Methods and compositions for treating alopecia using fibroblasts and fibroblast-derived products

Abstract

Disclosed herein are methods and compositions for the treatment and prevention of alopecia, including alopecia areata.Embodiments are directed to methods for stimulating or promoting hair follicle regeneration.Some embodiments are directed to methods for treating alopecia areata, comprising administering fibroblasts and / or fibroblast-derived products to subjects suffering from alopecia areata.In some embodiments, the fibroblast-derived products are fibroblast-derived conditioned medium, exosomes, or apoptotic bodies, which are formulated for topical use to treat or prevent alopecia areata.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 193,403, filed May 26, 2021, which is incorporated by reference in its entirety.

[0002] [Technical field] Embodiments of the present disclosure encompass at least the fields of cell biology and regenerative medicine. [Background technology]

[0003] Hair loss, also called alopecia or baldness, refers to the loss of hair from parts of the head or body. The extent of hair loss can vary from a small area to the entire body. Alopecia can be caused by psychological distress [1,2] or may be drug-induced [3-7] or caused by other causes. Common types of alopecia include androgenetic alopecia (including male pattern and female pattern hair loss), alopecia areata, and thinning hair known as telogen effluvium [8-17]. Causes of male pattern hair loss include a combination of genetics and androgen, causes of female pattern hair loss are unknown, causes of alopecia areata are autoimmune, and causes of telogen effluvium are generally physical or psychological stressful events [18,19].

[0004] The hair follicle is a regenerative organ that allows stem cells to regenerate on a large scale. It consists of the outer root sheath, the inner root sheath, and the hair shaft. Proliferating hair matrix cells give rise to the inner root sheath and the hair shaft, which are surrounded by the dermal papilla, which is made up of specialized mesenchymal cells. The dermal papilla directs the formation of the hair follicle, while the characteristics of the hair follicle are acquired by information from the epithelium. The lower part of the hair follicle undergoes a growth cycle that includes anagen (active growth), catagen (destruction), and telogen (rest). These various phases last for different periods of time, depending on the location and function of the hair follicle. During anagen, hair matrix cells rapidly proliferate and migrate upwards before differentiating into the cell types of the inner root sheath and the hair shaft. During catagen, the lower hair follicle undergoes apoptotic death and the dermal papilla migrates upwards until it reaches the area below the bulge. The dermal papilla remains there for the duration of the telogen phase. When the dermal papilla recruits stem cells from the bulge, anagen begins anew and the hair follicle regenerates through proliferation and differentiation.

[0005] Alopecia management is commonly achieved through medications and surgery

[20] . The drugs currently used to treat alopecia are minoxidil, finasteride, and dutasteride [21-23]. Surgical management of alopecia involves hair transplantation. Hair transplantation is usually performed under local anesthesia. The surgeon transplants healthy hair from the occipital or temporal region to the thinning area. The procedure takes 4-8 hours and additional sessions can be performed to thicken the hair. Traditional hair transplantation is hindered by the lack of sufficient hair in the non-balding areas. Stem cell therapy presents a new strategy for the treatment and management of alopecia

[24] . However, in the context of traditional hair transplantation, the administration of hair follicle stem cells to balding scalp areas is highly inefficient because the skin in such areas lacks the growth factors required for the growth of new hair follicles.

[0006] There is a need for further and improved methods and compositions for the treatment of alopecia, including androgenetic alopecia and alopecia areata. Summary of the Invention

[0007] Aspects of the present disclosure relate to fibroblasts and fibroblast-derived products and their use in treating and preventing alopecia. Certain embodiments relate to a method for treating alopecia, including androgenetic alopecia and alopecia areata, comprising providing fibroblasts or fibroblast-derived products to a subject with alopecia. Also disclosed is a method for preventing alopecia, including androgenetic alopecia and alopecia areata, comprising providing fibroblasts or fibroblast-derived products to a subject at risk of developing alopecia. The fibroblasts of the present disclosure may be subjected to conditions sufficient to enhance regenerative activity and / or angiogenic activity. Pharmaceutical compositions comprising fibroblasts or fibroblast-derived products for use in treating or preventing alopecia, including compositions formulated for topical administration such as soaps, shampoos, ointments, etc., are also contemplated herein. Thus, embodiments of the present disclosure provide a new strategy for treating and preventing alopecia, including androgenetic alopecia and alopecia areata.

[0008] The embodiments of the present disclosure include a method for treating alopecia, a method for preventing alopecia, a method for slowing hair loss, a method for stimulating hair growth, and a method for reducing inflammation. The methods of the present disclosure may include one or more of diagnosing a subject for alopecia, diagnosing a subject for androgenetic alopecia, diagnosing a subject for alopecia areata, diagnosing a subject for skin inflammation, providing a subject with fibroblasts, providing a fibroblast-derived product, and providing a fibroblast-derived exosome to a subject. Also disclosed are compositions comprising one or more of fibroblasts, dermal fibroblasts, fibroblast-derived products, fibroblast-derived conditioned medium, and fibroblast-derived exosomes. Any one or more of the steps or components described above may be excluded from certain embodiments of the present disclosure.

[0009] Disclosed herein is a method for treating or preventing alopecia in a subject, comprising providing an effective amount of fibroblasts or fibroblast-derived products to the subject in some embodiments. In some embodiments, the alopecia is associated with inflammation of the dermis. In some embodiments, the alopecia is androgenetic alopecia, male pattern alopecia, female pattern alopecia, alopecia areata, or telogen effluvium. In some embodiments, the alopecia is androgenetic alopecia. In some embodiments, the alopecia is alopecia areata. In some embodiments, the fibroblasts or fibroblast-derived products are administered topically. In some embodiments, the fibroblasts or fibroblast-derived products are administered intradermally and / or transdermally. In some embodiments, the fibroblasts or fibroblast-derived products are administered to an area of ​​the scalp of the subject. In some embodiments, the method further comprises providing a regenerative light source to an area of ​​the scalp of the subject. In some embodiments, the regenerative light source is a laser light.

[0010] In some embodiments, the method includes providing an effective amount of fibroblasts to the subject. In some embodiments, the fibroblasts are allogeneic, xenogeneic, or autologous to the subject. In some embodiments, the fibroblasts are derived from skin, fat, bone marrow, omental tissue, blood, deciduous teeth, fallopian tubes, testicular tissue, ovarian tissue, hair follicles, endometrial tissue, or combinations thereof. In some embodiments, the fibroblasts are dermal fibroblasts. In some embodiments, the fibroblasts have been previously subjected to conditions sufficient to enhance regenerative activity. In some embodiments, the conditions are sufficient to upregulate HIF1α expression in the fibroblasts. In some embodiments, the conditions are sufficient to upregulate HIF1α by at least 25% relative to an untreated control. In some embodiments, the conditions are sufficient to enhance nuclear translocation of HIF1α in the fibroblasts. In some embodiments, the conditions include an agent capable of mimicking hypoxia. In some embodiments, the conditions include culturing the fibroblasts with carbon monoxide. In some embodiments, the conditions include exposing the fibroblasts to a gas composition comprising about 0% to about 79% by weight nitrogen, about 21% to about 99.999999% by weight oxygen, and about 0.0000001% to about 0.3% by weight carbon monoxide. In some embodiments, the gas composition comprises 0% nitrogen and about 99.999999% oxygen. In some embodiments, the gas composition comprises between about 0.005% and about 0.05%. In some embodiments, the fibroblasts have been previously subjected to conditions sufficient to enhance survival and / or activity of the fibroblasts. In some embodiments, the conditions include treatment with an epigenetic regulator. In some embodiments, the epigenetic regulator is a histone deacetylase inhibitor. In some embodiments, the histone deacetylase inhibitor is valproic acid, vorinostat, entinostat, panobinostat, trichostatin A, mocetinostat, belinostat, FK228, MC1568, tubastatin, sodium butyrate, or sulforaphane.In some embodiments, the epigenetic regulator is a DNA methyltransferase inhibitor. In some embodiments, the DNA methyltransferase inhibitor is 5-azacytidine. In some embodiments, the condition comprises culturing the fibroblasts with a GSK-3 inhibitor. In some embodiments, the GSK-3 inhibitor is lithium or a lithium salt.

