Composition containing amniotic fluid stem cells or derivatives thereof and application thereof

Abstract

The present invention provides a composition comprising amniotic fluid stem cells derived from an audina animal. Also provided is a use of the composition for skin regeneration, skin treatment, promotion of hair growth, and prevention of hair loss, comprising applying an effective amount of the composition to an individual in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 373,964, filed on August 30, 2022. The contents of that application are incorporated herein by reference in their entirety. Background of the Invention 1. Technical Field

[0004] The present invention relates to a composition comprising amniotic fluid stem cells or derivatives thereof, and in particular to a composition comprising amniotic fluid stem cells or derivatives thereof derived from artiodactyl animals.

[0005] 2. Description of the Prior Art

[0006] The skin is the first barrier for animals and humans to resist external invasion. It plays an important role in physical and chemical defense. In addition, the skin is also the first line of defense for immune defense.

[0007] The skin is made up of three main layers: the epidermis, dermis and subcutaneous tissue. The three main layers contain specific cells and it is well known that the epidermis of the skin sheds millions of cells every day as we age, creating a need for cell replacement. In addition, pioneer cells and stem cells proliferate and differentiate as part of the body's natural repair and regeneration mechanisms. However, with aging, the proliferation of cells may not be able to keep up with the natural repair rate, and this is when wrinkles naturally appear. For example, with aging, skin wrinkles, skin folds or skin ridges become noticeable, such as crow's feet around the eyes and nasolabial folds on the face. It has been found that the loss of collagen is one of the main causes of wrinkles.

[0008] In addition, skin cells may also be damaged by other physical impacts, such as lacerations, abrasions, burns, diabetic ulcers, and bedsores caused by long-term bed rest. Wound healing is a complex and slow process, during which new epithelial tissue and connective tissue are formed through migration and proliferation.

[0009] The skin of an adult is essentially covered with hair follicles, with the scalp covered with approximately 100,000 to 150,000 follicles. It is believed that a hair follicle will produce approximately 20 individual hair shafts during its lifetime as it undergoes a cycle of hair production, shedding, regression, and new growth.

[0010] Hair has many important physiological functions, such as protection against external factors (including UV radiation), production of sebum, apocrine sweat and pheromones, and temperature regulation. For an adult scalp, at any given time, approximately 80 to 90% is in the growth phase; 10 to 20% is in the resting phase; and 1 to 2% is in the regression phase. Hair loss is a common phenomenon in the elderly population. Hair follicle stem cells (HFSCs) have been found to be very important for stimulating hair growth / promoting hair growth. HFSCs can be triggered to become dormant and activated by both endogenous and exogenous signals. Imbalance in stem cell differentiation and altered stem cell activity are considered important factors in the hair loss process. Therefore, there are many studies on treatments that can promote the activation of HFSCs.

[0011] As is well known, the use of human stem cells in this field raises many issues, indicating that there is a strong need for further research in this field to explore these issues. Some of the main obstacles raised by the use of embryonic cells are ethical questions, the risk of genetic defects, and the risk of transmitting viral and carcinogenic diseases.

[0012] Therefore, there is an unmet need in the art for compositions derived from stem cell lines and / or their derivatives that eliminate the risks of the above difficulties, and methods for using stem cells and / or their derivatives derived from reliable sources in the preparation of skin care, hair promotion and cosmetic products. Summary of the invention

[0013] The object of the present invention is to provide a composition comprising amniotic fluid stem cells or derivatives thereof and uses thereof, wherein the composition comprises stem cells or derivatives thereof and a cosmetically or pharmaceutically acceptable carrier, and the stem cells are derived from the amniotic fluid of artiodactyl animals.

[0014] The present invention provides a composition comprising: stem cells or derivatives thereof; and a cosmetically or pharmaceutically acceptable carrier, wherein the stem cells are derived from the amniotic fluid of an artiodactyl animal.

[0015] In at least one embodiment, the artiodactyl includes, but is not limited to, pigs, wild boars, hippos, antelopes, deer, giraffes, camels, llamas, alpacas, sheep, goats, and cattle. In some embodiments, the artiodactyl is a deer.

[0016] In at least one embodiment, the stem cell derivative may be, but is not limited to, a secretion body, a conditioned medium, an exosome, an extracellular carrier, a secreted substance, or any combination thereof. In some embodiments, the derivative is a conditioned medium.

[0017] In at least one embodiment, the stem cells are negative for CD31, CD34, CD45 or any combination thereof. In some embodiments, the stem cells are derived from amniotic fluid of a deer and are negative for CD31, CD34 and CD45.

[0018] In at least one embodiment, the stem cell is positive for CD9, CD29, CD44, CD73, CD90, CD105, Nestin, Sox2, NANOG, or any combination thereof. In some embodiments, the stem cell is derived from amniotic fluid of a deer and is positive for CD9, CD29, CD44, CD73, CD90, CD105, Nestin, Sox2, and NANOG.

[0019] In at least one embodiment, the stem cells have osteogenic differentiation, adipogenic differentiation, chondrogenic differentiation or any combination thereof.

[0020] In at least one embodiment, the composition can be selected from the group consisting of gel, lotion, ointment, emulsion, paste, cream, injection, capsule, oral solution, tablet, film, lozenge, granule, spray and pill.

[0021] In at least one embodiment, the stem cell or its derivative is the sole active ingredient in the composition.

[0022] The present invention further provides the use of the composition for skin regeneration (skin rejuvenation) or skin treatment (skin treatment), which comprises applying an effective amount of the composition of the present invention to an individual in need. In at least one embodiment, the use can be a therapeutic use or a non-therapeutic use (e.g., cosmetic use). The present invention also provides a method for skin regeneration or skin treatment, which comprises applying an effective amount of the composition of the present invention to an individual in need. In addition, the present invention provides a composition for skin regeneration or skin treatment. The present invention further provides the use of the composition in the preparation of a medicament for skin regeneration or skin treatment.

[0023] In at least one embodiment, the composition of the present invention promotes the synthesis of procollagen, collagen or elastin in an individual.

[0024] In at least one embodiment, the skin regeneration includes preventing, treating or reducing age-related features on the skin of the individual. In some embodiments, the age-related features may be, but are not limited to, wrinkles, age spots, rough skin texture, dull skin, fine lines, visible pores, or loss of skin structure due to decreased collagen synthesis, elastin or cell synthesis.

[0025] In at least one embodiment, the skin treatment comprises restoring damage to a subject's skin, reducing scarring of damaged skin in a subject, enhancing recovery of a subject's skin after cosmetic or dermatological surgery, or preventing or reducing wrinkles on a subject's skin.

