Mesenchymal stem cells for use in the treatment of skin defects

Abstract

The present invention relates to a composition comprising integrin alpha10 selected mesenchymal stem cells (MSCs) for use in the treatment and / or regeneration of skin defect wounds.

Description

[Technical field]

[0001] The present invention relates to a composition comprising integrin alpha10-selected mesenchymal stem cells (MSCs) for use in the treatment of skin defect wounds. [Background technology]

[0002] Chronic skin wounds are considered a major challenge for the health care system. The incidence of chronic ulcers as a result of diabetes, vascular insufficiency, and pressure ulcers is rising in line with the increase in the average age. Leg ulcers are estimated to cost the EU a total health care cost of about 1-2% of the budget. In the UK, the costs associated with pressure ulcers are about 4% of the total health care cost. Furthermore, wounds that are difficult to treat may be accompanied by pain that may persist for months or years, limited mobility, reduced quality of life, limited productivity, and economic stress. On the other hand, burn patients may suffer life-threatening injuries and long-term effects. Current management policies are associated with a significant reduction in mortality, although a corresponding improvement in functional or aesthetic outcome for extensive burns exceeding 50-60% of the total surface area has yet to be achieved.

[0003] Cell therapy is a new approach to replace damaged skin and promote regeneration, improving wound healing and coverage of large burn wounds with or without meshed autologous skin grafts (El-Serafi et al. 2017; El-Serafi et al. 2018). Cultured autologous keratinocytes were investigated as an improved therapeutic drug. Unfortunately, many difficulties were encountered; for example, they are difficult to isolate, have a slow proliferation rate, and require a skin biopsy to isolate the cells, which may not be available in severe cases (Karlsson et al. 2020). Moreover, the epidermal layer formed by these cells is usually brittle due to limited flexibility. As an alternative potential source, adipose-derived stem cells (ADSCs) offer the possibility of differentiation into various cell lineages, including epidermal cells (Kosaric et al. 2019; Raghuram et al. 2020). Unfortunately, efficient in vitro differentiation of ADSCs into epidermal cells represents an unmet challenge. On the other hand, the local application of ADSCs and other stem cell types may be associated with improved healing properties.

[0004] MSCs were first used as cellular medicines in humans in 1995. After several years of utilizing MSCs as therapeutic agents, many questions remain unanswered regarding their behavior, such as the heterogeneity of the MSC population in the final product, the appropriate conditions to activate their immunomodulatory capacity in vivo, the outcome of the banking procedure, the best route for their delivery, and their response to stressful conditions (Garcia-Bernal et al. 2021). Summary of the Invention

[0005] The present invention relates to a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs), and thus a homogenous composition of MSCs, for use in the treatment and / or regeneration of mammalian skin defect wounds. Indeed, the inventors have found that topical application of integrin alpha 10 selected MSCs, also referred to herein as XSTEM, to wounds results in excellent wound healing in a porcine model, with the newly formed skin tissue being highly similar to normal skin. There was regeneration of the basement membrane, keratinization of the skin, and formation of high levels of collagen structured in bundles and resembling collagen naturally occurring in skin. Integrin alpha 10 selected MSCs also caused less scarring compared to transplanted skin cells. Most interestingly, integrin alpha 10 selected MSCs produced more robust and complete re-epithelialization compared to other cells tested, including autologous keratinocytes or stem cells, the latter being a heterogeneous cell composition that also includes MSCs that have not been selected for a specific marker, particularly not for integrin alpha 10 expression.

[0006] Selection of MSCs based on expression of integrin alpha10 on their surface results in a homogenous cell composition with superior immunomodulatory, anti-inflammatory, and skin regenerative capabilities compared to cell compositions containing non-selected MSCs.

[0007] In one aspect of the disclosure, there is provided a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for use in the treatment and / or regeneration of a skin defect wound in a mammal.

[0008] Another aspect of the present disclosure provides a use of a cosmetic composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for reducing scar formation resulting from healing of a skin defect wound in a mammal.

[0009] In another aspect of the disclosure, there is provided a use of a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for the non-therapeutic treatment of a skin defect wound in a mammal. [Brief description of the drawings]