[0011] In some embodiments, the method includes providing an effective amount of a fibroblast-derived product to the subject. In some embodiments, the fibroblast-derived product is obtained from fibroblasts derived from skin, fat, bone marrow, omentum tissue, blood, milk teeth, fallopian tubes, testicular tissue, ovarian tissue, hair follicles, endometrial tissue, or combinations thereof. In some embodiments, the fibroblast-derived product is obtained from dermal fibroblasts. In some embodiments, the fibroblast-derived product is obtained from fibroblasts that are allogeneic, xenogeneic, or autologous to the subject. In some embodiments, the fibroblast-derived product comprises conditioned medium from a culture of fibroblasts. In some embodiments, the conditioned medium is obtained from a culture of fibroblasts in EMEM, α-MEM, IMDM, DMEM, or RPMI. In some embodiments, the conditioned medium is generated by culturing adherent fibroblasts in a suspension that includes nutrients for the fibroblasts. In some embodiments, the liquid suspension includes growth factors. In some embodiments, the liquid suspension includes stem cell exosomes. In some embodiments, the stem cell exosomes are mesenchymal stem cell-derived exosomes. In some embodiments, the mesenchymal stem cells are derived from umbilical cord, bone marrow, skin, fallopian tube, adipose tissue, endometrial tissue, peripheral blood, menstrual blood, hair follicles, or combinations thereof. In some embodiments, the liquid suspension comprises a neutralizing agent capable of inhibiting the activity of one or more inflammatory mediators. In some embodiments, the neutralizing agent is a monoclonal antibody, an antisense oligonucleotide, or a gene editing system. In some embodiments, the neutralizing agent is an antibody capable of binding to interleukin-1, interleukin-6, interleukin-8, interleukin-9, interleukin-11, interleukin-12, interleukin-15, interleukin-17, interleukin-18, interleukin-21, interleukin-23, interleukin-27, interleukin-33, TNFα, interferon gamma, TNFβ, or lymphotoxin.In some embodiments, the liquid suspension comprises VEGF, EGF, PGDF-BB, IGF-1, HGF-1, NGF, BDNF, IL-3, IL-4, IL-10, IL-13, IL-20, IL-35. In some embodiments, the fibroblast-derived product is a fibroblast-derived microvesicle. In some embodiments, the fibroblast-derived product is a fibroblast-derived exosome. In some embodiments, the exosomes are enriched from conditioned medium from fibroblasts. In some embodiments, the exosomes are enriched by a) functionalizing a support with a single-stranded oligonucleotide to produce a functionalized support; b) incubating the functionalized support with a ligand having a tag complementary to the single-stranded oligonucleotide to obtain an immobilized ligand; c) incubating the immobilized ligand with conditioned medium to allow capture of exosomes by binding between the immobilized ligand and the exosomes, to obtain a substrate for the captured exosomes; and d) incubating the captured exosomes with a restriction enzyme. In some embodiments, the ligand is an antibody, a peptide, or an aptamer. In some embodiments, the support is a magnetic bead, a membrane, a cell culture plate, a test tube, a slide, a microplate, a microchannel, a pillar, or a disk-shaped piece. In some embodiments, the support is functionalized with a ligand via covalent binding or biotinylation. In some embodiments, the restriction enzyme is a DNAse. In some embodiments, the ligand is an antibody capable of binding to an exosome-specific tetraspanin. In some embodiments, the ligand is an antibody capable of binding to MHC class I and II, HSP70, Annexin V, flotillin, or EpCAM. In some embodiments, the conditioned medium is derived from a culture of fibroblasts in EMEM, α-MEM, IMDM, DMEM, or RPMI. In some embodiments, the fibroblast-derived product is apoptotic vesicles derived from fibroblasts. In some embodiments, the fibroblast-derived product is a nucleic acid derived from fibroblasts. In some embodiments, the fibroblast-derived product is obtained from fibroblasts that have been subjected to conditions sufficient to enhance regenerative activity.In some embodiments, the conditions are sufficient to upregulate HIF1α expression in fibroblasts. In some embodiments, the conditions are sufficient to upregulate HIF1α by at least 25% relative to untreated controls. In some embodiments, the conditions are sufficient to enhance nuclear translocation of HIF1α in fibroblasts. In some embodiments, the conditions include an agent capable of mimicking hypoxia. In some embodiments, the conditions include culturing the fibroblasts with carbon monoxide. In some embodiments, the conditions include exposing the fibroblasts to a gas composition comprising about 0% to about 79% by weight nitrogen, about 21% to about 99.999999% by weight oxygen, and about 0.0000001% to about 0.3% by weight carbon monoxide. In some embodiments, the gas composition comprises 0% nitrogen and about 99.999999% oxygen. In some embodiments, the gas composition comprises between about 0.005% and about 0.05%. In some embodiments, the fibroblast-derived product is obtained from fibroblasts subjected to conditions sufficient to enhance fibroblast survival and / or activity. In some embodiments, the conditions include treatment with an epigenetic regulator. In some embodiments, the epigenetic regulator is a histone deacetylase inhibitor. In some embodiments, the histone deacetylase inhibitor is valproic acid, vorinostat, entinostat, panobinostat, trichostatin A, mocetinostat, belinostat, FK228, MC1568, tubastatin, sodium butyrate, or sulforaphane. In some embodiments, the epigenetic regulator is a DNA methyltransferase inhibitor. In some embodiments, the DNA methyltransferase inhibitor is 5-azacytidine. In some embodiments, the conditions include culturing the fibroblasts with a GSK-3 inhibitor. In some embodiments, the GSK-3 inhibitor is lithium or a lithium salt.

[0012] In some embodiments, the method further comprises providing to the subject an effective amount of diphenylcyclopropenone. In some embodiments, the method further comprises providing to the subject an effective amount of a c-Met activator (e.g., HGF). In some embodiments, the method further comprises providing to the subject one or more agents capable of stimulating HGF production.

[0013] In some embodiments, the subject has a decreased number of cells expressing FoxP3 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of cells expressing interleukin-10 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of cells expressing interleukin-4 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of cells expressing interleukin-13 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of cells expressing interleukin-35 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of regulatory T cells compared to an age-matched control subject. In some embodiments, the subject has a decreased number of myelosuppressor cells compared to an age-matched control subject. In some embodiments, the subject has a decreased number of B cells expressing TIM-1 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of B cells expressing IL-10 compared to an age-matched control subject. In some embodiments, the subject has a decreased number of regulatory B cells compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interferon gamma compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing TNFα compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-1 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-2 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-6 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-8 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-11 compared to an age-matched control subject.In some embodiments, the subject has an increased number of cells expressing interleukin-12 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-15 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-17 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-18 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-21 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-23 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-27 compared to an age-matched control subject. In some embodiments, the subject has an increased number of cells expressing interleukin-33 compared to an age-matched control subject. In some embodiments, the subject has an increased number of natural killer cells compared to an age-matched control subject. In some embodiments, the subject has increased numbers of neural killer T cells compared to an age-matched control subject. In some embodiments, the subject has increased numbers of Th1 cells compared to an age-matched control subject. In some embodiments, the subject has increased numbers of Th17 cells compared to an age-matched control subject.

[0014] Also disclosed herein, in some embodiments, is a method for treating or preventing androgenetic alopecia in a subject, comprising providing an effective amount of fibroblast-derived exosomes to the subject. In some embodiments, is a method for treating or preventing androgenetic alopecia in a subject, comprising providing an effective amount of fibroblast-derived conditioned medium to the subject.

[0015] Also disclosed herein, in some embodiments, is a method for treating or preventing alopecia areata in a subject, comprising providing an effective amount of fibroblast-derived exosomes to the subject. In some embodiments, is a method for treating or preventing alopecia areata in a subject, comprising providing an effective amount of fibroblast-derived conditioned medium to the subject.

[0016] Any method related to a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as the "use" of any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0017] It is specifically contemplated that limitations discussed with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. Any embodiment discussed with respect to one aspect of the present disclosure may also be applied to other aspects of the present disclosure, and vice versa. For example, any step of a method described herein may be applied to any other method. Furthermore, any method described herein may have the exclusion of any step or combination of steps. Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of different examples or embodiments discussed elsewhere in this application, for example, in the summary, detailed description, and claims.

[0018] The foregoing has outlined, rather broadly, the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as defined by the appended claims. The novel features believed characteristic of the designs disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] [I. Definition Example] Following long-standing patent law practice, the words "a" and "an" herein, including in the claims, when used in conjunction with the word comprising, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

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

[0021] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the art of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0022] As used herein, "allogeneic" refers to tissues or cells or other materials from another body that are derived from one or more individuals of the same species, but that are or may become immunologically incompatible in their natural environment.

[0023] As used herein, a "cell line" refers to a cell population formed by one or more subcultures of a primary cell culture. Each round of re-cultivation is called a passage. When cells are subcultured, they are referred to as subcultured cells. A particular cell population, or cell line, may be referred to or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be referred to as a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, is referred to as a P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the secondary culture, the cells become a tertiary culture (P2 or passage 2). It will be understood by those skilled in the art that there may be many population doublings during the period of subculture. The proliferation of cells (e.g., number of population doublings) during the period between passages depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passages.

[0024] As used herein, "conditioned medium" refers to a medium in which a particular cell or cell population has been cultured for a period of time, after which the medium has been removed and separated from the cells. When cells are cultured in medium, they may secrete cellular factors that provide trophic factors to other cells. Such trophic factors include, but are not limited to, hormones, cytokines, extracellular matrices (ECM), proteins, vesicles, antibodies, granules, and the like. In this example, the medium containing the cellular factors is the conditioned medium. Conditioned medium "from" a cell or cell population refers to conditioned medium obtained from a cell or cell population as described above. Thus, in one example, conditioned medium from fibroblasts refers to medium in which fibroblasts have been cultured for a period of time and then removed and the medium separated from the fibroblasts.

[0025] As used herein, a "trophic factor" refers to a substance that promotes and / or supports the survival, growth, proliferation, and / or maturation of a cell. Alternatively, or in addition, a trophic factor stimulates an increase in the activity of a cell.

