. IB) in response to LPS and IL-6 stimulation, respectively. To evaluate their effects on DC maturation, fibroblasts were cocultured with dendritic cells. Fibroblasts inhibited LPS-induced dendritic cell maturation, as indicated by the downregulation of CD40 (FIG. 1C), CD80 (FIG. ID), CD86 (FIG. IE), and IL-12 (FIG. IF), along with the upregulation of inhibitory molecules IL-10 (FIG. 1G), IL-IRA (FIG. 1H), and PD-L1 (FIG. II).

[0105] In FIG. 2, it is shown that fibroblast therapy restored hematological balance and mitigated systemic inflammation in vivo. Fibroblasts demonstrated systemic immunomodulatory effects by improving red blood cell count (FIG. 2A), hemoglobin levels (FIG. 2B), and platelet numbers (FIG. 2C) in mice with a skin inflammatory disease. The treatment significantly suppressed immune cell activation, as evidenced by a reduction in circulating neutrophils (FIG. 2D) and monocytes (FIG. 2E). The white blood cell (WBC) differential count is provided in FIG. 2F.

[0106] Fibroblasts demonstrated inhibitory effects on immune cell populations within the spleen (FIG. 3). Treatment reduced the populations of neutrophils (FIG. 3A), CD3+T cells (FIG. 3B), CD4+ T cells (FIG. 3C), CD8+T cells (FIG. 3D), and CD25+T cells (FIG. 3E), indicating a broad immunomodulatory impact on innate and adaptive immune responses.

[0107] In FIG. 4, it is demonstrated that in vivo fibroblast treatment exerted immunomodulatory effects in the inguinal lymph nodes in a mouse model of skin inflammatory disease, leading to a reduction in CD3+T cells (FIG. 4A), CD4+T cells (FIG. 4B), CD8+T cells (FIG. 4C), and CD25+T cells (FIG. 4D).

[0108] Fibroblasts modulate both systemic and tissue-specific inflammation in vivo (FIG. 5). In a mouse model of acute skin inflammatory disease, fibroblast treatment reduced circulating IL-17F (FIG. 5A) while enhancing IL- 10 production in the skin (FIG. 5B), indicating a shift toward an anti-inflammatory environment. In a chronic skin inflammatory disease model induced by cyclic immune activation, systemic cytokine levels remainedunchanged; however, fibroblasts significantly decreased local expression of pro-inflammatory cytokines IL-23 (FIG. 5C), TNF-a (FIG. 5D), and IL-12p70 (FIG. 5E).

[0109] FIG. 6 demonstrates that fibroblasts reduce tissue inflammation and immune cell infiltration in vivo. Histological analysis showed that fibroblasts mitigated skin inflammation by reducing both epidermal thickening (H&E and Ki67+ keratinocytes) (FIGS. 6A, 6B, and 6E) and immune cell infiltration (CD3+ T lymphocytes (FIG. 6C) and F4 / 80+ macrophages (FIG. 6D)), contributing to the restoration of normal epidermal and dermal architecture.EXAMPLE 2EXAMPLES OF MATERIALS AND METHODS

[0110] Examples of Protocols for Figure 1

[0111] Isolation of monocytes

[0112] Human blood was collected in EDTA tubes and kept on ice until processing. Briefly, 10 mL of blood was placed in a 15 mL conical tube containing 4 mL of Ficoll. Cells were centrifuged at 700 g for 30 min. The mononuclear layer was collected and washed 3 times in PBS, and the pellet was diluted in RPMI media with 10% FCS. Cells were allowed to attach for 2 hours in a T75 flask. After PBS wash, monocytes were removed by trypsinization for further experiments. To stimulate tumor necrosis factor (TNF)-OC production, monocytes (IxlO5cells / well in 96-well plates) were treated with 10 ng / mL lipopolysaccharides (LPS, Thermo Fisher Scientific, Waltham, MA, USA). Fibroblasts pretreated with 5 ng / mL of LPS were added to the culture at 25,000 or 50,000 cells per well. In a separate experiment, 40 pg / mL IL- 6 (R&D Systems, Minneapolis, MN, USA) was added to induce IL- 17 release from monocytes, followed by fibroblast treatment as previously described. After 48 hours of incubation, the supernatant was collected to assess the production of TNF-oc or IL- 17 using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) [1].