[0010] [Figure 1] Macroscopic images of wounds immediately after the surgical wound was created, i.e., before the application of cells and after 1 and 2 weeks. No signs of inflammation, tearing or infection were detected in any of the wounds. The wound healing patterns of XSTEM with and without autologous stem cells (500,000 cells) and autologous keratinocytes were particularly interesting. The vehicle was 2.5% human serum albumin in saline. [Diagram 2] Hematoxylin and eosin staining of skin wound biopsies 2 weeks after cell application. XSTEM of 500,000 cells (E) epidermal structure was comparable to normal skin biopsy (A). On the other hand, epidermal thickness was reduced when cells were combined with autologous keratinocytes (F). XSTEM of 1 million cells showed that cells were associated with a disorganized epidermis and hyperkeratosis. Epidermis development using autologous 500,000 stem cells (D), autologous 7 x 105 keratinocytes (C) and vehicle (B) was less efficient. Scale bar set at 100 μm. [Diagram 3] Masson's trichrome staining of skin wound biopsies 2 weeks after cell application. The collagen fascicular cell pattern and staining intensity of XSTEM of 500,000 cells (E) was comparable to normal skin biopsy (A). The intensity of collagen staining was higher than controls when cells were combined with autologous keratinocytes (F) or 500,000 autologous stem cells (D). XSTEM of 1 million cells was associated with a less organized epidermis and less intense collagen staining (G), while autologous 7 x 105 keratinocytes (C) produced less collagen compared to vehicle (B). Scale bar set at 100 μm. [Figure 4A]Clinical assessment of epithelialization. XSTEM with 500,000 cells showed a better trend towards epithelialization compared to vehicle after 1 and 2 weeks, reflecting faster wound healing. Furthermore, XSTEM with 500,000 cells had a better trend towards epithelialization than most of the groups studied, especially at early time points (7 days). Changes in wound size, mainly caused by wound edge contraction, did not appear to be affected by any of the cell types studied. [Figure 4B] Clinical assessment of wound size. XSTEM with 500,000 cells showed a better trend towards epithelialization compared to vehicle after 1 and 2 weeks, reflecting faster wound healing. Furthermore, XSTEM with 500,000 cells had a better trend towards epithelialization than most of the groups studied, especially at early time points (7 days). Changes in wound size, mainly caused by wound edge contraction, did not appear to be affected by any of the cell types studied. [Figure 5A] Secretion of factors promoting wound healing. Concentrations of secreted PGE2 (A) and IDO (B) were measured over 72 h after stimulation of XSTEM with various concentrations of the pro-inflammatory cytokines IFNγ and TNFα (40 ng / mL and 60 ng / mL of each cytokine, respectively). Unstimulated (0 ng / mL) XSTEM served as a control. Concentrations of VEGF (C) and HGF (D) secreted by XSTEM in the culture supernatant were higher in cell preparations containing a higher cell number. [Figure 5B] Secretion of factors promoting wound healing. Concentrations of secreted PGE2 (A) and IDO (B) were measured over 72 h after stimulation of XSTEM with various concentrations of the pro-inflammatory cytokines IFNγ and TNFα (40 ng / mL and 60 ng / mL of each cytokine, respectively). Unstimulated (0 ng / mL) XSTEM served as a control. Concentrations of VEGF (C) and HGF (D) secreted by XSTEM in the culture supernatant were higher in cell preparations containing a higher cell number. [Figure 5C]Secretion of factors promoting wound healing. Concentrations of secreted PGE2 (A) and IDO (B) were measured over 72 h after stimulation of XSTEM with various concentrations of the pro-inflammatory cytokines IFNγ and TNFα (40 ng / mL and 60 ng / mL of each cytokine, respectively). Unstimulated (0 ng / mL) XSTEM served as a control. Concentrations of VEGF (C) and HGF (D) secreted by XSTEM in the culture supernatant were higher in cell preparations containing a higher cell number. [Figure 5D] Secretion of factors promoting wound healing. Concentrations of secreted PGE2 (A) and IDO (B) were measured over 72 h after stimulation of XSTEM with various concentrations of the pro-inflammatory cytokines IFNγ and TNFα (40 ng / mL and 60 ng / mL of each cytokine, respectively). Unstimulated (0 ng / mL) XSTEM served as a control. Concentrations of VEGF (C) and HGF (D) secreted by XSTEM in the culture supernatant were higher in cell preparations containing a higher cell number.

[0011] definition "Anti-integrin alpha10 antibody" or "anti-integrin alpha10 subunit antibody" are used interchangeably herein to refer to an antibody capable of at least recognizing and binding to the integrin alpha10 subunit of the heterodimeric protein integrin alpha10beta1. These antibodies may be antibodies that recognize an epitope of the heterodimeric protein integrin alpha10beta1, the epitope including amino acid residues of both the integrin alpha10 subunit and the integrin beta1 subunit.

[0012] As used herein, "integrin α10" or "integrin alpha10" refers to the α10 subunit of the heterodimeric protein integrin α10β1. This designation does not exclude the presence of an integrin β1 subunit that binds to the integrin α10 subunit and forms the quaternary structure of the integrin α10β1 heterodimer. The human integrin α10 chain sequence is known and published in GenBank™ / EBI Databank Accession No. AF074015 and described in Camper 1998. "Alpha" and "α", as well as "alpha10" and "alpha10" are equivalent terms.