[0026] The term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes unspecified elements, steps, or ingredients. The phrase "consisting essentially of" limits the scope of the described subject matter to those materials or steps specified and that do not materially affect its basic and novel characteristics. It is contemplated that an embodiment described in the context of "consisting of" can also be implemented in the context of the term "consisting of" or "consisting essentially of."

[0027] The terms "reduce," "inhibit," "reduce," "suppress," "diminish," "prevent," and grammatical equivalents (including "lower," "lesser," etc.) in relation to the expression of any symptom in an untreated subject versus a treated subject mean that the amount and / or magnitude of the symptom in the treated subject is less than the amount and / or intensity of the symptom in an untreated subject by any amount recognized as clinically appropriate by medically trained personnel. In one embodiment, the amount and / or magnitude of the symptom in the treated subject is at least 10% less, at least 25% less, at least 50% less, at least 75% less, and / or at least 90% less than the amount and / or magnitude of the symptom in an untreated subject.

[0028] As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and / or "effective amount," and refers to an amount of a compound that elicits a biological, cosmetic, or clinical response desired by the skilled artisan in an individual in need thereof. As an example, an effective amount is an amount sufficient to reduce the immunogenicity of a cell population. The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by one of skill in the art using the guidance provided herein. For example, the effective amount can be extrapolated from the in vitro and in vivo assays described herein. One of skill in the art will recognize that an individual's condition can be monitored throughout the course of treatment, and the effective amount of the compound or composition disclosed herein administered can be adjusted accordingly.

[0029] As used herein, the terms "therapy," "treatment," or "treating" refer to an intervention that seeks to alter the natural course of the individual or cell being treated, and may be performed prophylactically or during the pathological course of a disease or condition. Treatment is performed to achieve one or more of a variety of desired results, such as, for example, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, remission, or improved prognosis.

[0030] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0031] Various aspects of the present disclosure can be described in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered as specifically disclosing all possible subranges as well as individual numerical values ​​within the range as if they were explicitly stated. For example, the description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. When a range is present, the range can include the endpoints of the range.

[0032] The term "subject" as used herein may be used interchangeably with the terms "individual" or "patient" and generally refers to an individual in need of treatment. The subject may be a mammal, such as a human, dog, cat, horse, pig, or rodent. The subject may be, for example, a patient having or suspected of having a disease or condition related to bone. For subjects having or suspected of having a condition directly or indirectly related to bone, the condition may be of one or more types. The subject may have or be suspected of having a disease. The subject may be asymptomatic. The subject may be of any gender. The subject may be of a certain age, for example, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years old or older.

[0033] The term "fibroblast-derived product" (also "fibroblast-associated product" or "fibroblast derivative") as used herein refers to a molecular or cellular agent derived or obtained from one or more fibroblasts. In some cases, the fibroblast-derived product is a molecular agent. Examples of fibroblast-derived molecular products include conditioned medium from fibroblast cultures, microvesicles obtained from fibroblasts, exosomes obtained from fibroblasts, apoptotic vesicles obtained from fibroblasts, nucleic acids (e.g., DNA, RNA, mRNA, miRNA, etc.) obtained from fibroblasts, proteins (e.g., growth factors, cytokines, etc.) obtained from fibroblasts, and lipids obtained from fibroblasts. In some cases, the fibroblast-derived product is a cellular agent. Examples of cellular fibroblast-derived products include cells (e.g., stem cells, hematopoietic cells, neural cells, etc.) produced by differentiation and / or dedifferentiation of fibroblasts.

[0034] "Carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline in water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Aqueous saline solutions, dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository, using conventional binders and carriers, such as triglycerides. The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those formed with free carboxyl groups from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions contain a therapeutically effective amount of the compound, preferably in purified form, together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should be appropriate for the mode of administration.

[0035] II. Methods and Compositions for Treating or Preventing Alopecia Aspects of the present disclosure relate to methods and compositions for treating or preventing alopecia.Alopecia (also "hair loss") describes any loss of hair from any part of the head of a subject.Various types of alopecia are recognized in the art, including but not limited to androgenetic alopecia (e.g., male pattern alopecia, female pattern alopecia), alopecia areata, and telogen effluvium.In some embodiments, a method for treating alopecia areata (AA) is disclosed.

[0036] As disclosed herein, fibroblasts and / or fibroblast-derived products (e.g., exosomes, conditioned media, etc.) are useful for treating and preventing alopecia. Accordingly, embodiments of the present disclosure are directed to methods for treating or preventing alopecia, comprising providing an effective amount of fibroblasts or fibroblast-derived products to a subject. In some embodiments, the methods of the present disclosure comprise administering fibroblasts to a subject. In some embodiments, the methods of the present disclosure comprise administering a fibroblast-derived product to a subject. In certain embodiments, methods are disclosed that comprise administering fibroblast-derived exosomes, or a composition comprising fibroblast-derived exosomes, to a subject for treating or preventing alopecia. Administration of such compositions includes, for example, topical administration, transdermal administration, and intradermal administration. Pharmaceutical compositions comprising fibroblasts or fibroblast-derived products include, for example, soaps, shampoos, ointments, and other such formulations.

[0037] Fibroblast or fibroblast-derived product can be provided to subject in combination with one or more therapeutic agents or therapies.In one example, fibroblast or fibroblast-derived product of the present disclosure is administered to subject in combination with diphenylcyclopropenone (e.g., simultaneously with diphenylcyclopropenone, before diphenylcyclopropenone, or after diphenylcyclopropenone).In another example, fibroblast or fibroblast-derived product of the present disclosure is administered to subject in combination with HGF production stimulant (e.g., simultaneously with HGF production stimulant, before HGF production stimulant, or after HGF production stimulant).

[0038] Fibroblasts of the present disclosure, including fibroblasts administered to a subject and fibroblasts cultured to obtain fibroblast-derived products, can be subjected to conditions sufficient to enhance the regenerative activity of fibroblasts. In one example, fibroblasts are subjected to such conditions before administration to a subject. In another example, fibroblasts are subjected to such conditions before obtaining fibroblast-derived products from fibroblasts. Such conditions include, for example, conditions sufficient to upregulate HIF1α expression in fibroblasts compared to control untreated fibroblasts. In some embodiments, fibroblasts are subjected to conditions sufficient to upregulate HIF1α expression by at least, at most, or by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more, or any range or value derivable therein. Such conditions may be sufficient to enhance nuclear translocation of HIF1α in cells (i.e., increase the amount of HIF1α in the nucleus of cells) compared to control untreated fibroblasts. Such conditions may consist of hypoxic conditions and / or treatment with one or more agents to simulate hypoxia. In some embodiments, the fibroblasts of the present disclosure are cultured with carbon monoxide. In some embodiments, culturing the fibroblasts with carbon monoxide comprises exposing the fibroblasts to a gas composition comprising about 0% to about 79% by weight nitrogen (or any range or value therein), about 21% to about 99.999999% by weight oxygen (or any range or value therein), and 0.0000001% to about 0.3% by weight carbon monoxide (or any range or value therein).

[0039] Fibroblasts of the present disclosure, including fibroblasts administered to a subject and fibroblasts cultured to obtain fibroblast-derived products, may be subjected to conditions that enhance fibroblast survival and / or activity. In one example, the fibroblasts are subjected to such conditions before administration to a subject. In another example, the fibroblasts are subjected to such conditions before obtaining fibroblast-derived products from the fibroblasts. In some embodiments, such conditions include treatment with an epigenetic modifier. In some embodiments, the epigenetic modifier is a histone deacetylase inhibitor, such as, for example, valproic acid, vorinostat, entinostat, panobinostat, trichostatin A, mocetinostat, belinostat, FK228, MC1568, tubastatin, sodium butyrate, or sulforaphane. In some embodiments, the epigenetic modifier is a DNA methyltransferase inhibitor, such as, for example, 5-azacytidine. In some embodiments, such conditions include treatment with a glycogen synthase kinase-3 (GSK-3) inhibitor, such as a GSK-3β inhibitor, hi some embodiments, the GSK-3 inhibitor is lithium or a lithium salt.

[0040] Embodiments of the present disclosure include the use of fibroblasts and / or fibroblast-derived products (e.g., exosomes) to reduce T cell infiltration near the hair bulb in AA patients. Local immunotherapy with diphenylcyclopropenone (DCP) is known to have therapeutic effects in AA patients [25-41]. Using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) with RNA extracted from scalp biopsies of AA patients and healthy controls before and after successful treatment with DCP, researchers detected T cell responses in all types of untreated AA accompanied by increased steady-state mRNA levels of interferon (IFN)-γ, interleukin (IL)-1β, and IL-2. After DCP administration, the expression of IFN-γ decreased but was still higher than the constitutive levels seen in the control group. Meanwhile, the mRNA expression of IL-2, IL-8, IL-10, and tumor necrosis factor α increased. These results suggest the presence of cytokines involved in the pathogenesis of AA. There is a TH1-type cytokine pattern in untreated AA that is altered by cytokines secreted during DCP treatment. IL-10 has recently been reported as an immunomodulator of TH1 responses, and it has therefore been hypothesized that after DCP application, basal keratinocytes or lesional T cells secrete bioactive IL-10, resulting in an inhibitory effect on lesional T lymphocytes

[42] .