[0113] Dendritic cell culture and maturation

[0114] Human monocyte-derived dendritic cells (DCs) were generated using the commercially available kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). DCs (IxlO5cells / well) were co-cultured with quiescent or preactivated HDFs (1x105 cells / well) in the presence of LPS (5 ng / mL). After 48 hours, cells were analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA) for surface markers including CD40, CD80, CD86, and programmed cell death- 1 (PD-L1) with subsequent analysisperformed using Flowjo software (Ashland, OR, USA), or for IL- 12, IL- 10, and IL-1 receptor antagonist (IL-IRa) using ELISA kits (R&D Systems, Minneapolis, MN, USA) [1].

[0115] Examples of Protocols for Figures 2-4, 5, and 6

[0116] A mouse model of acute skin inflammatory disease

[0117] C57BL / 6J female mice (10 weeks of age, 20-25 g) were randomly assigned to receive imiquimod (IMQ) with plasma Lyte-A (PLA) or fibroblasts via the tail vein. To induce psoriasis, mice were anesthetized with isoflurane, and a rectangular area of 3 cm x 4 cm on the back skin was shaved and treated with Nair™ hair-removal cream on Day 0. Psoriasis was induced via topical application of 31.25 mg of IMQ cream (5% IMQ, Taro Pharmaceutical, Hawthorne, NY, USA) to the lower dorsal skin for 7 consecutive days [2]. For cell-based treatment, fibroblast spheroids cultured in Coming Elplasia 12K flasks (seeded at 20x106cells / flask) were washed twice with PBS, resuspended in PLA, and 577 spheroids were administered via the tail vein at a dosage of IxlO6cells / 100 mL / mouse. 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 at StillMeadow, Inc. (Sugar Land, TX, USA).

[0118] Clinical assessment of psoriasis

[0119] The clinical features of IMQ-induced psoriasis were evaluated daily using the Psoriasis Area and Severity Index (PASI) as described previously [3]. Briefly, double-folded skin thickness was measured using a digital micrometer (Rexbeti, Auburn, Washington, USA). Psoriatic skin lesions, including thickness, erythema, and scaling, were assessed by two observers daily on a scale of 0 to 4 (0-no incidence; 1 -slight; 2-moderate; 3-marked; 4-severe), respectively. No significant deviation was noted between observers. The scores from two observers were then averaged to determine the mean PASI for each mouse. Fibroblast spheroids were administered when the average PASI score reached 2, indicating mild psoriasis. Euthanasia occurred on Day 8.

[0120] Blood profile and analysis

[0121] Approximately 100 pL of blood was drawn into EDTA-coated microcentrifuge tubes (BD, Franklin Lakes, NJ, USA), and the complete blood count (CBC) profile was determined at Baylor College of Medicine Pathology Core and Lab (Houston, TX, USA). An additional 250 p L of blood was collected into serum- separator tubes and allowed to clot at room temperature (RT) for 30 min. Approximately 10 mg of skin tissue was weighed and placed in 500 pL lysis buffer containing RIPA buffer and a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The tissue was homogenized thoroughly and incubatedon ice for 30 min to allow complete lysis. The lysates were centrifuged at 12,000 x g for 15 min at 4°C, and the supernatant was transferred to a new tube. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions. Mouse serum and skin cytokine levels were determined with MILLIPLEX® Mouse Cytokines / Chemokines Magnetic Bead Panel using Luminex multiplex bead assay (Millipore Sigma, St Louis, MO, USA) according to the manufacturer's instructions, and data were analyzed using the xPONENT software.