[0013] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0014] The term "some embodiments" can include one or more than one embodiments.

[0015] The use of the terms "a" or "an" as used throughout the text or in conjunction with the term "comprising" in the claims and / or this specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0016] As used herein, the terms "isolate", "sort" and "select" refer to the act of identifying a cell as a specific type of cell and separating it from cells that do not belong to the same cell type or another differentiation state. Moreover, these terms may also refer to the act of identifying cells by the presence of a specific marker. For example, the present invention relates to mesenchymal stem cells (MSCs) selected with integrin alpha 10. Usually, isolation refers to the first step of separation, which can be, for example, mechanical, while "selection" is more specific and is performed, for example, with antibodies. The skilled artisan will understand that the procedure of "isolating", "sorting" or "selecting" cells results in the enrichment of said cells.

[0017] As used herein, the term "integrin alpha 10 enriched MSCs" refers to MSCs enriched with integrin alpha 10. 高 MSCs”, “Integrin alpha 10 selected mesenchymal stem cells” and “Mesenchymal stem cell enriched for integrin alpha 10 high As described in Example 1, the MSCs used in the present invention are selected using a procedure to enrich for MSCs expressing integrin alpha 10, for example by selecting those MSCs expressing integrin alpha 10 using an anti-integrin alpha 10 antibody. One skilled in the art will appreciate that cells selected for a particular property, for example, MSCs expressing integrin alpha 10 or MSCs expressing integrin alpha 10, can be enriched for integrin alpha 10. 高 It will be appreciated that MSCs may form a specific homogenous cell population.

[0018] As used herein, "mesenchymal stem cells" or "MSCs" refer to multipotent stromal cells as defined by the International Society for Cellular Therapy's Mesenchymal and Tissue Stem Cells Committee (see Dominici M et al., Cytotherapy. 8(4):315-7 (2006)). MSCs must be plastically adherent when maintained in standard culture conditions, and must express CD105, CD73, and CD90, and must lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19, and HLA-DR surface molecules. MSCs must have the ability to differentiate in vitro into osteoblasts, adipocytes, or chondroblasts.

[0019] As used herein, the term "skin defect wound" refers to, but is not limited to, damage or trauma to internal or external tissue, preferably to the epidermis and / or dermis of the skin. The wound may be an acute or chronic wound. For example, the acute wound may be an incision, a laceration, abrasion or burn, a puncture wound, a penetration wound, or a wound resulting from a skin disease such as psoriasis, acne, and eczema. By way of example, the chronic wound may be a venous ulcer, a diabetic ulcer, a decubitus ulcer, a corneal ulcer, a digestive ulcer, or a wound resulting from ischemia and radiation poisoning.

[0020] As used herein, "treatment and / or regeneration of a skin defect wound" refers to promoting, accelerating, and / or improving healing at the wound site, i.e., the formation of functional skin at the wound site. Thus, "treatment and / or regeneration of a skin defect wound" ideally results in the formation or regeneration of the dermis and epidermis, including the basal layer, that exhibit the characteristics of functional skin.

[0021] The terms "difficult to heal wound", "refractory wound" and "chronic wound" are used interchangeably herein. As used herein, the terms "difficult to heal wound" and "chronic wound" refer to wounds that have not healed. For example, wounds that do not heal within about 4-6 weeks are considered chronic. A "chronic wound" is a wound that does not progress through an orderly and timely repair sequence, does not respond to treatment, and / or where the demands of treatment exceed the patient's physical health, tolerance or endurance. Many wounds initially considered acute wounds eventually become chronic wounds due to factors that are not yet fully understood. One important factor is the formation of biofilms due to the translocation of planktonic bacteria within the wound. For example, chronic wounds may have an epithelial layer that does not cover the entire wound surface and may undergo bacterial colonization, resulting in the formation of biofilms that are resistant to treatment with antimicrobial agents.

[0022] Generally, chronic wounds are classified into three broad categories based on their primary cause: venous insufficiency, arterial insufficiency, and diabetic complications, or pressure ulcer-related. Difficult-to-heal wounds due to venous insufficiency account for 70%-90% of difficult-to-heal wounds and are more prevalent in the elderly. Venous insufficiency leads to venous hypertension, which blocks blood flow and causes subsequent ischemia. Venous insufficiency can occur as a result of obstruction to venous outflow or reflux due to valvular damage. Reperfusion of tissues after a period of ischemia can cause reperfusion injury, which causes tissue damage leading to wound formation.

[0023] Typical chronic wounds include first-degree burns, which can be shallow, reddened areas of skin; second-degree burns, which can be blistered areas that may heal naturally after the blister fluid is removed; third-degree burns, which are burns across the skin and usually require surgical intervention for wound healing; scalding burns, which can occur from hot water, grease, or radiator fluids; thermal burns, which can occur after contact with a flame and are usually deep burns; chemical burns, which can occur from acids or alkalis and are usually deep burns; electrical burns; and may include "burn ulcers", which include contact burns, which are usually deep and can occur from muffler tailpipes, hot irons and stoves, or other substances.