[0026] In some embodiments, the skin treatment comprises controlling, treating and / or preventing scar formation, abnormal scarring, abnormal wound healing, widened scars, hypertrophied scars, keloids, keloid scars or wound healing complications.

[0027] In further embodiments, the skin treatment comprises primary healing, wound closure, secondary healing, epithelialization, re-epithelialization, tertiary wound closure, or delayed primary closure.

[0028] In other embodiments, the compositions described herein are used to increase or decrease the inflammatory phase, proliferative phase, maturation phase, hemostasis, inflammation, collagen, coagulation, thromboxane A2, prostaglandin 2a, bleeding, vasodilation, histamine, platelets, chemokine, epidermal growth factor, fibronectin, fibrinogen, clot formation, platelet degranulation, collagenase, fibroblasts, collagen deposition or insulin-like growth factor at the site of administration to the subject.

[0029] In at least one embodiment, the wrinkles are caused by age, obesity, injury, hormonal deficiency, drug side effects, or any combination thereof. In at least one embodiment, the wounds are caused by cuts, burns, acute trauma, chronic wounds, surgical wounds, or other chemical or physical influences.

[0030] The present invention also provides the use of the composition for promoting hair growth or preventing hair loss, which comprises administering an effective amount of the composition of the present invention to an individual in need. In at least one embodiment, the use can be therapeutic or non-therapeutic (e.g., cosmetic). The present invention also provides a method for promoting hair growth or preventing hair loss, which comprises administering an effective amount of the composition of the present invention to an individual in need. In addition, the present invention provides a composition for promoting hair growth or preventing hair loss. The present invention further provides the use of the composition in the preparation of a medicament for promoting hair growth or preventing hair loss.

[0031] In at least one embodiment, the hair loss is caused by a condition selected from the group consisting of: androgenic alopecia, alopecia areata, and telogen effluvium.

[0032] In at least one embodiment, the composition promotes the growth or migration of hair follicle dermal papilla cells of a subject.

[0033] In at least one embodiment, the composition is administered topically, subcutaneously, intradermally or intramuscularly to an individual. In some embodiments, the administration may be by direct injection, conditioned medium or topical administration to one or more skin areas selected from the group consisting of the scalp, face, neck, collarbone, chest, back, arms, hands, legs, feet and soles.

[0034] In at least one embodiment, the stem cell or its derivative is the sole active ingredient in the composition of the present invention.

[0035] In at least one embodiment, the composition of the present invention can be used alone or in combination with: gauze, facial mask, ointment, plug, cosmetic composition, cosmetically acceptable excipient or cosmetically acceptable carrier, thickener, filler, moisturizer, emulsifier, wetting agent, surfactant, buffer or pH adjuster, film forming agent, preservative, antioxidant, fragrance, solvent, propellant, colorant or any combination thereof.

[0036] In at least one embodiment of the present invention, the stem cells of an artiodactyl animal are obtained by the following steps:

[0037] (a) Obtaining amniotic fluid samples from artiodactyls by amniocentesis;

[0038] (b) filtering the amniotic fluid through a sterile cell filter;

[0039] (c) centrifuging and obtaining stem cells from the amniotic fluid;

[0040] (d) culturing the stem cells in a culture medium;

[0041] (e) removing non-attached cells and allowing attached cells to grow as a colony; and when the attached cells reach confluence,

[0042] (f) Trypsinizing the attached cells and subculturing the attached cells for expansion or preservation.

[0043] In some embodiments of the present invention, the deer stem cells are obtained by the following steps:

[0044] (a) Obtaining amniotic fluid samples from deer by amniocentesis, where amniotic fluid is extracted using a long-needle syringe under ultrasound guidance, where the deer is more than 3 months pregnant;

[0045] (b) filtering fresh deer amniotic fluid with a sterile cell filter, centrifuging the amniotic fluid sample at 200 × g for at least 5 minutes, and removing the supernatant;

[0046] (c) culturing deer stem cells in a culture medium or a commercially available stem cell culture medium, wherein the culture medium is an alpha-modified minimum essential medium supplemented with about 10% to 20% fetal bovine serum and, if necessary, antibiotics;

[0047] (d) removing non-attached cells by changing the culture medium twice a week and growing the attached cells as colonies for at least 5 to 7 days until 80% to 90% confluence is reached; and

[0048] (e) Trypsinize adherent cells and plate at a density of approximately 1000 to 9000 cells / cm 2 The seeding density was set to allow the attached cells to proliferate.

[0049] In some embodiments of the present invention, the basal medium can be MEM, DMEM, IMDM, or RPMI 1640. In some embodiments of the present invention, the α-modified minimal essential medium is further supplemented with about 4 ng / ml basic fibroblast growth factor.

[0050] In some embodiments, the attached cells are mammalian adherent cells. Any seeding density can be used that allows the cells to form a confluent monolayer immediately or after a period of culture. In some embodiments, the seeding density can be about 10 cells / cm 2 Up to 100,000 cells / cm 2 , or about 100 cells / cm 2 to approximately 75,000 cells / cm 2 , or about 500 cells / cm 2 to approximately 50,000 cells / cm 2 , or about 500 cells / cm 2 Up to about 10,000 cells / cm 2 , or about 500 cells / cm 2 to approximately 5,000 cells / cm 2 , or about 500 cells / cm 2 About 2,500 cells / cm2 , or about 1,000 cells / cm 2 to approximately 25,000 cells / cm 2 , or about 2,000 cells / cm 2 Up to 10,000 cells / cm 2 , or about 3,000 cells / cm 2 About 5000 cells / cm 2 In some embodiments, the adherent cells may be stem cells, somatic cells, precursor cells, mature cells and / or cells from multiple germ layers, but the present invention is not limited thereto.

[0051] In some embodiments of the present invention, the essential medium may be supplemented with antibiotics, such as penicillin, streptomycin, tetracyclines and / or gentamicin, but the present invention is not limited thereto.

[0052] In some embodiments of the present invention, the essential medium can be supplemented with antifungal agents, such as polyenes (amphotericin B), flucytosine, imidazoles, triazoles (ketoconazole, fluconazole, itraconazole and voriconazole), griseofulvin and ciclopirox, but the present invention is not limited thereto.

[0053] In at least one embodiment, the composition may further include glutamic acid, an antioxidant and / or nicotinamide. In some embodiments, the antioxidant may be vitamin E, vitamin A, vitamin C or any combination thereof.