[0122] Flow cytometry

[0123] Spleens and inguinal lymph nodes were harvested and placed in cold RPMI-1640 medium supplemented with 10% FBS and 1% penicillin- streptomycin. Tissues were then digested in the medium containing 150 mg / mL Liberase (Millipore Sigma, St Louis, MO, USA) for 1 hour at 37°C and homogenized by gently pressing through a 70 pm cell strainer (Greiner Bio-One, Kremsmunster, Austria) using the plunger of a syringe. The homogenate was collected into a 15 mL conical tube, and the cell suspension was centrifuged at 300 x g for 7 minutes at 4°C. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 3-5 minutes at RT. Cells were washed twice with PBS by centrifugation at 300 x g for 5 minutes at 4°C and resuspended in 1 mL of PBS containing 2% FBS. Cells were blocked with antimouse CD16 / CD32 (TruStain FcX™, BioLegend, San Diego, CA, USA) and stained with fluorophore-conjugated antibodies, including Ly6G (eF780-conjugated, Thermo Fisher Scientific, Waltham, MA, USA), CD3e (APC-conjugated, Thermo Fisher Scientific, Waltham, MA, USA), CD4 (FITC-conjugated, BD Biosciences, Franklin Lakes, New Jersey, USA), and CD25 (BV421 -conjugated, BD Biosciences, Franklin Lakes, New Jersey, USA). Following 10 min incubation at 4°C in the dark, cells were washed twice with PBS and resuspended in 100 mL PBS containing 2% PFA. Stained cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). The percentage of neutrophils and T cell subtypes within the splenocyte and inguinal lymph node population was determined, and data were analyzed using FlowJo software (Tree Star, Inc.). The absolute number of cells was calculated by multiplying the total tissue number, determined by cell counting using a NucleoCounter NC- 3000 instrument, by the percentage of the cell population identified through flow cytometry [4].

[0124] Histology

[0125] Mouse skin sections (5 pm) were stained with primary antibodies (1:200) for CD3 (T lymphocytes, Abeam, Waltham, MA, USA), F4 / 80 (macrophages, Thermo Fisher Scientific, Waltham, MA, USA), and Ki-67 (cell proliferation marker, Abeam, Waltham, MA, USA). Afterward, sections were incubated with secondary antibodies (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at RT. For CD3 and F4 / 80 staining, sections were incubated with avidin-biotin-peroxidase (ABC) complex (Vector Laboratories, Burlingame, CA, USA) for 45 min at RT, developed using 3,3 '-diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA, USA), and counterstained with nuclear fast red (Vector Laboratories, Burlingame, CA, USA). For Ki67, sections were stained with DAPI (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. Three random images were obtained for each section (Echo, San Diego, CA, USA). The immunopositive area (expressed as a percentage of the total analyzed area) was quantified using NIH Image J software 1.53 [2].

[0126] Examples of Protocols for Figure 5

[0127] A mouse model of chronic skin inflammatory disease

[0128] SKH-1 hairless mice (10-week-old males) were randomly assigned to the following groups: 1) IMQ+Control and 2) IMQ+fibroblasts. To induce psoriasis, mice were anesthetized with isoflurane, and the back skin was treated with Nair™ hair-removal cream on Day 0. Psoriasis was induced via topical application of 31.25 mg of IMQ cream (5% IMQ, Taro Pharmaceutical, Hawthorne, NY, USA) to the lower dorsal skin according to the cyclic IMQ regimen. A cyclic IMQ regimen consisting of one cycle for psoriasis induction (Days 1-6) and two cycles for relapse (Days 9-12 and Days 15-18, respectively) was used to mimic the chronic, relapsing nature of the disease [5]. Fibroblast spheroids (at a dosage of IxlO6cells / 100 mL / mouse) were administered intravenously via the tail vein on Days 3 and 13. The clinical features of IMQ-induced psoriasis were evaluated daily using the PASI as described previously.