[0024] The terms "severe hereditary bullous disease" and "epidermolytic bullous disease" are used interchangeably herein.

[0025] As used herein, "preventing" or "prevention" includes delaying, halting, or reducing the risk of the onset of a disease, disorder, or condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Mesenchymal stem cells selected with integrin alpha 10 In some embodiments of the present disclosure, at least 50% of the MSCs express the integrin alphal0 subunit.

[0027] Example 1 describes a method for producing integrin alpha 10 selected MSCs as presented in the present disclosure. An important advantage of integrin alpha 10 selected MSCs is that they have been selected using the criteria of integrin alpha 10 protein expression and are therefore homogenous cultures and / or populations of MSCs. These cells have been shown to exhibit robust expression of stem cell markers, see for example WO 2018 / 138322. The skilled artisan will know that several methods can be used to select and thereby enrich cells. In the present invention, integrin alpha 10 expressing MSCs are enriched during the isolation / selection procedure. For this purpose, anti-integrin alpha 10 antibodies can be used. Isolation and selection of MSCs can be performed as described in WO 2018 / 138322, which is incorporated herein by reference.

[0028] As disclosed in Example 1, in the selection step, the selected MSCs are selected by the expression of integrin alpha 10 using anti-integrin alpha 10 antibody, and express integrin alpha 10. More specifically, the selected cells are MSCs that express heterodimer integrin alpha 10 beta 1 (alpha 10 beta 1), since integrin alpha 10 subunit is expressed together with integrin beta 1 subunit. The selection step is followed by an expansion step, in which the integrin alpha 10 expression of each of the selected MSCs may vary, i.e., not all MSCs may always express integrin alpha 10 during expansion, i.e., at the time of administration. However, at least 50% of the administered cells express integrin alpha 10 subunit at the time of administration of the MSCs to the patient.

[0029] In some embodiments of the present disclosure, at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example at least 100% of the MSCs express integrin alpha 10 subunit.

[0030] In some embodiments of the present disclosure, the MSCs are MHC class II, CD45, CD34, CD11b and / or CD19 negative.

[0031] In some embodiments of the present disclosure, the MSCs express CD73, CD90 and / or CD105.

[0032] In some embodiments of the present disclosure, the composition secretes growth factors, anti-inflammatory factors and / or immunomodulatory factors.

[0033] In some embodiments of the present disclosure, the composition secretes a growth factor, an anti-inflammatory factor and / or an immunomodulatory factor at a higher concentration compared to a composition in which less than 50% of the MSCs express the integrin alpha 10 subunit.

[0034] In some embodiments of the present disclosure, the composition secretes a growth factor, an anti-inflammatory factor and / or an immunomodulatory factor at a higher concentration compared to a composition in which less than 50% of the MSCs express the integrin alpha 10 subunit.

[0035] In some embodiments of the present disclosure, the growth factor, anti-inflammatory factor and / or immunomodulatory factor is indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF) and / or hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are growth factors that promote tissue regeneration and skin regeneration. The integrin alpha 10 selected MSCs of the present disclosure secrete these factors in greater amounts and / or concentrations compared to MSC populations not selected for integrin alpha 10 expression, and are therefore more effective in treating chronic wounds and regenerating skin at skin defect wound sites as defined herein.

[0036] In some embodiments of the present disclosure, the MSCs are selected from the group consisting of mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal stromal cells, or a mixture thereof.

[0037] In some embodiments of the present disclosure, MSCs are induced to express the integrin alphal0 subunit.

[0038] In some embodiments of the present disclosure, the MSCs are cultured in a medium comprising mammalian serum and FGF-2.

[0039] In some embodiments of the present disclosure, MSCs are cultured in a medium that includes platelet lysate and / or platelet lysate components.

[0040] In some embodiments of the present disclosure, MSCs are cultured in a medium comprising FGF-2 and platelet lysate and / or platelet lysate components.

[0041] In some embodiments of the present disclosure, MSCs are cultured in a medium comprising mammalian serum and platelet lysate and / or platelet lysate components.

[0042] In some embodiments of the present disclosure, MSCs are cultured in a medium containing TGFβ.

[0043] In some embodiments of the present disclosure, MSCs are cultured in a medium comprising FGF2.

[0044] In some embodiments of the present disclosure, MSCs are cultured in serum-free medium that includes platelet lysate and / or platelet lysate components.

[0045] In some embodiments of the present disclosure, MSCs are cultured in serum-free medium containing growth factors.