[0054] In at least one embodiment, the stem cells derived from amniotic fluid have better efficacy in promoting skin regeneration or skin treatment, promoting hair growth or preventing hair loss than stem cells derived from adult tissues such as adipose tissue or bone marrow. For example, in the process of wound healing, stem cells derived from adult tissues may cause scar formation due to the promotion of myofibroblast differentiation and collagen synthesis. In contrast, the amniotic fluid stem cells or their derivatives (e.g., secretions, conditioned medium, exosomes, extracellular carriers or secreted substances) can achieve scarless wound healing because they have anti-fibrotic properties by inhibiting myofibroblast differentiation and collagen synthesis. In some embodiments, the dAFSC shows better efficacy in promoting skin regeneration or skin treatment, promoting hair growth or preventing hair loss than stem cells derived from human amniotic fluid. For example, the dAFSC inhibits more myofibroblast differentiation and collagen synthesis than other stem cells from other human tissues.

[0055] These and other objects of the present invention will no doubt become embodied to those skilled in the art having ordinary knowledge in the art after reading the following detailed description of the preferred embodiments illustrated in the figures and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] This patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] The present invention will be more readily understood by referring to the following description in conjunction with the accompanying drawings.

[0058] Figure 1 The morphology of fibroblast-like cells derived from deer amniotic fluid is shown under a microscope (100x) according to at least one embodiment of the present invention.

[0059] Figure 2A and Figure 2B The expression of biomarkers CD9, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, Nestin, Sox2, GAPDH and NANOG in cells detected using reverse transcription and polymerase chain reaction (RT-PCR) according to at least one embodiment of the present invention is shown.

[0060] Figure 3A , Figure 3B , Figure 3C and Figure 3D Flow cytometric analysis of antigens detected in cells is shown in accordance with at least one embodiment of the present invention. Figure 3A The quantitative results are as follows Figure 3C As shown, Figure 3BThe quantitative results are as follows Figure 3D shown.

[0061] Figure 4A and Figure 4B It is shown that according to at least one embodiment of the present invention, the cell ( Figure 4A ) differentiate into osteoblasts ( Figure 4B ) images.

[0062] Figure 4C and Figure 4D It is shown that according to at least one embodiment of the present invention, the cell ( Figure 4C ) differentiate into adipocytes ( Figure 4D ) images.

[0063] Figure 4E and Figure 4F According to at least one embodiment of the present invention, the cell ( Figure 4C ) differentiated into chondrocytes (100X image) Figure 4E ) and 400X images ( Figure 4F ).

[0064] Figure 5 The figure shows the cell proliferation (%) and procollagen synthesis rate (%) of human skin fibroblasts treated with different concentrations of dASFC-CM according to at least one embodiment of the present invention. PC: positive control; NC: negative control.

[0065] Figure 6 The wound healing area (%) of human skin fibroblasts treated with different concentrations of dASFC-CM according to at least one embodiment of the present invention is shown. PC: positive control group; NC: negative control group.

[0066] Figure 7 The cell viability (%) of human skin fibroblasts treated with different concentrations of dASFC-CM (%) according to at least one embodiment of the present invention is shown. NC: negative control group.

[0067] Figure 8 The relative survival rates (%) of NC (negative control group), PC (positive control group) and dAFSC-CM 10% in an in vitro skin irritation test according to at least one embodiment of the present invention are shown. PC: positive control group; NC: negative control group.

[0068] Fig. 9 The figure shows the average tissue viability (%) of NC (negative control group), PC (positive control group) and dAFSC-CM 10% in an in vitro eye irritation test according to at least one embodiment of the present invention.

[0069] Fig.10 The relative cell growth (%) of human hair follicle dermal papilla cells (HFDPC) treated with different concentrations of dASFC-CM according to at least one embodiment of the present invention is shown. Ctrl: negative control group.

[0070] Fig.11 The effect of dAFSC-CM on cell migration of HFDPCs is shown. According to at least one embodiment of the present invention, HFDPCs were treated with dAFSC-CM at different concentrations (1%, 2%, 5%, 10% and 15%) and different culture times (0, 5, 9 and 24 hours). Ctrl: Negative control group. DETAILED DESCRIPTION

[0071] The following examples are provided to explain the present invention in detail. A person skilled in the art with general knowledge can easily understand the advantages and effects of the present invention after reading the disclosure of this specification sheet, and can also be implemented or applied in other different embodiments. Therefore, the following embodiments for performing the present invention can be modified and / or changed without violating the scope of its different aspects and applications, and any element or method within the scope of the present invention can be combined with any other element or method disclosed in any embodiment of the present invention.

[0072] The articles "a", "an" and "the" refer to one or more than one (i.e., at least one) grammatical object of the article. The term "or" is used interchangeably with the term "and / or", unless otherwise clearly stated. The term "includes / comprising" is used in the present invention to represent the phrase "includes / comprising but not limited to", and can be used interchangeably with it. As used in the present invention, the term "about" refers to the typical tolerance range of the art. For example, "about" can be understood as about 2 standard deviations from the mean. When "about" appears before a series of numbers or ranges, it should be understood that "about" can modify each number in the series or range. For example, the numerical value is intended to cover changes from the numerical value ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5% or ± 0.1%. As used in the present invention, the numerical range is inclusive and combinable, and any numerical value falling within the numerical range of the present invention can be regarded as a maximum or minimum value to derive a sub-range therefrom. For example, it should be understood that the numerical range "20 to 30%" includes any sub-ranges between a minimum of 20% and a maximum of 30%, such as sub-ranges from 20% to 25%, from 25% to 30%, and from 22.5% to 27.5%. Such numerical variations may occur due to, for example, experimental errors, typical errors in measurement or processing of manufacturing compounds, compositions, concentrates or preparations, differences in the source, manufacture or purity of the starting materials or ingredients used in the present invention, or similar considerations.

[0073] As used herein, the term "effective amount" refers to an amount of an active agent or pharmaceutical composition sufficient to bring about a cosmetic or therapeutic effect to an individual in need thereof. The effective amount may be varied by a person of ordinary skill in the art having ordinary knowledge, depending on the use of excipients, the route of administration, the possibility of co-administration with other treatment methods, or the condition to be treated, but the present invention is not limited thereto.

[0074] As used in the present invention, the term "administer" refers to introducing an active ingredient into an individual by a method or route so that at least a portion of the active ingredient is localized in a desired location to produce a desired effect. For example, the active ingredient of the present invention can be administered to an individual by injection or topical administration, but the present invention is not limited thereto.

[0075] As used herein, "subject" refers to any vertebrate, including but not limited to humans or mammals, such as deer, mules, elks, and black-tailed deer. In some preferred embodiments, the subject is a mammal, such as a human or a non-human mammal, such as a domesticated mammal, such as a dog, cat, horse, rat, mouse, etc., or a livestock mammal, such as a cow, sheep, pig, deer, etc.