[0129] Statistical analysis

[0130] Statistical analysis was performed using GraphPad Prism version 10 (GraphPad Software, La Jolla, CA, USA). Results are expressed as the Mean ± Standard Error of the Mean (SEM). Normality tests were performed, and statistical differences between groups were analyzed using one-way ANOVA followed by Holm-Sidak's multiple comparison test for parametric data or Kruskal-Wallis test followed by Dunn's multiple comparison test for nonparametric data. A p-value of less than 0.05 was considered statistically significant.The 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.References for Example 2 only[1] T.E. Ichim, P. O’Heeron, J. Perez, P. Liu, W.-P. Min, S. Kesari, Fibroblasts as anAlternative to Mesenchymal Stem Cells with Successful Treatment and Immune Modulation in EAE Model of Multiple Sclerosis, bioRxiv (2020) 2020.06. 04.133249.[2] M. Jabeen, A.S. Boisgard, A. Danoy, N. El Kholti, J.P. Salvi, R. Boulieu, B. Fromy, B.Verner, M. Lamrayah, Advanced Characterization of Imiquimod-Induced Psoriasis- Like Mouse Model, Pharmaceutics 12(9) (2020).[3] S.D. Neu, A. Strzepa, D. Martin, M.G. Sorci-Thomas, K.A. Pritchard, Jr., B.N. Dittel,Myeloperoxidase Inhibition Ameliorates Plaque Psoriasis in Mice, Antioxidants (Basel) 10(9) (2021).[4] S. Fujiyama, C. Nakahashi-Oda, F. Abe, Y. Wang, K. Sato, A. Shibuya, Identification and isolation of splenic tissue-resident macrophage sub-populations by flow cytometry, Int Immunol 31(1) (2019) 51-56.[5] K. Fenix, D.K. Wijesundara, A. J. Cowin, B. Grubor-Bauk, Z. Kopecki, ImmunologicalMemory in Imiquimod-Induced Murine Model of Psoriasiform Dermatitis, Int J Mol Sci 21(19) (2020).* * *

[0131] 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 embodiments, 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 and modifications 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. Pan, L., et al., Immunological pathogenesis and. treatment of systemic lupus erythematosus. World J Pediatr, 2020. 16(1): p. 19-30.2. Tsokos, G.C., et al., New insights into the immunopathogenesis of systemic lupus erythematosus. Nat Rev Rheumatol, 2016. 12(12): p. 716-730.3. Dolff, S., et al., Disturbed Thl, Th2, Thl7 and T(reg) balance in patients with systemic lupus erythematosus. Clin Immunol, 2011. 141(2): p. 197-204.4. Lee, H.Y., et al., Altered frequency and migration capacity of CD4+CD25+ regulatory T cells in systemic lupus erythematosus. Rheumatology (Oxford), 2008. 47(6): p. 789-94.5. Zhang, Z., V.C. Kyttaris, and G.C. Tsokos, The role of IL-23 / IL-17 axis in lupus nephritis. J Immunol, 2009. 183(5): p. 3160-9.

Claims

WHAT IS CLAIMED IS:

1. A method of treating systemic lupus erythematosus (SLE) in an individual, comprising the step of administering to the individual a therapeutically effective amount of fibroblasts and / or fibroblast-derived products.

2. The method of claim 1, wherein the fibroblast-derived products comprise fibroblast spheroids, fibroblast exosomes, fibroblast microvesicles, fibroblast lysates, fibroblast apoptotic bodies, fibroblast-conditioned media, fibroblast fragments, or a combination thereof.

3. The method of claim 1 or 2, wherein following the administering, there is a reduction of the proliferation of SLE-associated pathogenic immune cells.

4. The method of claim 3, wherein the SLE-associated immune cells express pathogenic levels of IL-1, IL2, IL-6, IL-10, IL-21, IL-22, IL-17, IL-12, TNE, INE-y, TGE-pl, or a combination thereof.

5. The method of claim 3 or 4, wherein the SLE-associated immune cells are pathogenic T cells, pathogenic dendritic cells, pathogenic macrophages, pathogenic neutrophils, or a combination thereof.

6. The method of any one of claims 1-5, wherein the administering is intravenously, intramuscular, local injection, or by subcutaneous injection.

7. The method of any one of claims 1-6, wherein the fibroblasts or fibroblast-derived products are autologous with respect to the individual.

8. The method of any one of claims 1-6, wherein the fibroblasts or fibroblast-derived products are allogeneic with respect to the individual.

9. The method of any one of claims 1-6, wherein the fibroblasts or fibroblast-derived products are xenogeneic with respect to the individual.

10. The method of any one of claims 1-6, wherein the fibroblasts or fibroblast-derived products are syngeneic with respect to the individual.

11. A method of administering, optionally via intravenous injection or subcutaneous injection, in an individual, comprising the step of providing to the individual an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials; and(2) one or more types of non-pathogenic immune cells and / or one or more derivative agents of the immune cells.

12. The method of claim 11, wherein the administering is into the bloodstream of the individual.

13. The method of claim 11 or 12, wherein following the administering step, there is modulation of the proliferation of one or more types of pathogenic immune cells.