[0041] In one embodiment of the present disclosure, fibroblasts are administered to a subject at a frequency and concentration sufficient to reduce interferon gamma expression. In another embodiment, fibroblasts and / or fibroblast-derived products (e.g., exosomes) are administered with DCP to enhance the effectiveness of DCP treatment in increasing hair regrowth / reducing hair loss. In another embodiment of the present invention, fibroblast-derived products such as exosomes are administered at a frequency and concentration sufficient to reduce interferon gamma expression. Certain exemplary methods for exosome generation for immunomodulatory therapy are art-recognized and contemplated herein

[43] .

[0042] In one embodiment of the present disclosure, oxidative / inflammatory stress is utilized as a marker of hair loss propensity to guide the dosage and / or frequency of administration of fibroblasts and / or fibroblast-derived products. Means for assessing oxidative / inflammatory stress have been described in the art and are contemplated herein [44-66]. In some circumstances, the present disclosure provides for administration of fibroblasts and / or fibroblast-derived products in conjunction with antioxidants.

[0043] In some embodiments, a stimulator of HGF (i.e., an agent capable of stimulating HGF production) is added to promote hair follicle cell proliferation [67-70].

[0044] [III. Fibroblasts and cultured cells] Aspects of the present disclosure include cells useful in therapeutic methods and compositions. Cells disclosed herein include, for example, fibroblasts, stem cells (e.g., hematopoietic stem cells or mesenchymal stem cells), and endothelial progenitor cells. A type of cell (e.g., fibroblasts) may be used alone or in combination with other types of cells. For example, fibroblasts can be isolated and provided to a subject alone or in combination with one or more stem cells. In some embodiments, disclosed herein are fibroblasts capable of alopecia, including androgenetic alopecia and alopecia areata. In some embodiments, the fibroblasts of the present disclosure adhere to plastic. In some embodiments, the fibroblasts express CD73, CD90, and / or CD105. In some embodiments, the fibroblasts are CD14, CD34, CD45, and / or HLA-DR negative. In some embodiments, the fibroblasts have the ability to differentiate into osteogenic, chondrogenic, and adipogenic cells.

[0045] The compositions of the present disclosure can be obtained from isolated fibroblasts or populations thereof that can proliferate and differentiate into ectoderm, mesoderm, or endoderm. In some embodiments, the isolated fibroblasts express at least one of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, or Stella markers. In some embodiments, the isolated fibroblasts do not express at least one of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, or CD90 cell surface proteins. Such isolated fibroblasts can be used as a source of conditioned medium. The cells can be cultured alone or in the presence of other cells to further upregulate the production of growth factors in the conditioned medium.

[0046] In some embodiments, the fibroblasts of the present disclosure express telomerase, Nanog, Sox2, β-III-tubulin, NF-M, MAP2, APP, GLUT, NCAM, NeuroD, Nurr1, GFAP, NG2, Olig1, alkaline phosphatase, vimentin, osteonectin, osteoprotegerin, adipsin, erythropoietin, SM22-α, HGF, c-MET, α-1-Antriptrypsin, ceruloplasmin, AFP, PEPCK1, BDNF, NT-4 / 5, TrkA, BMP2, BMP4, FGF2, FGF4, PDGF, PGF, TGFα, TGFβ, and / or VEGF.

[0047] Fibroblasts can be grown and utilized by administration per se, or can be cultured in a growth medium to obtain a conditioned medium. The term growth medium generally refers to a medium sufficient for the culture of fibroblasts. In particular, one medium for culturing the cells disclosed herein comprises Dulbecco's Modified Essential Medium (DMEM). One example is DMEM-low glucose (also referred to herein as DMEM-LG) (Invitrogen®, Carlsbad, Calif.). DMEM-low glucose is preferably supplemented with 15% (v / v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone). TM The medium is supplemented with 0.01% (v / v) 2-mercaptoethanol (Sigma, St. Louis, MO), 0.0 ...

[0048] The medium may be serum-containing, serum-free, or xeno-free. From the viewpoint of preventing contamination with components derived from different animals, serum derived from the same animal as the stem cells may be used. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and also includes a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). The medium may or may not contain a serum substitute. Serum substitutes may include materials that suitably contain albumin (e.g., albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin, or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol giserol, or equivalents thereof. Serum substitutes may be prepared, for example, by the methods disclosed in WO 98 / 30679, which is incorporated herein in its entirety. Alternatively, more conveniently, any commercially available material can be used, including Knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), Glutamax (Gibco), etc. One or more of the media components can be added at a concentration of at least, at most, 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, μg / ml, mg / ml, or any range derivable therein.

[0049] In some cases, different growth media are used or different supplements are provided, which are usually referred to as supplements to growth media. Also in the context of the present invention, the term standard growth conditions as used herein refers to the culture of cells at 37° C. in a standard atmosphere with 5% CO2, with relative humidity maintained at about 100%. It should be understood that while the above conditions are useful for culture, such conditions can be changed by a person skilled in the art who understands the options available in the art for culturing cells, such as by changing temperature, CO2, relative humidity, oxygen, growth medium, etc.

[0050] Also disclosed herein are cultured cells. Various terms are used for cultured cells. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions ("in culture" or "culture"). Primary cell culture refers to the culture of cells, tissues, or organs taken directly from an organism prior to the first partial culture. Cells expand in culture when placed in a growth medium under conditions that promote cell growth and / or division, resulting in a larger population of cells. When cells are expanded in culture, the rate of cell growth is sometimes measured by the time required for the number of cells to double, i.e., the "doubling time."

[0051] Fibroblasts used in the disclosed methods can undergo at least 25, 30, 35, or 40 doublings before reaching a senescent state. 14 Methods are provided for inducing cells that can double to reach more than about 10 cells in culture. 3 About 10 6 cells / cm 2 When sown at 10 14 , 10 15 , 10 16 , or 10 17The present invention relates to a method for inducing cells capable of doubling sufficiently to produce 10 or more cells. Preferably, these numbers of cells are produced within 80, 70, or 60 days. In some embodiments, the fibroblasts used are isolated and expanded and have one or more markers selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, and HLA-C. In some embodiments, the fibroblasts do not produce one or more of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, or HLA-DQ.

[0052] When referring to cultured cells, including fibroblasts and vertebral cells, the term senescence (also called "replicative senescence" or "cellular senescence") refers to a property resulting from finite cell culture, i.e., the inability to proliferate beyond a finite number of population doublings (sometimes called the Hayflick limit). Although cellular senescence was first reported using fibroblast-like cells, most normal human cell types that can be successfully grown in culture undergo cellular senescence. The in vitro life span of different cell types varies, but the maximum life span is usually less than 100 population doublings (the number of doublings before all cells in the culture become senescent and, consequently, the culture can no longer divide). Senescence is not dependent on calendar time, but rather is measured by the number of cell divisions, or population doublings, that the culture has undergone. Thus, cells rendered quiescent by the withdrawal of essential growth factors can resume growth and division upon reintroduction of the growth factors, and subsequently undergo the same number of doublings as comparable cells grown continuously. Similarly, cells can be frozen in liquid nitrogen, undergo various numbers of cell doublings, and then thawed and cultured, undergoing virtually the same number of cell doublings as cells cultured without freezing. Senescent cells are not dead or dying cells; they are resistant to programmed cell death (apoptosis) and can remain non-dividing for up to three years. These cells are alive and metabolically active, but do not divide.

[0053] In some cases, fibroblasts are obtained from a biopsy, and the donor providing the biopsy may be the individual being treated (autologous) or a different donor than the individual being treated (allogeneic). When allogeneic fibroblasts are utilized for an individual, the fibroblasts may be from one or more donors. In one embodiment, the fibroblasts are used from a young donor. In another embodiment, genes are introduced into the fibroblasts to allow for enhanced proliferation and overcoming the Hayflick limit. After the cells are induced, they can be grown in culture using standard cell culture techniques.