[0076] The terms "include", "comprising", "having", "containing", "including" and any other variations thereof are intended to cover non-exclusive inclusions in the present invention. For example, when describing an object "comprising" a limitation, other components, elements, components, structures, regions, parts, devices, systems, steps or connections may be additionally included unless otherwise specified, and other limitations should not be excluded.

[0077] As used herein, the term "effective amount" refers to the amount of an active agent or pharmaceutical composition sufficient to produce a cosmetic or therapeutic effect in an individual in need thereof. For example, a "cosmetically effective amount" may be the amount of a composition administered at a specific dose sufficient to achieve the desired appearance, feel and / or protective effect. In some embodiments, the effective amount of the composition promotes the prevention or reduction of the appearance and / or symptoms associated with an undesirable condition, such as wrinkles, fine lines, thinning of the skin, loss of skin elasticity or loss of skin softness, or other skin characteristics associated with aging, ultraviolet light, exposure to chemicals, adverse climate (such as temperature, humidity), dietary intake, biological agents, environmental oxidants, etc. The effective amount may be varied by a person of ordinary skill in the art with common knowledge, depending on the use of excipients, the route of administration, the possibility of co-use with other therapeutic treatments, or the condition to be treated, but the present invention is not limited thereto.

[0078] As used in the present invention, the term "administering" refers to introducing an active ingredient into an individual by a method or route so that at least a portion of the active ingredient is located at a desired site to produce a desired effect. For example, the active ingredient of the present invention can be administered to an individual by injection or topical administration, but the present invention is not limited thereto. Administration of the composition of the present invention can be performed in the systemic or local environment of an individual. For example, the site of topical administration can be any site in the body where desired or beneficial tissue development occurs, such as a joint, a surgical site, a segmented bone gap or a site of non-union of a fracture, a wound, an ulcer, or an inflammatory rash. In addition, the composition of the present invention can be administered to an individual in, on, or as part of an implantable device. For example, the device can be a sponge, a biocompatible polymer, a bioerodible polymer, a filler, a gel, a bone matrix, an artificial bone matrix, a bolt, a screw, an endotracheal tube, a stent, a contact lens, a pacemaker, a central IV tube, a catheter, or an intracranial device.

[0079] As used herein, "skin" as an enveloping organ refers to the largest and fastest growing organ of the body and is classified as a major component of the integumentary system, one of the ten major organ systems found in higher animals. Due to its protective function, the skin is particularly susceptible to disease and trauma. The skin plays a variety of critical roles, including regulating temperature, providing a dynamic barrier to the outside world, repair processes that occur throughout life, and serving as a conduit to support a vast network of sensory receptors. The skin performs a variety of functions that are important for survival or physical health. The skin heals to prevent blood loss after injury, regulates body temperature by dissipating heat, and serves as a layer of cold resistance, absorption, secretion, thermal regulation, sensory detection and orientation, and barrier protection.

[0080] As used herein, "age-related features" or "wrinkles" refer to fine lines, small wrinkles, creases, folds or ridges, or wrinkles particularly on the face, neck, arms or any part of the skin.

[0081] As used in the present invention, "derivatives" of cells include but are not limited to: multipotent stem cells, pluripotent stem cells, adult stem cells, tissue specific stem cells, secretomes, conditioned medium, exosomes, extracellular-vehicles, secreted substances or any combination thereof.

[0082] As used herein, the term "cosmetically or pharmaceutically acceptable carrier" refers to a cosmetically or pharmaceutically acceptable material, vehicle or composition, such as a solid or liquid filler, adhesive, diluent, preservative, biocompatible solvent, disintegrant, lubricant, suspending agent, flavor enhancer, capsule material, thickener, acid, surfactant, complexing agent, wetting agent or any combination thereof. In some embodiments, each element is "cosmetically or pharmaceutically acceptable", which means that it is compatible with other raw materials of cosmetic or pharmaceutical preparations and can be used in contact with organs or tissues of an individual (e.g., mammals) without causing substantial toxicity, allergic reactions, irritation, immunogenicity or other complications or problems. References include, for example, Remington: The Science and Practice of Pharmacy, 22nd ed.; Allen Ed.: Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients, 7th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2012; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. As used herein, the term "carrier" may be a solid, a solution, or a mixture in which the stem cells and / or their derivatives are suspended for topical treatment procedures, transplantation, or any other subsequent use. Such carriers include, but are not limited to, gels, ointments, pastes, or aerosol sprays. In some embodiments, a "cosmetically acceptable carrier" refers to a medium that is compatible with keratin materials (e.g., human skin).

[0083] As used herein, the term "wound" may be a site to be treated on the outside of an individual's body. A wound may be a site of severe injury, such as a burn, an acute traumatic wound, a chronic wound, or a surgical wound, where the outer skin of the site is damaged and / or lacks the outer skin. Embodiments of wounds may include both open and closed wounds. For example, wounds include burns, incisions, excisions, lacerations, abrasions, puncture or penetration wounds, surgical wounds, contusions, hematomas, crush injuries, and ulcers, but the present invention is not limited thereto.

[0084] Wounds and Wound Classification

[0085] In addition to the definitions provided above, the term "wound" may include, for example, injuries to the skin and subcutaneous tissue caused by different means and with various characteristics (e.g., pressure sores caused by prolonged bed rest and wounds caused by trauma). According to the depth of the wound, it can be classified into 4 levels: Level 1: Wounds limited to the epithelium; Level 2: Wounds extending to the dermis; Level 3: Wounds extending to the subcutaneous tissue; Level 4 (or full-thickness wounds): Wounds with exposed bones (e.g., bone pressure points such as the greater trochanter or sacrum). The term "partial-thickness wound" refers to wounds including levels 1 to 3. Examples of partial-thickness wounds include: burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers. The term "deep wound" refers to wounds including levels 3 to 4. The methods of the present invention contemplate the treatment of all types of wounds, including deep wounds and chronic wounds. The term "chronic wound" refers to unhealed wounds. Optionally, the chronic wound is selected from the group consisting of venous ulcers, pressure sores, vasculitic ulcers, diabetic ulcers and decubitus ulcers. Chronic skin wounds include, for example, pressure sores, diabetic ulcers, venous ulcers, vasculitic ulcers, arterial ulcers and mixed ulcers. Chronic wounds may be arterial ulcers, including ulcers caused by complete or partial arterial occlusion. Chronic wounds may be venous stasis ulcers, including ulcers caused by venous valve dysfunction and related vascular diseases. Chronic wounds may be trauma-induced ulcers, diabetic ulcers or vasculitic ulcers.