[0046] In some embodiments of the present disclosure, MSCs are cultured in serum-free medium containing the growth factors FGF2 and / or TGFβ.

[0047] In some embodiments of the present disclosure, the MSCs are allogeneic or autologous.

[0048] In some embodiments of the present disclosure, the MSCs and the mammal are from the same species.

[0049] In some embodiments of the present disclosure, the MSCs and the mammal are from different species.

[0050] In some embodiments of the present disclosure, MSCs are isolated from tissue containing mesenchymal stem cells, selected for expression of integrin alpha10 beta1, and expanded in culture.

[0051] In some embodiments of the present disclosure, selection of MSCs was performed using anti-integrin alpha10 antibody.

[0052] In some embodiments of the present disclosure, the MSCs are derived from adipose tissue, bone marrow, synovium, peripheral blood, umbilical cord blood, Wharton's jelly, and / or amniotic fluid.

[0053] In some embodiments of the present disclosure, the MSCs are derived from adipose tissue.

[0054] In some embodiments of the present disclosure, the MSCs are derived from bone marrow.

[0055] In some embodiments of the present disclosure, the MSCs are derived from fetal, neonatal, juvenile or adult MSCs and / or progenitor cells.

[0056] In some embodiments of the present disclosure, the MSCs are not derived from embryonic cells or embryos.

[0057] In some embodiments of the present disclosure, the MSCs are in an in vitro cell culture. In some embodiments of the present disclosure, the MSCs are in an in vitro cell suspension.

[0058] In some embodiments of the present disclosure, the composition comprising integrin alpha10 selected MSCs is a cosmetic composition.

[0059] In some embodiments of the present disclosure, the composition comprising integrin alpha10-selected MSCs is a non-therapeutic composition.

[0060] Treatment of skin defects In one aspect, the present disclosure provides a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for use in the treatment and / or regeneration of a skin defect wound in a mammal.

[0061] Another aspect of the present disclosure provides a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for use in preventing fibrosis in mammalian skin.

[0062] A further aspect of the present disclosure provides a method of regenerating skin at the site of a skin defect in a mammal and / or reducing scar formation resulting from healing of the skin defect, said method comprising administering to the skin defect a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs).

[0063] The treatment disclosed herein targets multiple skin defect wounds, which can be classified into non-extensive categories such as difficult-to-heal or chronic wounds, skin disorders caused by external factors, inflammatory skin diseases, post-treatment disorders of the skin, and genetic or developmental disorders of the skin, such as genetic disorders of the skin.

[0064] In some embodiments of the present disclosure, a composition is provided comprising integrin alpha 10 selected MSCs for use in treating and / or regenerating mammalian skin defect wounds, such as difficult-to-heal wounds, such as chronic wounds.

[0065] The inventors have demonstrated the ability of integrin alpha 10 selected MSCs to heal skin defect wounds in a pig model, which is the most accepted and widespread animal model for testing the healing of human skin defect wounds, such as difficult-to-heal wounds. Indeed, among the animals used in the study, pigs have the skin most similar to human skin. Furthermore, the wound healing process and process of acute and difficult-to-heal wounds are essentially the same, except for the possible presence of an underlying disease in the case of difficult-to-heal wounds. Furthermore, the size and depth of the full-thickness wounds created in our study resemble chronic wounds, since the large wound size does not allow for natural healing by contracture. Those skilled in the art will recognize that other animal models can be used to confirm the ability of integrin alpha 10 selected MSCs to heal skin defect wounds.

[0066] The integrin alpha10 selected MSCs of the present disclosure are suitable and successful for treating skin defect wounds, including difficult to heal wounds, due to their demonstrated ability to regenerate skin and secrete factors that promote wound healing by acting against inflammation and modulating the immune system and / or promoting tissue regeneration.

[0067] In some embodiments of the present disclosure, the difficult-to-heal wounds are difficult-to-heal wounds associated with venous insufficiency, arterial insufficiency and diabetic complications, rare diseases such as sickle cell anemia, or pressure sores.

[0068] In some embodiments of the present disclosure, the difficult-to-heal wound is a skin ulcer, wound, or skin defect wound resulting from vascular insufficiency or diabetic damage of blood vessels. For example, diabetic foot syndrome / diabetic foot ulcers and diabetic neuropathic ulcers are several types of difficult-to-heal wounds that can benefit from the treatments of the present disclosure.

[0069] In some embodiments of the present disclosure, the skin defect wound is a skin disorder caused by an external factor.

[0070] Skin disorders caused by external factors include, for example, pressure sores, skin diseases caused by friction or mechanical stress, skin diseases caused by foreign bodies, skin diseases induced or aggravated by exposure to cold air, heat or electricity induced skin diseases, skin diseases caused by light or ultraviolet light, skin diseases caused by ionizing radiation, allergic contact dermatitis, photoallergic contact dermatitis, irritant contact dermatitis, allergic contact urticaria, protein contact dermatitis, allergic contact sensitization, phototoxic reactions to skin contact with photoactive agents, and skin reactions to poisonous or toxic animals.