[0054] An example of a procedure for harvesting fibroblasts from a biopsy is shown. Skin tissue (dermal and epidermal layers) can be biopsied from the rear of the ear of a subject. In one embodiment, the starting material consists of three 3 mm punch skin biopsies taken using standard aseptic techniques. The biopsies are taken by a treating physician and placed in vials containing sterile phosphate buffered saline (PBS). The biopsies are shipped in a refrigerated shipper back to the manufacturing facility. In one embodiment, upon arrival at the manufacturing facility, the biopsies are inspected and, upon acceptance, are transported directly to the manufacturing area. After the process begins, the biopsy tissue is washed prior to enzymatic digestion. After washing, Liberase digestion enzyme solution is added without mincing and the biopsy tissue is incubated at 37.0±2° C. for 1 hour. The digestion time of the biopsy tissue is a critical process parameter that affects the viability and proliferation rate of cultured cells. Liberase is a collagenase / neutral protease enzyme cocktail, formulated by Lonza Walkersville, Inc. and available in unformulated form from Roche Diagnostics Corp. Alternatively, other commercially available collagenases, such as Serva Collagenase NB6 (Helidelburg, Germany), may be used. After digestion, the enzyme is neutralized by adding Initiator Growth Medium (IMDM, GA, 10% fetal bovine serum (FBS)), and the cells are pelleted by centrifugation and resuspended in 5.0 mL of Initiator Growth Medium. Alternatively, no centrifugation is performed and the enzyme is completely inactivated by the addition of Initiator Medium alone. Initiator Medium is added before seeding the cell suspension into T-175 cell culture flasks to initiate cell growth and expansion. T-75, T-150, T-185, or T-225 flasks can also be used instead of T-75 flasks. Cells are cultured at 37.0 ± 2°C, 5.0 ± 1.0% CO2 and fed fresh complete growth medium every 3-5 days. All feeds within the process are performed by removing half of the complete growth medium and replacing it with an equal amount of fresh medium. Alternatively, the entire volume can be fed. Cells prior to passage should not remain in the T-175 flask for more than 30 days. Confluence is monitored throughout the process to ensure appropriate seeding density when splitting cultures.When cells reach 40% or greater confluence in the T-175 flask, they are passaged by removing the spent medium, washing the cells, and treating with trypsin-EDTA to release the adherent cells in the flask into solution. The cells are then trypsinized and seeded into a T-500 flask for continued cell growth. Instead of the T-500 flask, one or two T-300 flasks, a single-layer cell stack (1CS), a single-layer cell factory (1CF), or a double-layer cell stack (2CS) can also be used. Morphological assessments are performed at each passage and before harvest to monitor the purity of the culture throughout the process. Morphology is assessed by comparing observed samples with visual standards for cell culture morphology. When grown in culture monolayer, the cells display a typical fibroblast morphology. The cells display an elongated spindle or fusiform appearance, sometimes with elongated extensions, and sometimes appear as larger, flattened stellate cells with a cytoplasmic leading edge. A mixture of these morphologies is also observed. Fibroblasts in non-confluent areas have a similar shape but may be randomly oriented. The presence of keratinocytes in cell cultures is also evaluated. Keratinocytes are round and irregular in shape and appear organized in a cobblestone pattern at high confluence. At low confluence, keratinocytes are observed as small colonies. Cells are cultured at 37.0 ± 2°C and 5.0 ± 1.0% CO2 and passaged every 3-5 days in T-500 flasks and every 5-7 days in 10-layer cell stacks (10CS). Cells should not remain in T-500 flasks for more than 10 days before passage. Quality control (QC) release testing for drug substance safety includes sterility and endotoxin testing. Once cell confluence in the T-500 flasks is 95% or greater, the cells are passaged into 10CS culture vessels. Alternatively, two 5-layer cell stacks (5CS) or a 10-layer cell factory (10CF) can be used instead of the 10CS. Passage into the 10CS is performed by removing the spent medium, washing the cells, and treating with trypsin-EDTA to release the adherent cells in the flask into solution. The cells are then transferred to the 10CS.Complete growth medium is added to neutralize the trypsin, and the cells are pipetted from the T-500 flask into a 2L bottle containing fresh complete growth medium. The contents of the 2L bottle are transferred to a 10CS and seeded to full thickness. The cells are then cultured at 5.0±1.0% CO2 and 37.0±2°C and fed fresh complete growth medium every 5-7 days. Pre-passaged cells should not remain in the 10CS for more than 20 days. In one embodiment, the passaged dermal fibroblasts are substantially depleted of immunogenic proteins present in the medium by incubating the expanded fibroblasts for a period of time in Primary Harvest, a protein-free medium. When the cells reach 95% or greater confluence in the 10CS, the cells are harvested. Harvesting is performed by removing the spent medium, washing the cells, treating them with Trypsin-EDTA to release the attached cells into solution, and then adding complete growth medium to neutralize the trypsin. Cells are harvested by centrifugation, resuspended, and then subjected to in-process QC tests to measure total viable cell count and cell viability.

[0055] The fibroblasts may be derived from a variety of sources, including, for example, dermal fibroblasts, placental fibroblasts, adipose fibroblasts, bone marrow fibroblasts, foreskin fibroblasts, umbilical cord fibroblasts, hair follicle-derived fibroblasts, nail-derived fibroblasts, endometrium-derived fibroblasts, keloid-derived fibroblasts, and fibroblasts derived from by-products associated with plastic surgery. In some embodiments, the fibroblasts are dermal fibroblasts.

[0056] In some embodiments, the fibroblasts are engineered or stimulated to produce one or more factors. In some embodiments, the fibroblasts are engineered or stimulated to produce one or more factors, such as leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6. glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M- CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor β (TGFβ-1) and / or TGFβ-3. Factors from engineered or stimulated fibroblasts are present in the conditioned medium and can be harvested for therapeutic use.

[0057] In some embodiments, fibroblasts are transfected with one or more angiogenic genes to enhance their ability to promote angiogenesis. "Angiogenic gene" refers to a gene that encodes a protein or polypeptide that can stimulate or enhance angiogenesis in a culture system, tissue, or organism.Examples of angiogenic genes useful for transfection of fibroblasts include activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, bFGF, B61, bFGF-inducible activity, cadherin, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, connexin, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, E DM, EGF, EMAP, endoglin, endothelin, endostatin, endothelial cell growth inhibitory factor, endothelial cell viability maintaining factor, endothelial differentiation glycosphingolipid G protein-coupled receptor-1 (EDG1), ephrin, Epo, HGF, TGF-β, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin, fibronectin receptor, factor X, HB-EGF, HBNF, HGF, HUAF, cardiac vascular cell growth inhibitory factor, IL1, IGF-2 IFN-γ, α1β1 integrin, α2β1 integrin, K-FGF, LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP2, MMP3, MMP9, urokinase plasminogen activator, neuropilin, neurotherin, nitric oxide donor, nitric oxide synthase (NOSs), notch, occludin, zona occludin, oncostatin M, PDGF, PDGF-B, PDGF receptor, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PK R1, PKR2, PPARγ, PPARγ ligand, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell-derived migration factor, sphingosine-1-phosphate-1 (SIP1), Syk, SLP76, tachykinin, TGF-β, Tie1, Tie2, TGF-β, TGF-β receptor, TIMPs, TNF-α, transferrin, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF(164), VEGI, and EGFR-VEGF.Fibroblasts transfected with one or more angiogenic factors can be used in the disclosed methods of treating or preventing diseases.

[0058] Under appropriate conditions, fibroblasts may produce interleukin-1 (IL-1) and / or other inflammatory cytokines. In some embodiments, the fibroblasts of the present disclosure are modified (e.g., by gene editing) to prevent or reduce expression of IL-1 or other inflammatory cytokines. For example, in some embodiments, the fibroblasts are fibroblasts that have a deleted or non-functional IL-1 gene such that the fibroblasts cannot express IL-1. Such modified fibroblasts may be useful in the therapeutic methods of the present disclosure by having limited pro-inflammatory capabilities when provided to a subject. In some embodiments, the fibroblasts are treated with TNF-α (e.g., cultured with TNF-α), thereby inducing growth factor expression and / or fibroblast proliferation.

[0059] In some embodiments, the fibroblasts of the present disclosure are used as progenitor cells that differentiate after introduction into an individual. In some embodiments, the fibroblasts are subjected to differentiation into a different cell type (e.g., hematopoietic cells) prior to introduction into an individual.

[0060] As disclosed herein, the fibroblasts may secrete one or more factors before or after introduction into an individual. Such factors include, but are not limited to, growth factors, trophic factors, and cytokines. In some embodiments, the secreted factors may have a therapeutic effect in an individual. In some embodiments, the secreted factors activate the same cells. In some embodiments, the secreted factors activate adjacent and / or distal endogenous cells. In some embodiments, the secreted factors stimulate cell proliferation and / or cell differentiation. In some embodiments, the fibroblasts secrete a cytokine or growth factor selected from human growth factors, fibroblast growth factors, nerve growth factors, insulin-like growth factors, hematopoietic stem cell growth factors, members of the fibroblast growth factor family, members of the platelet-derived growth factor family, vascular or endothelial cell growth factors, and members of the TGFβ family.

[0061] In some embodiments, the fibroblasts of the present disclosure are cultured with an mRNA degradation inhibitor. In some embodiments, the fibroblasts are cultured under conditions suitable for supporting fibroblast reprogramming. In some embodiments, such conditions include temperature conditions between 30°C and 38°C, between 31°C and 37°C, or between 32°C and 36°C. In some embodiments, such conditions include up to 4.6 g / l, 4.5 g / l, 4 g / l, 3 g / l, 2 g / l, or 1 g / l glucose. In some embodiments, such conditions include about 1 g / l glucose.

[0062] Aspects of the present disclosure include generating conditioned medium from fibroblasts. The conditioned medium can be obtained from culture with fibroblasts. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. In some embodiments, the fibroblasts are cultured for about 3 days before harvesting the conditioned medium. The conditioned medium can be obtained by separating the cells from the medium. The conditioned medium may be centrifuged (e.g., at 500×g). The conditioned medium may be filtered through a membrane. The membrane may be a membrane of greater than 1000 kDa. The conditioned medium may be subjected to liquid chromatography, such as HPLC. The conditioned medium may be separated by size exclusion.