14. The method of any one of claims 11-13, wherein the providing of (1) and (2) occurs generally simultaneously.

15. The method of any one of claims 11-13, wherein the providing of (1) and (2) occurs at different times.

16. The method of claim 15, wherein (1) occurs before (2).

17. The method of claim 15, wherein (1) occurs after (2).

18. The method of any one of claims 11-17, wherein the fibroblasts are activated and / or the fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, or fibroblast-derived materials come from activated fibroblasts.

19. The method of claim 18, wherein the fibroblasts are activated ex vivo with one or more nucleic acids, one or more transcription factor activators, one or more growth factors, one or more cytokines, one or more chemokines, and / or with co-culture with non-fibroblast cells.

20. The method of claim 19, wherein the non-fibroblast cells are one or more types of non- pathogenic immune cells.

21. The method of claim 20, wherein the immune cells are T cells, dendritic cells, natural killer cells, natural killer T cells, macrophages, microglia, B cells, neutrophils, or a combination thereof.

22. The method of claim 20, wherein fibroblast cells are used to regulate dendritic cell function to reduce pathogenic immune cells associated with SLE.

23. A method of administering via intravenous injection, or subcutaneous injection in an individual, comprising the step of providing to the individual an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials; wherein the fibroblasts are activated with one or more agents and / or the fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, or fibroblast-derived materials come from activated fibroblasts are activated with one or more agents; and(2) one or more types of immune cells or derivative agent.

24. The method of claim 22, wherein the fibroblasts are activated ex vivo with one or more nucleic acids, one or more transcription factor activators, one or more growth factors, one or more cytokines, one or more chemokines, and / or with co-culture with non-fibroblast cells.

25. The method of claim 22, wherein the fibroblasts are activated in vivo with one or more nucleic acids, one or more transcription factor activators, one or more growth factors, one or more cytokines, one or more chemokines, and / or with co-culture with non-fibroblast cells.

26. The method of claim 22, wherein the providing is into the blood stream of the individual.

27. The method of claim 22 or 23, wherein following the providing step, there is modulation of the expression of pathogenic levels of one or more of IL-1, IL-2, IL-6, IL-10, IL-21, IL-22, IL-17, IL-12, TNF, INF- y, TGF- pl.

28. A method of epigenetic reprogramming of localized damaged tissue and / or epithelial cells in an individual, comprising the step of providing to the individual an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials; wherein the fibroblasts are activated with one or more agents and / or the fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, or fibroblast-derived materials come from activated fibroblasts are activated with one or more agents; and(2) one or more types of immune cells or derivative agent.

29. The method of claim 28, wherein the providing is into the bloodstream of the individual.

30. A method of enabling the activation and migration of localized stem cell niches to replace pathogenetic fibroblasts and / or pathogenic epithelial cells in the damaged tissues of an individual with SLE, comprising the step of providing to the individual an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials; wherein the fibroblasts are activated with one or more agents and / or the fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, or fibroblast-derived materials come from activated fibroblasts are activated with one or more agents; and(2) one or more types of immune cells or derivative agent.

31. The method of claim 30, wherein the providing is into the bloodstream of the individual.

32. A method of eliciting an immune response in an individual with SLE, comprising the step of providing to the individual an effective amount of:(1) fibroblasts, fibroblast spheroids, fibroblast exosomes, fibroblast lysates, fibroblast apoptotic bodies, and / or fibroblast-derived materials; and(2) one or more adjuvants.

33. The method of claim 32, wherein the adjuvant is chemical product-based, viral productbased, bacterial product-based, or a mixture thereof.

34. The method of claim 33, wherein the adjuvant is an aluminum salts.

35. The method of any one of claims 32-34, wherein the fibroblast cells are genetically modified to overexpress specific immunosuppressive molecules.

36. The method of claim 35, wherein the immunosuppressive molecule is one or more chemokines, one or more cytokines, or a mixture thereof.

37. The method of claim 36, wherein the chemokine is CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, or a combination thereof.

38. The method of claim 36 or 37, wherein the cytokine is IL-lra, IL-4, IL-5, IL-6, IL-10, IL- 11, IL-13, TGF-beta, or a combination thereof.

39. The method of any one of claims 32-38, wherein the fibroblast cells are genetically modified.

Images