[0071] In some embodiments of the present disclosure, the skin disorder caused by an external factor is a burn, a bed sore due to prolonged bed rest, a trauma-induced skin defect wound, or an amputation.

[0072] In some embodiments of the present disclosure, the skin injury caused by external factors is burn.For example, the burn can be a second degree burn, such as deep burn or scald.The burn can be caused by exposure to low temperature, heat, electricity, light, UV radiation, ionizing radiation, allergens, chemicals such as acid and base, and other external agents.

[0073] In some embodiments of the present disclosure, the skin defect wound is an inflammatory skin disease.

[0074] Examples of inflammatory skin diseases that may benefit from the treatment of the present disclosure are dermatitis, eczema, atopic dermatitis, papillary squamous cell disease, urticaria, angioedema or other scleral disorders, inflammatory erythema and other reactive inflammatory skin diseases, immune bullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory skin diseases.

[0075] In some embodiments of the present disclosure, the skin defect wound is a post-treatment defect of the skin.

[0076] Examples of post-treatment disorders of the skin that may benefit from the treatment of the present disclosure are insufficient surgical scarring of the skin, skin flap necrosis, myocutaneous flap necrosis, skin graft failure, and composite graft failure.

[0077] In some embodiments of the present disclosure, the skin post-treatment disorder is a surgical incision.

[0078] In some embodiments of the present disclosure, the skin defect is a genetic and / or developmental disorder affecting the skin.

[0079] Examples of genetic and / or developmental disorders affecting the skin that may benefit from treatment of the present disclosure are genetic skin diseases such as epidermolysis bullosa, or dermatitis pemphigoid.

[0080] In some embodiments of the present disclosure, the epidermolysis bullosa is selected from the group consisting of simple epidermolysis bullosa, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive epidermolysis bullosa, chondrotic epidermolysis bullosa, epidermolysis bullosa, and epidermolysis bullosa.

[0081] Regardless of the underlying cause of the skin defect wound, compositions comprising integrin alpha10-selected MSCs of the present disclosure can be used to treat or regenerate skin defect wounds having the physical characteristics described herein.

[0082] In some embodiments of the present disclosure, the skin defect wound is an external defect. Thus, the skin defect wound may be a defect of the dermis and / or the epidermis.

[0083] Additionally, skin defect wounds may extend below the skin surface, which is also called subcutaneous or subcutaneous.

[0084] In some embodiments of the present disclosure, the skin defect wound is a dermal and / or epidermal and / or subcutaneous defect.

[0085] In some embodiments of the present disclosure, the skin defect is an open defect.

[0086] In some embodiments of the present disclosure, the skin defect wound is an inflamed wound or an ulcer.

[0087] In some embodiments of the present disclosure, the skin defect is an acute skin defect wound.

[0088] In some embodiments of the present disclosure, the skin defect is a chronic skin defect wound. As provided herein, the chronic skin defect wound is a skin defect wound that is difficult to heal. If an acute skin defect wound does not heal within 4 to 6 weeks under normal circumstances, it may become a chronic skin defect wound.

[0089] In some embodiments of the present disclosure, the skin defect is a full thickness skin defect, for example, a skin defect that affects both the dermis and the epidermis is considered to be a full thickness skin defect.

[0090] In some embodiments of the present disclosure, the skin defect is a partial thickness skin defect, for example, a skin defect that affects only a portion of the dermis and epidermis is considered to be a partial thickness skin defect.

[0091] In some embodiments of the present disclosure, the skin defect wound has a surface area of ​​at least 0.3 cm x 0.3 cm and covers up to the entire body of the mammal being treated. The compositions comprising integrin alpha 10-selected MSCs of the present disclosure can be used to treat skin defect wounds of any size.

[0092] Those skilled in the art, including trained physicians, will recognize that skin defect wounds can vary depending on the cause of the wound and complications that arise during the healing process. For example, wounds as small as 0.3 cm x 0.3 cm can cause difficulties for patients, as is common in patients with diabetic complications, and can pose a risk of infection if not healed adequately. On the other hand, in certain conditions, up to 100% of the patient's skin area may be affected, such as in the case of severe burns. The integrin alpha 10-selected MSCs described herein can be used to treat wounds of any size.

[0093] In some embodiments of the present disclosure, administration of a composition comprising integrin alpha 10 selected MSCs according to the present disclosure results in increased collagen production at the administration site. Furthermore, the collagen produced appears to be arranged into bundle-like structures typical of functional skin.