[0063] In some embodiments, the present disclosure utilizes fibroblast-derived exosomes as a therapeutic tool. Fibroblast-derived exosomes may be used in addition to or in place of fibroblasts in various methods and compositions disclosed herein. Exosomes, also referred to as "microparticles" or "particles," may include vesicles or flattened spheres bounded by a lipid bilayer. Microparticles may include diameters of 40-100 nm. Microparticles may be formed by inward budding of endosomal membranes. Microparticles may have a density of about 1.13-1.19 g / ml and may float on a sucrose gradient. Microparticles may be enriched in cholesterol and sphingomyelin, as well as lipid raft markers such as GM1, GM3, flotillin, src protein kinase Lyn, and the like. Microparticles may include one or more proteins present in fibroblasts, such as proteins characteristic or specific to fibroblasts or fibroblast-conditioned medium. They may include RNA, such as miRNA. The microparticles may have one or more genes or gene products found in fibroblasts or in media conditioned by culturing fibroblasts. The microparticles may contain molecules secreted by fibroblasts. Such combinations of microparticles and molecules (including, inter alia, proteins or polypeptides) contained therein may be used to supplement or replace fibroblast activity, for example, for the treatment or prevention of alopecia, including androgenetic alopecia and alopecia areata. The microparticles may contain cytoplasmic proteins found in the cytoskeleton (e.g., tubulin, actin and actin-binding proteins), intracellular membrane fusion and trafficking (e.g., annexins and rab proteins), signaling proteins (e.g., protein kinases, 14-3-3 and heterotrimeric G proteins), metabolic enzymes (e.g., peroxidases, pyruvate and lipid kinases, and enolase-1), and the tetraspanin family (e.g., CD9, CD63, CD81 and CD82). In particular, the microparticles may contain one or more tetraspanins.

[0064] [IV. Isolation and purification of membrane vesicles] The exosomes of the present disclosure can be obtained from fibroblasts (e.g., from conditioned medium from fibroblast cultures) and purified for use in the disclosed methods and compositions. In some embodiments, the exosomes can be purified by anion exchange, optionally under pressure. In some embodiments, the exosomes of the disclosure are purified by high performance liquid chromatography (HPLC). Different types of supports can be used to perform anion exchange chromatography. More preferably, these can include cellulose, poly(styrene-divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycol-methacrylate copolymers, or mixtures thereof, such as agarose-dextran mixtures. Thus, in a specific embodiment, the present disclosure relates to a method for preparing membrane vesicles, in particular exosomes, from a biological sample, such as a tissue culture containing fibroblasts, comprising at least one step of treating the biological sample by anion exchange chromatography on a support selected from cellulose, poly(styrene-divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymer, alone or in mixtures, optionally functionalized.

[0065] Moreover, in order to improve the chromatographic separation capacity, it is preferred within the scope of the present disclosure to use supports in the form of beads. In some embodiments, these beads have a homogeneous and calibrated diameter with a sufficiently high porosity to allow the penetration of the objects under chromatography (e.g., exosomes). Thus, taking into account the diameter of exosomes (generally between 50 and 100 nm), in certain aspects of the present disclosure, it is preferred to use gels with high porosity, in particular between 10 nm and 5 μm, for example between about 20 nm and about 2 μm, for example between about 100 nm and about 1 μm. For anion exchange chromatography, it is necessary to functionalize the support used with a group capable of interacting with anionic molecules. In general, this group may consist of a tertiary or quaternary amine, which defines a weak or strong anion exchanger, respectively. In some embodiments, it is particularly advantageous to use a strong anion exchanger. In this way, according to the present disclosure, a chromatographic support as described above functionalized with a quaternary amine is used. Thus, according to more specific embodiments, anion exchange chromatography is carried out on a support functionalized with a quaternary amine. In some embodiments, the support is selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran, and silica, either alone or in mixtures, and functionalized with quaternary amines. Examples of supports functionalized with quaternary amines include gels SOURCE Q, MONO Q, Q SEPHAROSE®, POROS®, and the like. TM HQ and POROS TM .QE, FRACTOGEL TM TMAE type gel, TOYOPEARL SUPER TM Q gel etc.

[0066] Supports that can be used for anion exchange chromatography include poly(styrene-divinylbenzene). An example of this type of gel that can be used within the scope of the present invention is SOURCE Q gel, e.g. SOURCE 15 Q (Pharmacia). This support has the advantage of having very large internal pores, which, on the one hand, offer a small resistance to the circulation of liquid through the gel, while allowing rapid diffusion of exosomes to the functional groups, a parameter that is particularly important considering the size of the exosomes. The biological compounds retained in the column can be eluted in various ways. For example, they can be eluted using a gradient of saline, the concentration of which varies from 0 to 2 M. The various fractions thus purified are detected by measuring the optical density (OD) at the column outlet using a continuous spectrophotometer. As an example, under the conditions used in the examples, the fractions constituting membrane vesicles were eluted at an ionic strength of about 350 to 700 mM, depending on the type of vesicle.

[0067] In this chromatography step, different types of columns can be used, depending on the requirements and the amount to be processed. For example, depending on the preparation, it is possible to use columns of about 100 μl to 10 ml or more. Thus, the carriers available have a capacity that reaches, for example, 25 mg / ml of protein. Thus, a 100 μl column has a capacity of about 2.5 mg of protein, and considering the sample in question, allows the processing of about 2 liters of culture supernatant (for example, after 10-20 times concentration, showing a volume of 100-200 ml per preparation). It is understood that larger amounts can also be processed, for example by increasing the volume of the column. Furthermore, in order to carry out the invention, it is also possible to combine an anion exchange chromatography step with a gel permeation chromatography step. Thus, according to a specific embodiment of the present disclosure, a gel permeation chromatography step is added to the anion exchange step, either before or after the anion exchange chromatography step. In this embodiment, the permeation chromatography step may be performed after the anion exchange step. In a specific embodiment, the anion exchange chromatography step is replaced by a gel permeation chromatography step. The present application demonstrates that membrane vesicles can be purified using gel permeation liquid chromatography, particularly when this process is combined with anion exchange chromatography or other processing steps of the biological sample as detailed below.

[0068] To carry out the gel permeation chromatography step, a support can be used that is selected from silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymers or mixtures thereof, such as agarose-dextran mixtures. By way of example, SUPERDEX TM 200HR (Pharmacia), TSK G6000 (TosoHaas), or SEPHACRYL TMSupports such as PBS (Pharmacia) can be used. The process according to the invention can be applied to different biological samples. In particular, these can consist of biological fluids from subjects (bone marrow, peripheral blood, etc.), culture supernatants, cell lysates, pre-purification solutions or other compositions that contain membrane vesicles. In this regard, in a specific embodiment of the present disclosure, the biological sample is the culture supernatant of fibroblasts producing membrane vesicles.

[0069] Furthermore, according to certain embodiments of the present disclosure, the biological sample is treated to be enriched in membrane vesicles (enrichment step) prior to the chromatography step. Thus, in certain embodiments, the present disclosure provides a method for preparing membrane vesicles from a biological sample, characterized in that it comprises at least: 1) a concentration step to prepare a sample enriched in membrane vesicles, and 2) a step of treating the sample with anion exchange chromatography and / or gel permeation chromatography.

[0070] In one embodiment, the biological sample is a culture supernatant that has been treated to enrich for membrane vesicles.In particular, the biological sample may be composed of a pre-purified solution obtained from the culture supernatant of a population of cells (e.g., fibroblasts) that produce membrane vesicles, or from a biological liquid, by treatments such as centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography, in particular clarification and / or ultrafiltration and / or affinity chromatography.Thus, a preferred method for preparing membrane vesicles according to the present invention more particularly comprises the following steps: a) culturing a population of cells that produce membrane vesicles (e.g., exosomes) under conditions that allow the release of vesicles, b) enriching the sample with membrane vesicles, and c) anion exchange chromatography and / or gel permeation chromatography treatment of the sample.

[0071] As indicated above, the sample (e.g., supernatant) concentration step may include one or more centrifugation, clarification, ultrafiltration, nanofiltration, and / or affinity chromatography steps on the supernatant. In a first specific embodiment, the concentration step includes (i) removal of cells and / or cell debris (clarification), optionally followed by (ii) concentration and / or affinity chromatography steps. In another specific embodiment, the concentration step includes an affinity chromatography step, optionally preceded by a removal of cells and / or cell debris (clarification) step. A particular concentration step according to the present disclosure includes (i) removal of cells and / or cell debris (clarification), (ii) concentration, and (iii) affinity chromatography. Cells and / or cell debris can be removed, for example, by centrifuging the sample at low speed, preferably below 1000 g, for example between 100 and 700 g. Preferred centrifugation conditions for this step are, for example, about 300 g or 600 g for 1 to 15 minutes.

[0072] The cells and / or cell debris can also be removed by filtration of the sample, possibly combined with the above-mentioned centrifugation. Filtration can in particular be carried out by successive filtrations using filters of decreasing porosity. For this purpose, filters with a porosity of 0.2 μm or more, for example between 0.2 and 10 μm, can be used. In particular, it is possible to use successive filters with a porosity of 10 μm, then 1 μm, 0.5 μm and 0.22 μm.