[0086] As used herein, "administer", "treat", "supplement", "injection" or "provide" refers to a technique for delivering a substance (i.e., stem cells) to the whole body or a part thereof or any combination thereof. When a therapeutically effective amount of the present invention is administered parenterally or intravenously, it is usually formulated into a unit dosage form (e.g., emulsions, pills, and ointments).

[0087] It has been found that when stem cells are supplemented or treated with stem cells in individuals in need, stem cells can migrate / circulate to the target area, where they differentiate into fat cells, hair follicle cells, skin cells, bone cells and cartilage cells, and form structures including fat, hair, skin and bones, restoring various functions.

[0088] As used in the present invention, "stem cells" may include cells derived from amniotic fluid, bone marrow, umbilical cord blood, umbilical cord, placental tissue, adipose tissue, peripheral blood, dental pulp and deer antlers, but the present invention is not limited thereto. Stem cells can self-proliferate and differentiate into at least two cell lineages. Further, as used in the present invention, "stem cells" may also refer to cells that have the ability to replicate indefinitely, usually throughout the life cycle of an organism, and under certain conditions or given specific signals, stem cells can differentiate into many different cell types that make up the organism. In other words, stem cells have the potential to develop into mature cells with characteristic shapes and specialized functions, such as keratinocytes, sebocytes, transmission amplifier cells or melanocytes. Stem cells may be present in the basal layer of the epithelium, the pits of the interfollicular epidermis (IFE), the sebaceous glands and the bulges of the hair follicles.

[0089] As used in the present invention, "cell" refers to the smallest structural unit of living matter that can function autonomously. The cell is surrounded by a semipermeable membrane and consists of one or more nuclei, cytoplasm and various organelles. Cells include all somatic cells obtained or derived from living or dead animals at any stage of development, as well as germ cells, including sperm and eggs (animal reproductive bodies with nutrient and protective envelopes, consisting of eggs or embryos). The present invention includes two general categories of cells: prokaryotes and eukaryotes. The cells intended for use in the present invention include all cell types from all organisms in all kingdoms: plants, animals, protists, fungi, archaea and true bacteria. Stem cells are cells that produce many different levels of specialized cells through continuous division. For example, hematopoietic stem cells produce red blood cells and white blood cells. From conception to death, humans contain stem cells, but the differentiation ability of adult stem cells decreases.

[0090] As used in the present invention, the term "differentiation" in relation to cells refers to the process by which cell structure and function become specialized. During embryonic development, differentiation gradually limits development potential and increases functional specialization, forming specialized cells, tissues and organs. The term "differentiable" refers to the ability of a cell to differentiate into a desired cell type. As used in the present invention, the term "differentiates" refers to specialization (differentiation) or return to a more primitive cell type (dedifferentiation).

[0091] As used in the present invention, "medium", "basal medium" or "media" refers to the optimal culture medium for a variety of animal cells, including neurons, stem cells, mesenchymal stem cells, primary epithelial cells, keratinocytes, cervical epithelial cells, renal epithelial cells and established cell lines, but not limited thereto. In at least one embodiment, the culture can be expansion or differentiation.

[0092] "Growth media" refers to compositions used for the growth of microorganisms or cell culture. Different types of media can be used to grow different types of cells. The biggest differences are between media used for cell culture (cell culture uses specific cell types derived from plants or animals) and media used for the growth of microorganisms. The reason for these differences is that cells derived from whole organisms and in culture often cannot grow in the absence of certain conditions, such as hormones or growth factors that are often present in the body. In the case of animal cells, these conditions are usually met by adding serum to the culture medium. These media usually appear red or pink due to pH indicators. Growth media for embryonic stem cells optionally contain minimal essential media, namely Eagle's: amino acids, salts (ferric nitrate nonahydrate, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate), vitamins (ascorbic acid, folic acid, nicotinamide, riboflavin, B-12) or Dulbecco's: added iron, glucose; non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, L-glutamic acid, fetal bovine serum and leukemia inhibitory factor (LIF). In the case of microorganisms, there are no such restrictions because microorganisms are generally single-celled organisms. Another major difference is that animal cells in culture are generally grown on surfaces that they are in contact with, and the medium is provided in the form of a liquid and covers the cells. Bacteria such as Escherichia coli (E. coli, the most commonly used microorganism in laboratories) can be grown in solid media or liquid media, and liquid nutrient media are generally called nutrient broth. Depending on the growth medium required by the microorganism, it is either nutrient broth or Luria-Bertani medium (LB medium). Bacteria grown in liquid culture generally form a colloidal suspension. When agar (a substance that forms a gel) is added to the liquid culture medium, the liquid culture medium can be poured into a culture dish, where the agar solidifies (called an agar plate) and provides a solid medium in which the microorganism can be cultured.

[0093] A person of ordinary skill in the art with general knowledge can determine a growth medium suitable for initial stem cell preparation. Commonly used growth medium for stem cells includes, but is not limited to, Iscove's Modified Dulbecco's (IMDM) medium, DMEM, KO-DMEM, DMEM / F12, RPMI 1640 medium, McCoy's 5A medium, minimal essential medium alpha medium (α-MEM), F-12K nutrient mixed medium (Kaighn's modified, F-12K), X-vivo 20, Stemline, CC100, H2000, Stemspan, MCDB 131 medium, Eagle basal medium (BME), Glasgow minimal essential medium, modified Eagle medium (MEM), Opti-MEM I reduced serum medium, Waymouth's MB 752 / 1 medium, Williams medium E, medium NCTC-109, neuroblastoma medium, BGJb medium, Brinster's BMOC-3 medium, CMRL medium, non-CO 2 Dependent culture medium, Leibovitz's L-15 medium, etc.

[0094] In at least one embodiment of the present invention, the composition or cosmetic composition comprises an effective amount of a matrix derived from amniotic fluid stem cells, wherein the amniotic fluid is obtained from deer.

[0095] The subject of the present invention is a human, deer, dog, cat, horse, rat, mouse, etc. or a livestock mammal, such as a cow, sheep, or pig.

[0096] The gestation period of this deer is 4, 5, 6, 7, 8 or 9 months.

[0097] The composition or cosmetic composition derived from deer amniotic fluid exhibits properties that influence skin regeneration and its biological renewal, so that the composition or cosmetic composition can reconstruct age-related degeneration.

[0098] Furthermore, the composition or cosmetic composition produced from deer amniotic fluid exhibits properties as a pharmaceutical agent useful for treating difficult-to-heal wounds (eg, caused by diabetes) and skin damage after chemotherapy.