[0094] In some embodiments of the present disclosure, administration of a composition comprising integrin alpha10 selected MSCs according to the present disclosure results in regeneration of the basement membrane at the site of administration.

[0095] In some embodiments of the present disclosure, administration of a composition comprising integrin alpha10 selected MSCs according to the present disclosure results in keratinization of the skin at the site of administration.

[0096] Dosage form A composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in the treatment and / or regeneration of skin defects in a mammal, in some embodiments, is adapted for topical administration.

[0097] As used herein, "local administration" or "administering locally" means direct administration of an agent at or near a site on or within the body of an animal where the biological effect of the agent is desired.

[0098] In some embodiments of the present disclosure, the composition is administered topically to a skin defect wound.

[0099] In some embodiments of the present disclosure, the compositions disclosed herein may be administered by injection. For example, the skin defect may be covered with a film suitable for covering the skin defect of a mammal, such as a propylene film or a film made of any other material, and an enclosure may be formed between the skin defect and the film. The compositions disclosed herein may then be injected into the skin defect, such as the enclosure between the skin defect and the film, by passing a needle laterally through the skin. Such procedures are known to those skilled in the art.

[0100] In some embodiments of the present disclosure, the compositions disclosed herein are administered by injection into the skin.

[0101] In some embodiments of the present disclosure, the compositions disclosed herein are administered by subcutaneous injection.

[0102] In some embodiments of the present disclosure, the skin defect wound is covered prior to being treated with the compositions disclosed herein.

[0103] In some embodiments of the present disclosure, the skin defect wound is covered after being treated with the compositions disclosed herein.

[0104] 8. The composition for use according to any one of the preceding items, wherein the composition comprises 100,000 to 2,000,000 MSCs, such as 200,000 to 1,000,000 MSCs, for example about 200,000 MSCs, such as about 300,000 MSCs, for example about 400,000 MSCs, such as about 500,000 MSCs, for example about 600,000 MSCs, such as about 700,000 MSCs, for example about 800,000 MSCs, such as about 900,000 MSCs, for example about 1,000,000 MSCs, such as about 1,200,000 MSCs, for example about 1,500,000 MSCs.

[0105] Depending on the size of the skin defect wound to be treated, different amounts of the MSCs of the present disclosure can be administered.

[0106] In some embodiments of the present disclosure, the composition is 2 20,000–150,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000–130,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000-120,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000-100,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000-90,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000-80,000 MSCs per cm of the skin defect to be treated. 2 30,000-75,000 MSCs per cm of the skin defect to be treated, for example. 2 30,000-70,000 MSCs per cm of the skin defect to be treated. 2 30,000-60,000 MSCs per cm of the skin defect to be treated. 2 40,000-60,000 MSCs per cm of the skin defect to be treated. 2 40,000-70,000 MSCs per cm of the skin defect to be treated. 2 40,000–75,000 MSCs per cm of the skin defect to be treated, for example. 2 40,000-80,000 MSCs per cm of the skin defect to be treated. 2 50,000-100,000 MSCs per cm of skin defect to be treated, for example. 2 50,000-120,000 MSCs per cm of the skin defect to be treated, for example. 2 50,000-130,000 MSCs per cm of the skin defect to be treated, for example.2 The cells are administered to contain 50,000 to 150,000 MSCs per well.

[0107] In some embodiments of the present disclosure, the composition is administered in the form of a cell suspension in a pharma- ceutically acceptable liquid medium.

[0108] In some embodiments of the present disclosure, the pharma- ceutically acceptable liquid medium comprises human serum albumin.

[0109] In some embodiments of the present disclosure, the pharma- ceutically acceptable liquid medium comprises human serum albumin, such as clinical grade human serum albumin diluted in saline.

[0110] In some embodiments of the present disclosure, the composition is administered in the form of a cell suspension in 2.5% clinical grade human serum albumin diluted in saline.

[0111] Alternatively, or additionally, in addition to administering the MSCs or compositions of the present disclosure locally by injection, the skin defect wound can be treated with a dressing or reservoir loaded with integrin alpha 10 selected MSCs. Thus, in some embodiments of the present disclosure, the composition is administered locally to the skin defect wound on the dressing or reservoir.

[0112] In some embodiments of the present disclosure, the MSCs used in the treatment and / or regeneration of skin defect wounds are allogeneic.

[0113] In some embodiments of the present disclosure, the MSCs used in the treatment and / or regeneration of skin defect wounds are autologous.

[0114] In some embodiments of the present disclosure, the composition further comprises a hydrogel, which may help protect the site of the skin defect from infection and thus aid in the healing process.

[0115] A variety of hydrogels known to those skilled in the art for use in moisturizing skin may be used, for example, the hydrogel may be fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose or pectin, or any functionally equivalent hydrogel.