[0073] A concentration step can also be carried out to reduce the amount of sample to be processed at the chromatographic stage. In this way, the concentration can be achieved by centrifuging the sample at high speed, for example at 10,000 to 100,000 g, in order to precipitate the membrane vesicles. This can consist of a series of differential centrifugations, the last of which can be carried out at 70,000 g. The membrane vesicles in the resulting pellet are taken up in a smaller amount in a suitable buffer and subjected to the subsequent steps. The concentration step can also be carried out by ultrafiltration. In fact, this ultrafiltration allows to concentrate the supernatant and to carry out an initial purification of the vesicles. According to one embodiment, the biological sample (for example the supernatant) is subjected to ultrafiltration, such as tangential ultrafiltration. Tangential ultrafiltration consists in concentrating and fractionating the solution between two compartments (filtrate and reflux) separated by a membrane of a determined cut-off threshold. The separation is achieved by applying a flow to the filtrate compartment and a transmembrane pressure between this compartment and the filtrate compartment. A variety of systems can be used to perform ultrafiltration, including spiral membranes (Millipore, Amicon), flat membranes, hollow fibers (Amicon, Millipore, Sartorius, Pall, GF, Sepracor), etc. Within the scope of the present disclosure, it is advantageous to use membranes with a cut-off threshold of less than 1000 kDa, for example between 300 kDa and 1000 kDa, for example between 300 kDa and 500 kDa.

[0074] The affinity chromatography step can be carried out in various ways, using different chromatographic supports and materials. Advantageously, it is a non-specific affinity chromatography, aiming to retain (i.e. bind) certain contaminants present in the solution, without retaining the target (i.e. exosomes). It is therefore a negative selection. In some embodiments, affinity chromatography on dyes is used, allowing the removal (i.e. retention) of contaminants such as proteins and enzymes, for example albumins, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or also cofactors. In some embodiments, the support used in this chromatography step is a support used for ion exchange chromatography functionalized with a dye. As a specific example, the dye is Blue SEPHAROSE TM (Pharmacia), YELLOW 86, GREEN 5, and BROWN 10 (Sigma). The support may be agarose. It should be understood that any other support and / or dye or reactive group that allows for the retention (binding) of contaminants from the processed biological sample may be used in the present disclosure.

[0075] V. Administration of Therapeutic Compositions Therapies provided herein may include administering therapeutic agents (e.g., fibroblasts, fibroblast-derived exosomes, fibroblast-derived products, etc.), alone or in combination. Therapeutic agents may be administered in any suitable manner known in the art. For example, the first and second treatments (e.g., fibroblast-derived products and diphenylcyclopropenone) may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second treatments are administered in separate compositions. In some embodiments, the first and second treatments are administered in the same composition.

[0076] The embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapeutic agents may be administered in one composition or in two or more compositions, such as two compositions, three compositions, or four compositions. Various combinations of agents may be employed.

[0077] The therapeutic agents of the present disclosure (e.g., fibroblasts, fibroblast-derived products, diphenylcyclopropenone) may be administered by the same or different routes of administration. In some embodiments, the therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0078] Therapeutic agents include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount administered, as well as the particular route and formulation, are within the judgement of those skilled in the clinical art. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0079] The dosage depends on the desired therapeutic effect, both in number of treatments and unit dosage. Effective amount is understood to refer to the amount required to achieve a specific effect. In the implementation of certain embodiments, it is contemplated that a dosage ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, dosages include 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 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0080] In certain embodiments, an effective amount of the pharmaceutical composition may provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount provides a blood level of about 4 μM to 100 μM. Or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose may provide the following blood levels of drug resulting from the therapeutic agent being administered to a subject: 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 μM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to a metabolic therapeutic agent, in which case blood levels may refer to the amount of the therapeutic agent. Alternatively, to the extent that a therapeutic agent is not metabolized by the subject, blood levels discussed herein may refer to the therapeutic agent that is not metabolized.

[0081] Precise amounts of therapeutic compositions also depend on the judgment of the practitioner and are peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (alleviation of symptoms or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy the subject may be undergoing.

[0082] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg of body weight can be expressed in equivalent concentration units of μg / ml or mM (blood concentration), such as 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Conversion factors and physiological assumptions applicable to uptake and concentration measurements are well known, and one skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0083] In some embodiments, about 10 per 100 kg 5 About 10 13 of cells are administered to a human per infusion. In some embodiments, about 1.5×10 cells per 100 kg. 6 ~Approx. 1.5×10 12 In some embodiments, about 1×10 cells per 100 kg are injected. 9 ~Approx. 5×10 11 In some embodiments, about 4×10 cells per 100 kg are injected. 9 ~about 2×10 11 In some embodiments, about 5×10 cells per 100 kg are injected. 8 cells ~ approx. 1 x 10 1 In some embodiments, a single dose of cells is provided. In some embodiments, multiple doses are provided. In some embodiments, multiple doses are provided over 3-7 consecutive days. In some embodiments, 3-7 doses are provided over 3-7 consecutive days. In some embodiments, 5 doses are provided over 5 consecutive days. In some embodiments, about 10 per 100 kg of cells are administered. 5 About 10 13 A single dose of between about 1.5×10 cells per 100 kg is provided in some embodiments. 8 ~Approx. 1.5×10 12 In some embodiments, a single dose of cells of about 1×10 per 100 kg is provided. 9 ~Approx. 5×10 11A single dose of cells is provided. In some embodiments, about 5×10 per 100 kg. 10 A single dose of cells is provided. In some embodiments, 1×10 per 100 kg 10 A single dose of cells is provided. In some embodiments, about 10 per 100 kg 5 cells ~ about 10 13 Multiple administrations of cells are provided. In some embodiments, about 1.5×10 per 100 kg. 8 ~Approx. 1.5×10 12 Multiple administrations of cells are provided. In some embodiments, about 1×10 per 100 kg 9 ~Approx. 5×10 11 Multiple doses of cells are provided over 3-7 consecutive days. In some embodiments, about 4×10 cells per 100 kg are administered. 9 The cells are administered multiple times over 3-7 consecutive days. In some embodiments, about 2×10 cells per 100 kg are administered. 11 Multiple doses of cells are provided over 3-7 consecutive days. In some embodiments, about 3.5×10 9 Five doses of cells are provided over five consecutive days. In some embodiments, about 4×10 9 Five doses of cells are provided over five consecutive days. In some embodiments, about 1.3×10 11 Five doses of cells are provided over five consecutive days. In some embodiments, about 2×10 11 Five doses of cells are provided over five consecutive days.

[0084] [VI. Disclosure Kit] Any of the cellular and / or non-cellular compositions described herein, or compositions similar thereto, may be included in the kit. In a non-limiting example, one or more reagents for use in the method for preparing fibroblasts or their derivatives (e.g., fibroblast-derived exosomes) may be included in the kit. Such reagents may include cells, vectors, one or more growth factors, vector(s), one or more costimulatory factors, media, enzymes, buffers, nucleotides, salts, primers, compounds, and the like. The components of the kit are provided in suitable containers.

[0085] Some components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components can be placed and preferably suitably dispensed. When multiple components are included in the kit, the kit generally also includes a second, third or other additional container into which the additional components can be placed separately. However, various combinations of components may be included in the vial. The kit of the present disclosure also generally includes a means for containing the components in a closed state for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held.

[0086] When the components of the kit are provided in one and / or more liquid solutions, the solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and / or other such device, or may be a substrate having multiple compartments for the desired reaction.

[0087] Some components of the kit may be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is also envisioned that the solvent may be provided in another container means. The kit may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent.

[0088] In specific embodiments, the reagents and materials include primers, nucleotides, appropriate buffers or buffering reagents, salts, etc. for amplifying the desired sequence, and in some cases, the reagents include equipment or reagents for isolating specific desired cells.

[0089] In certain embodiments, the kit includes one or more devices suitable for extracting one or more samples from an individual. The device may be a syringe, a fine needle, a scalpel, or the like. EXAMPLES

[0090] The following examples are included to illustrate certain embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the implementation of the disclosed method, and therefore can be considered to constitute certain aspects for its implementation. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0091] Example 1: Use of fibroblast conditioned medium in the treatment of androgenetic alopecia. Exosome preparation: Fibroblasts are cultured under conditions sufficient to promote the release of exosomes into the medium. Specifically, foreskin fibroblasts are cultured in 15 ml of αMEM medium containing 10% fetal bovine serum in a T175 flask. After 24 h of culture, the medium is replaced with phosphate-buffered saline and the cells are cultured for an additional 12 h. Exosomes are extracted by gradient centrifugation at 300g for 10 min, 2000g for 10 min, and 10000g for 30 min, followed by centrifugation at 100000g for 70 min to pellet the exosomes. Optionally, the exosome preparation is purified from the low mobility fraction, mainly from free proteins, by repeated 100000g centrifugation of the resuspended pellet.

[0092] Clinical trial for the treatment of androgenetic alopecia (AGA): Thirty male patients who meet the eligibility criteria and provide written informed consent will be enrolled. A dermatologist will perform a clinical diagnosis of AGA for all participants. Inclusion criteria are age 18-60 years, hair loss duration ≥6 months, and Hamilton-Norwood grade 3-6. Patients with scalp infections, malignant diseases, autoimmune diseases (Hashimoto's disease, rheumatoid arthritis, lupus, etc.), history of vascular surgery within the past 3 months, systemic diseases affecting hair growth, telangiectasia, history of blood diseases, and chronic users of oral or intravenous narcotics will be excluded from the study.