[0099] The cosmetic composition of the present invention is further used in cosmetic medicine, particularly as a medicament for stimulating skin regeneration after physical trauma such as burns, acne, cuts and lacerations.

[0100] As used herein, the term "NANOG" refers to a homeobox gene. NANOG is believed to be necessary for the unlimited proliferation of stem cells and the generation of cells of different types. This gene is a potential master gene that helps embryonic stem cells grow and immortalize them in the laboratory.

[0101] As used herein, the term "SOX2" refers to the gene encoding the sex-determining region Y (SRY) box 2 protein. This intronless gene encodes a member of the SRY-related HMG-box (SOX) transcription factor family, which is involved in the regulation of embryonic development and the determination of cell fate.

[0102] In at least one embodiment of the present invention, wrinkles are caused by age, obesity, injury, lack of hormones, side effects of drugs, or any combination thereof, but the present invention is not limited thereto. In addition, as humans age, wrinkles may naturally appear on the skin.

[0103] In at least one embodiment of the present invention, the wound is caused by a cut, a burn, an acute trauma, a chronic trauma, a surgical wound or other effects. In addition, skin wounds may also be caused by a variety of reasons, such as cuts, abrasions, scalds, diabetic ulcers, bedsores caused by long-term bed rest, etc. Wound healing is a complex and slow process. During the treatment, new epithelial tissue and connective tissue are formed through migration and proliferation.

[0104] In at least one embodiment of the present invention, the stem cells may be derived from amniotic fluid, bone marrow, umbilical cord blood, umbilical cord, placental tissue, adipose tissue, peripheral blood and dental pulp, but the present invention is not limited thereto. In at least one embodiment of the present invention, the stem cells are derived from deer and cultured for 4 to 14 days for expansion. In some embodiments, RT-PCR and / or flow cytometry are used to show that the stem cells from deer amniotic fluid are positive for CD9, CD90, CD29, CD44, CD73, CD105, Nestin, Sox2 and NANOG, and negative for CD31, CD34 and CD45.

[0105] In the present invention, the deer stem cells for skin care or hair growth promotion can be used alone or with gauze, a mask, an ointment or any combination thereof.

[0106] Example

[0107] Exemplary embodiments of the present invention are further described in the following examples, which should not be construed as limiting the scope of the present invention.

[0108] Materials and Methods

[0109] Cell culture

[0110] Amniotic fluid was collected from 5-month pregnant female deer and provided by Taiwan Deer Velvet Biotechnologies Co., Ltd. (Tainan, Taiwan, China). The procedure was the same as amniocentesis. Under the guidance of ultrasound, 20 ml of amniotic fluid was aspirated with a syringe with a long needle. Fresh amniotic fluid was filtered with a 70 μm sterile cell filter (Corning Falcon) and centrifuged at 200×g for 5 minutes. The resulting precipitate was washed with phosphate-buffered saline and re-centrifuged. The cells were cultured in minimal essential medium Eagle, α-modified (α-MEM) containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin and 100 μg / ml streptomycin (P / S), placed in a T75 flask, and cultured at 37°C with 5% CO 2 and humidified environment. Change the medium twice a week. Remove non-attached cells by changing the medium. Cell colonies appear after 5 to 7 days of culture and reach 80 to 90% confluence in 10 to 14 days. Dissociate the cells with trypsin / EDTA solution and pass them for expansion or storage.

[0111] RT-PCR

[0112] Follow the manufacturer's instructions for use Mini kit (Qiagen) was used to extract total RNA from deer amniotic fluid stem cells. cDNA was synthesized from RNA using QuantiNova reverse transcription kit (Qiagen) and PCR amplification was performed using DreamTaqGreen PCR premix solution (Thermo Scientific). The PCR reaction conditions were 95°C for 2 minutes, followed by 38 amplification cycles (95°C for 30 seconds, specific primer binding temperature for 30 seconds and 72°C for 30 seconds, and a final extension at 72°C for 15 minutes). All primers (as listed in Table 1 (SEQ ID NO: 1 to 20) or Table 2 (SEQ ID NO: 21 to 26)) were designed by "NCBI Primer Selection". The RT-PCR results are shown in Figure 2. Figure 2A and Figure 2B shown.

[0113] Table 1 Primers used in this study for detecting CD9, CD29, CD44, CD73, CD90, CD105, Nestin, SOX2, NANOG, and GAPDH by RT-PCR

[0114]

[0115]

[0116] Table 2 Primers used in this study that did not detect CD31, CD34, and CD45 by RT-PCR

[0117]

[0118] Flow cytometric analysis

[0119] Surface markers of deer amniotic fluid stem cells were analyzed by flow cytometry using human-Nestin-FITC antibody (R&D System) and mouse / human-SOX2-APC antibody (R&D System). Isotype-matched mouse IgG1-FITC (R&D System) and mouse IgG2A APC (R&D System) were used as negative controls.

[0120] Differentiation potential

[0121] To evaluate the differentiation potential of the three cell lines of deer amniotic fluid stem cells, three different types of induction were performed. Osteogenesis: Deer amniotic fluid stem cells were cultured on 6-well plates until 90% confluence. After washing once with PBS, the culture medium was replaced with an osteogenic medium consisting of DMEM containing 10% FBS, 50 μM ascorbic acid 2-phosphate, 100 nM dexamethasone, and 10 mM b-glycerophosphate (all Sigma-Aldrich). The culture medium was changed twice a week. After three weeks, Alizarin Red S staining was performed to detect calcium deposition. Adipogenesis: Deer amniotic fluid stem cells were cultured on 6-well plates until 90% confluence. After washing once with PBS, the medium was replaced with adipogenic medium, which consisted of DMEM containing 10% FBS, 500 μM 3-isobutyl-1-methyl-xanthine (IBMX), 1 μM dexamethasone, 60 μM indomethacin and 5 μg / ml insulin (all from Sigma-Aldrich). The medium was replaced twice a week. After three weeks, fat droplets were detected by Oil Red O staining. Chondrogenesis: 5 x 10 5 Deer amniotic fluid stem cells were added to a 15 ml polypropylene conical centrifuge tube and centrifuged at 200 x g for 5 minutes. Chondrogenic medium (MSCgoTM Chondrogenic Differentiation Medium, Biological Industries catalog number 05-220-1B) was added to the cell pellet and incubated at 37°C in 5% CO 2The cells were stored in an incubator for 3 weeks. The culture medium was changed twice a week. After 4 weeks, the round precipitates were embedded in paraffin and cut into 3 μm sections. The sections on the slides were stained with Alcian blue to detect the sulfated glycosaminoglycan (GAG) products that differentiated into cartilage.

[0122] Collagen synthesis

[0123] Human foreskin fibroblasts (HS68) were seeded in 96-well plates and cultured at 37°C in 5% CO 2 Incubate overnight in an incubator. Remove the culture medium. Then treat fibroblasts with different concentrations of deer amniotic fluid stem cell conditioned medium (dAFSC-CM), original culture medium (as negative control group, NC) and 100 ng / ml TGF-β (as positive control group, PC). The treated cells were stored at 37°C with 5% CO 2 The supernatant was transferred to a microplate spectrophotometer (Epoch TM ) to detect the concentration of type I collagen C-peptide. After the collagen in the supernatant was detected using a microtiter plate, MTT solution was added to the microtiter plate and stored at 37°C with 5% CO 2 Incubate for 2 hours. MTT is used to react with enzymes in mitochondria to form purple formazan After the reaction, DMSO was added to dissolve the crystals, and the OD value was measured at a wavelength of 570 nm using spectrophotometry.

[0124] Detection of healing area

[0125] Mouse fibroblasts (NIH / 3T3) were inoculated with SPLScar TM When cells reached confluence, remove SPLScar TM The fibroblasts were then treated with different concentrations of dAFSC-CM (deer amniotic fluid stem cell conditioned medium), medium (NC) and 10 ng / ml TGF-β (positive control group). The treated cells were stored at 37°C with 5% CO 2 The cells were incubated in an incubator for 8 hours. Images of cell migration were analyzed using ImageJ.

[0126] Cell viability test

[0127] Mouse fibroblasts (NIH / 3T3) were seeded into 96-well plates and incubated at 37°C and 5% CO 2 The cells were cultured overnight in an incubator. The culture medium was removed. Then, fibroblasts were treated with different concentrations of dAFSC-CM (deer amniotic fluid stem cell conditioned medium) and culture medium (NC). The treated cells were maintained at 37°C in a 5% CO 2 Incubate incubator for 24 hours. Replace the medium with MTT solution. MTT is used to react with enzymes in mitochondria to form purple formazan. After the reaction, DMSO was added to dissolve the crystals, and the OD value was measured by spectrophotometry at a wavelength of 570 nm.

[0128] In vitro skin irritation test

[0129] In EpiDerm TM dAFSC-CM (deer amniotic fluid stem cell conditioned medium) and 5% SDS (positive control group) were added on top of the tissue. All conditions were according to EpiDerm TM The skin irritation test standard procedure (OECD TG 439) was performed in triplicate to evaluate whether dAFSC-CM caused skin irritation. After exposure to dAFSC-CM for 60 minutes, the tissue was washed with PBS and dried. Then, the tissue was transferred to new culture medium and incubated at 37°C with 5% CO 2 The tissue was incubated with MTT solution (1 mg / mL) at 37°C in a 5% CO 2 The cells were incubated in an incubator for 3 hours to detect the relative survival rate (%) of the cells. MTT is used to react with enzymes in mitochondria to form a purple formazan After the reaction, isopropanol was added to dissolve the crystals, and the OD value was measured by spectrophotometry at a wavelength of 570 nm.

[0130] In vitro eye irritation test

[0131] In EpiOcular TM dAFSC-CM (deer amniotic fluid stem cell conditioned medium), sterile deionized water (NC) and methyl acetate (PC) were added on top of the tissue. All conditions were according to EpiOcular TM The eye irritation test standard procedure (OECD TG 492) was performed in triplicate to evaluate whether dAFSC-CM caused eye irritation. After exposure to dAFSC-CM for a certain period of time, the tissue was washed with PBS and dried. Then, the tissue was transferred to new culture medium and incubated at 37°C with 5% CO 2The tissue was incubated with MTT solution (1 mg / mL) at 37°C and 5% CO 2 The cells were incubated in an incubator for 3 hours to detect the relative survival rate (%) of the cells. MTT is used to react with enzymes in mitochondria to form a purple formazan After the reaction, isopropanol was added to dissolve the crystals, and the OD value was measured at a wavelength of 570 nm using spectrophotometry.

[0132] Human Hair Follicle Dermal Papilla Cells (HFDPC) for Cell Proliferation and Migration Assays

[0133] In the HFDPC proliferation assay, HFDPCs were seeded at 1,500 cells / well in a 96-well plate and cultured at 37°C for 4 hours to allow cell attachment. After washing twice with sterile PBS buffer, HFDPCs were cultured in αMEM medium containing 2% FBS and different concentrations of dAFSC-CM (0%, 1%, 2%, 5%, 10% and 15%) at 37°C for 48 hours. Cell growth was assessed using Cell Counting KIT-8 (Dojindo Laboratories, Gaithersburg, MD) according to the manufacturer's instructions, and the absorbance at 450 nm was read by a microplate reader. All experiments were repeated three times and statistically analyzed by Student's t test (p<0.05). In the HFDPC migration assay, HFDPC cells were seeded into a 24-well plate and cultured at 37°C for three days until 90% confluence. After washing twice with sterile PBS buffer, HFDPC cells were manually scratched with a 200 μl yellow pipette and re-cultured in αMEM medium containing 2% FBS and different concentrations of dAFSC-CM (0%, 1%, 2%, 5%, 10% and 15%) at 37° C. for 0, 5, 9 and 24 hours. Cell migration images were captured at the beginning and at fixed intervals, and the closed area was analyzed by ImageJ software.

[0134] Example 1: Characteristics of Deer Amniotic Fluid Stem Cells

[0135] like Figure 1 As shown in the figure, microscopic images show that cells derived from deer amniotic fluid present a morphology similar to fibroblasts after culture, indicating that deer amniotic fluid may contain stem cells. Figure 2A As shown in the figure, the RT-PCR results showed that the cells derived from the deer amniotic fluid were negative for CD45 (first lane), CD34 (second lane) and CD31 (third lane) after culture. Figure 2BAs shown in Figure 1, RT-PCR results showed that cells derived from deer amniotic fluid were positive for CD9, CD29, CD44, CD73, CD90, CD105, Nestin, Sox2 and NANOG after culture. The primer sequences of the above genes are listed in Tables 1 and 2. Figures 3A to 3D As shown, the expression of the used antigens is shown in red histograms compared to the isotype control (black histogram). The values ​​show the positive expression results (Nestin = 98.5 and SOX2 = 92.5, respectively). Figure 4A and Figure 4B As shown, under the conditions of osteogenic differentiation, the cultured cells showed strong positive staining with Alizarin Red S ( Figure 4A : Control group (not induced); Figure 4B : after induction). Figure 4C and Figure 4D As shown, cells cultured in adipogenic differentiation medium for 3 weeks showed fat droplets and were positive after Oil Red O staining ( Figure 4C : Control group (not induced); Figure 4D : after induction). Figure 4E and Figure 4F As shown, cells cultured in chondrogenic differentiation medium for 4 weeks were positive for Alcian blue staining. These results indicate that the cultured cells are deer amniotic fluid stem cells.