[0093] Other exclusion criteria were the use of topical or systemic medications for hair loss within the past six months, administration of chemotherapy or immunosuppressants, use of medications containing growth factors, hemoglobin level less than 10, thrombocytopenia (or platelet count less than 100,000), serum albumin less than 2.5 g / dl, and participation in other clinical trials (within the past three months).

[0094] Fifteen patients will receive 1 microgram of exosomal protein per milliliter every two weeks for five sessions, with the solution injected intradermally at the temples and foreheads, at 0.05 milliliters per site, 1-2 cm apart, and a further 15 patients will receive the same frequency at a concentration of 5 million fibroblasts per scalp.

[0095] At the beginning of the study before the intervention and 3 months after the final treatment session, standard photographs will be taken with a digital camera (Nikon D300s®, Tokyo, Japan), trichograms will be performed, and hair density and diameter will be measured at the sites marked with tattoos using a digital photo hair analyzer (KC Triple Scope®, KC Technology Co, Korea) with a 150x lens. In addition, patients will answer a patient satisfaction questionnaire at each visit on a scale of -2 to +2 (-2: much worse, -1: slightly worse, 0: no change, +1: slightly improved, +2: much improved).

[0096] Percentages and frequencies are used to describe qualitative data, and means and standard deviations to describe quantitative data. Comparison of quantitative data before and after testing is performed by paired t-tests (or non-parametric equivalent tests, depending on the data distribution and categorized information). All test estimates are performed at the 5% significance level.

[0097] After 5 weeks of treatment, all parameters improved significantly.

[0098] 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 design 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 herein. As will be readily understood by those skilled in the art from the present disclosure, any currently existing or later developed process, machine, manufacture, composition of matter, means, methods or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Thus, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

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Claims

**Claim 1**: A composition containing an effective amount of fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium, and / or fibroblast-derived products, wherein the composition is formulated for use in a method for treating or preventing alopecia in a subject, the fibroblasts express CD73, CD90, and / or CD105, the fibroblast-derived exosomes, the fibroblast-derived conditioned medium, and / or the fibroblast-derived products are obtained from fibroblasts that express CD73, CD90, and / or CD105. A composition characterized by the above. **Claim 2**: (a) The alopecia is related to dermal inflammation; and / or (b) The alopecia is alopecia areata or androgenetic alopecia; and / or (c) The composition is formulated for topical, intradermal, and / or transdermal administration; and / or (d) The composition is formulated for administration to the scalp area of the subject; and / or (e) The fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium, or fibroblast-derived products are allogeneic, xenogeneic, or autologous to the subject; and / or (f) The fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium, or fibroblast-derived products are derived from skin, fat, bone marrow, omentum, blood, deciduous teeth, fallopian tubes, testicular tissue, ovarian tissue, hair follicles, endometrial tissue, or combinations thereof; and / or (g) The fibroblasts are dermal fibroblasts. The composition according to claim 1. **Claim 3**: The composition according to claim 1 or 2, further comprising an effective amount of diphenylcyclopropenone in addition to the fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium, and / or fibroblast-derived products. **Claim 4**: A composition wherein the fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium, or fibroblast-derived products have been pre-exposed to conditions sufficient to enhance their regenerative activity, and the conditions are: (a) Conditions sufficient to upregulate HIF1α expression in the fibroblasts; and / or (b) Conditions sufficient to upregulate HIF1α by at least 25% compared to an untreated control; and / or (c) Conditions sufficient to enhance nuclear translocation of HIF1α in fibroblasts; and / or (d) Containing a drug that can mimic hypoxia; and / or (e) culturing fibroblasts with carbon monoxide; and / or (f) exposing fibroblasts to a gas composition comprising from about 0 wt% to about 79 wt% nitrogen, from about 21 wt% to about 99.999999 wt% oxygen, and from about 0.0000001 wt% to about 0.3 wt% carbon monoxide, A composition according to any one of claims 1 to 3, characterized by the above. **Claim 5**: The composition according to claim 4, wherein the condition comprises exposing the fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium or fibroblast-derived products to a gas composition comprising 0% nitrogen and about 99.999999% oxygen. **Claim 6**: The composition according to any one of claims 1 to 5, wherein the fibroblasts, fibroblast-derived exosomes, fibroblast-derived conditioned medium or fibroblast-derived products have been previously subjected to conditions sufficient to enhance the survival and / or activity of the fibroblasts. **Claim 7**: The condition is (a) treatment with an epigenetic regulator; and / or (b) culturing with a GSK-3 inhibitor, **Claim 8**: The epigenetic regulator is (a) a histone deacetylase inhibitor; and / or (b) a DNA methyltransferase inhibitor The composition according to claim 7. **Claim 9**: The composition according to claim 7, wherein the GSK-3 inhibitor is lithium or a lithium salt. **Claim 10**: The histone deacetylase inhibitor is valproic acid, vorinostat, entinostat, panobinostat, trichostatin A, mocetinostat, belinostat, FK228, MC1568, tubastatin, sodium butyrate, or sulforaphane, or The composition according to claim 8, wherein the DNA methyltransferase inhibitor is 5-azacytidine. **Claim 11**: The conditioned medium is (a) obtained from a culture of fibroblasts in EMEM, α-MEM, IMDM, DMEM, or RPMI; and / or (b) generated by culturing adherent fibroblasts in a suspension containing nutrients for fibroblasts, **Claim 12**: The liquid suspension is (a) a growth factor; and / or (b) stem cell exosomes; and / or (c) a neutralizing factor capable of inhibiting the activity of one or more inflammatory mediators; and / or (d) VEGF, EGF, PDGF-BB, IGF-1, HGF-1, NGF, BDNF, IL-3, IL-4, IL-10, IL-13, IL-20, IL-35 The composition according to claim 11, comprising **Claim 13** The composition according to claim 12, wherein the stem cell exosome is an exosome derived from mesenchymal stem cells. **Claim 14** The composition according to claim 13, wherein the mesenchymal stem cells are derived from umbilical cord, bone marrow, skin, fallopian tube, adipose tissue, endometrial tissue, peripheral blood, menstrual blood, hair follicle, or a combination thereof. **Claim 15** The neutralizing factor is (a) a monoclonal antibody, an antisense oligonucleotide, or a gene editing system; and / or (b) an antibody capable of binding to interleukin-1, interleukin-6, interleukin-8, interleukin-9, interleukin-11, interleukin-12, interleukin-15, interleukin-17, interleukin-18, interleukin-21, interleukin-23, interleukin-27, interleukin-33, TNFα, interferon γ, TNFβ, or lymphotoxin, the composition according to claim 12. **Claim 16** The fibroblast-derived product is (a) microvesicles; and / or (b) apoptotic vesicles derived from fibroblasts; and / or (c) nucleic acids derived from fibroblasts The composition according to any one of claims 1 to 15. **Claim 17** The composition according to any one of claims 1 to 16, wherein the exosome is concentrated from fibroblast-derived conditioned medium. **Claim 18** The exosome is (a) a step of functionalizing a support with a single-stranded oligonucleotide to produce a functionalized support; (b) incubating the functionalized support with a ligand having a tag complementary to the single-stranded oligonucleotide to obtain an immobilized ligand; (c) incubating the immobilized ligand with the conditioned medium to capture the exosome through the binding of the immobilized ligand and the exosome, and obtaining a substrate of the captured exosome; (d) a step of incubating the captured exosome with a restriction enzyme; Concentrated by (i) the ligand is an antibody, peptide, or aptamer; and / or (ii) the support is a magnetic bead, membrane, cell culture plate, test tube, slide, microplate, microchannel, pillar, or disk-shaped piece; and / or (iii) the support is functionalized with the ligand via a covalent bond or biotinylation; and / or (iv) the restriction enzyme is DNase; and / or (v) the ligand is an antibody capable of binding to exosome-specific tetraspanin; and / or (vi) the ligand is an antibody capable of binding to MHC class I and II, HSP70, annexin V, flotillin, or EpCAM; and / or (vii) the conditioned medium is derived from a culture of fibroblasts in EMEM, α-MEM, IMDM, DMEM, or RPMI, The composition according to claim 17.

19. The subject, as compared to an age-matched control subject, (a) cells expressing FoxP3, interleukin-10, interleukin-4, interleukin-13, and / or interleukin-35; and / or (b) regulatory T cells, myeloid-derived suppressor cells, B cells expressing TIM-1, B cells expressing IL-10, and / or regulatory B cells The composition according to any one of claims 1 to 18, wherein the number is decreased.

20. The subject, as compared to an age-matched control subject, (a) cells expressing interferon gamma, TNF-α, interleukin-1, interleukin-2, interleukin-6, interleukin-8, interleukin-11, interleukin-12, interleukin-15, interleukin-17, interleukin-18, interleukin-21, interleukin-23, interleukin-27, and / or interleukin-33; and / or (b) natural killer cells, natural killer T cells, Th1 cells, and / or Th17 cells The composition according to any one of claims 1 to 18, wherein the number is increased.