[0136] Example 2: In vitro functional assay of deer amniotic fluid stem cell conditioned medium (dASFC-CM)

[0137] like Figure 5 As shown in the results, 2.5%, 5% and 10% dAFSC-CM significantly promoted the proliferation of human fibroblasts, and the effects of 2.5%, 5% and 10% dAFSC-CM on the proliferation of human fibroblasts were 130%, 142.7%, 159.6% and 159.6%, respectively. Furthermore, 5% and 10% dAFSC-CM significantly promoted the synthesis of procollagen in human fibroblasts, and the synthesis rates of 5% and 10% dAFSC-CM on procollagen were 116.6% and 113%, respectively. Figure 6 It was shown that 5% and 10% dAFSC-CM significantly promoted wound healing, and the healing areas in 5% and 10% dAFSC-CM were 132% and 142%, respectively. Figure 7 The cell survival rate of fibroblasts treated with different concentrations of dASFC-CM (the horizontal axis is %) is shown, among which the untreated cells (negative control group, NC) are 100%. The cell survival rate test under different concentrations of dAFSC-CM did not show significant changes, which shows that dAFSC-CM does not produce cytotoxicity to fibroblasts. Figure 8 and Fig. 9 As shown, 10% dAFSC-CM did not affect the relative survival rate and the average tissue survival rate, indicating that dAFSC-CM is not irritating to the skin and eyes.

[0138] Fig.10 Compared with the control group, 1%, 2%, 5%, 10% and 15% dAFSC-CM significantly stimulated the proliferation of human hair follicle dermal papilla cells (HFDPCs), with relative cell growth rates of 126%, 147%, 165%, 166% and 163%, respectively. The data showed that 5% or more dAFSC-CM had a saturation effect on the growth of HFDPCs. Fig.11 For the cell migration test, the images of different concentrations (1%, 2%, 5%, 10% and 15%) of dAFSC-CM at 0, 5, 9 and 24 hours were compared, and the recovery rate of the scratch area was calculated by Image-J software. The data showed that compared with the negative control group, the wound coverage rate was fully improved after 5 hours of culture with 2%, 5%, 10% and 15% dAFSC-CM. After 9 hours of culture, 2% dAFSC-CM showed improved scratch wound coverage. In addition, after 5 hours of culture, 2% dAFSC-CM achieved a scratch wound coverage rate of 17%, and after 24 hours of culture, 2% dAFSC-CM achieved a scratch wound coverage rate of at least 86%.

[0139] The above description of the specific embodiments is only for illustrating the implementation mode required by the present invention, and is not intended to limit the scope of the present invention. Therefore, all modifications and changes made by a person skilled in the art with general knowledge should fall within the scope defined by the appended claims of the present invention.

[0140] Those skilled in the art will readily observe that various modifications and changes can be made to the apparatus and methods while maintaining the teachings of the present invention. Therefore, the above disclosure should be interpreted as being limited only by the scope and metes and bounds of the appended claims.

Claims

1. A composition, characterized in that: Include: Stem cells or their derivatives; and A cosmetically or pharmaceutically acceptable carrier, Among them, the stem cells are derived from the amniotic fluid of even-toed ungulates.

2. The composition according to claim 1, characterized in that The artiodactyl animal is selected from the group consisting of pigs, wild boars, hippopotamuses, antelopes, deer, giraffes, camels, llamas, alpacas, sheep, goats and cattle.

3. The composition according to claim 2, characterized in that The even-toed ungulate is a deer.

4. The composition according to claim 1, characterized in that The derivative is selected from the group consisting of secretomes, conditioned medium, exosomes, extracellular carriers, secreted substances and any combination thereof.

5. The composition according to claim 1, characterized in that The stem cells are negative for CD31, CD34, CD45, or any combination thereof.

6. The composition according to claim 1, wherein The stem cells are positive for CD9, CD29, CD44, CD73, CD90, CD105, Nestin, Sox2, NANOG, or any combination thereof.

7. Use of a composition for preparing a drug for skin regeneration or skin treatment, characterized in that: The composition comprises: Stem cells or their derivatives; and A cosmetically or pharmaceutically acceptable carrier, Among them, the stem cells are derived from the amniotic fluid of even-toed ungulates.

8. The use according to claim 7, characterized in that The even-toed ungulate is a deer.

9. The use according to claim 7, characterized in that The derivative is selected from the group consisting of secretomes, conditioned medium, exosomes, extracellular carriers, secreted substances and any combination thereof.

10. The use according to claim 7, characterized in that The composition promotes the synthesis of procollagen, collagen or elastin in the subject.

11. The use according to claim 7, characterized in that The skin regeneration includes preventing, treating or reducing age-related features on the skin of the individual.

12. The use according to claim 11, characterized in that The age-related characteristic is wrinkles, age spots, rough skin texture, dull skin, fine lines, visible pores, loss of skin structure, or any combination thereof.

13. The use according to claim 7, characterized in that The skin treatment includes restoring damage to the subject's skin, reducing scarring of damaged skin of the subject, enhancing recovery of the subject's skin after cosmetic or dermatological surgery, or preventing or reducing wrinkles on the subject's skin.

14. The use according to claim 13, characterized in that The damage is a wound caused by burns, acute trauma, chronic trauma, surgical wounds or other physical or chemical impacts, and the wrinkles are caused by age, obesity, injury, hormonal deficiency, drug side effects or any combination thereof.

15. Use of a composition for preparing a drug for promoting hair growth or preventing hair loss, characterized in that: The composition comprises: Stem cells or their derivatives; and A cosmetically or pharmaceutically acceptable carrier, Among them, the stem cells are derived from the amniotic fluid of even-toed ungulates.

16. The use according to claim 15, characterized in that The hair loss is caused by a condition selected from the group consisting of: androgenic alopecia, alopecia areata and telogen effluvium.

17. The use according to claim 15, characterized in that The even-toed ungulate is a deer.

18. The use according to claim 15, characterized in that The derivative is selected from the group consisting of secretomes, conditioned medium, exosomes, extracellular carriers, secreted substances and any combination thereof.

19. The use according to claim 15, characterized in that The composition promotes the growth or migration of hair follicle dermal papilla cells of the subject.

Images