[0116] In some embodiments of the present disclosure, the composition further comprises keratinocytes, such as autologous or allogeneic keratinocytes, which can promote epithelialization and help restore the vascular network at the site of a skin defect.

[0117] In some embodiments of the present disclosure, the composition further comprises an anti-inflammatory agent. The presence of an anti-inflammatory agent can help minimize inflammation at the skin defect site, which can also help promote healing.

[0118] In some embodiments of the present disclosure, the compositions further comprise an immunosuppressant to prevent cell rejection and / or therapeutic failure, which is believed to be particularly useful in the compositions of the present disclosure when allogeneic integrin alpha 10 selected MSCs are used alone or in combination with allogeneic keratinocytes.

[0119] Methods and Uses One aspect of the present disclosure provides a use of a composition comprising integrin alpha10-selected MSCs for the preparation of a medicament for treating a skin defect wound.

[0120] Another aspect of the present disclosure provides a method of treating a skin defect wound in a mammal in need thereof, comprising administering to the mammal a composition comprising integrin alpha 10-selected MSCs in an amount effective to treat the skin defect wound.

[0121] One aspect of the present disclosure relates to a non-therapeutic cosmetic use of a composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) to reduce scar formation resulting from the healing of a skin defect wound in a mammal.

[0122] In another aspect of the disclosure, there is provided a use of a composition comprising integrin alpha 10 selected mesenchymal stem cells (MSCs) for the non-therapeutic treatment of a skin defect wound in a mammal.

[0123] The composition comprising the integrin alpha 10 selected MSCs of the present disclosure may also be used to reduce or minimize scarring, for example, to reduce or minimize scar formation. As used herein, the term "reduce or minimize" preferably refers to the administration of the composition of the present disclosure to a subject to reduce or minimize the de novo formation of scarring. Furthermore, the reduction of scarring preferably also includes the amelioration or prevention of pain and / or pruritus during scar formation. To reduce scarring, the composition of the present disclosure is preferably administered locally to the site of a skin defect wound while the skin defect wound is in the process of healing.

[0124] In some embodiments of the present disclosure, a cosmetic composition is provided comprising integrin alpha10 selected MSCs, further comprising a cosmetically acceptable carrier.

[0125] In some embodiments of the present disclosure, a non-therapeutic composition is provided comprising integrin alpha10 selected MSCs, further comprising a cosmetically acceptable carrier. EXAMPLES

[0126] Example 1: Production of MSCs enriched for integrin alpha 10 the purpose This example demonstrates how integrin alpha10 selected MSCs can be isolated, selected, expanded and stored prior to use in a treatment model.

[0127] As used herein, the term "XSTEM" is synonymous with the term "integrin alpha10 enriched MSCs" and XSTEM is an internal designation used to refer to integrin alpha10 enriched MSCs.

[0128] Materials and Methods Integrin alpha 10 selected mesenchymal stem cells (MSCs) were isolated from human or animal adipose donor tissue or other MSC-containing sources. Adipose tissue was dissociated / digested and the adipose-derived stromal vascular fraction (SVF) was resuspended in MSC expansion medium and seeded into cell culture flasks, allowing MSCs to attach to the plastic and proliferate.

[0129] Plastic-adherent cells were analyzed for positive expression (≧95%) of cell surface markers CD73, CD90 and CD105 and negative expression (≦2%) of CD45, CD34, CD11b, CD19 and HLA-DR, as measured by flow cytometry. This specific antigen expression criterion is also part of the MSC definition defined by the International Society for Cellular Therapy (Dominici 2006). MSC preparations were expanded in monolayer culture in MSC expansion medium and integrin alpha10 expressing MSCs were selected using an antibody that specifically binds to integrin alpha10 (thereby recognizing the full receptor integrin alpha10 beta1, i.e. integrin α10β1) and magnetic bead separation or by FACS cell sorting. Integrin alpha10 selected MSCs were further expanded to check cell surface expression of defined MSC antigens and further demonstrate triple differentiation potential. Alpha10 selected MSCs were frozen alive in cryopreservation medium and frozen until use.

[0130] result: This procedure resulted in integrin alpha 10 selected MSCs (XSTEM) expanded and frozen in vials ready for administration, for example local administration.

[0131] conclusion This manufacturing process generates alpha10-selected MSCs that meet the minimum standards defining human MSCs and can be applied in cell therapy.

[0132] Example 2: Wound healing factors secreted by integrin alpha 10 selected MSCs the purpose This example shows the paracrine effect of integrin alpha 10 enriched MSCs (XSTEM) by secreting immunomodulatory, anti-inflammatory and regenerative factors. As used herein, the term "XSTEM" is synonymous with the term "integrin alpha 10 enriched MSCs".

[0133] Materials and Methods Integrin alpha 10-selected mesenchymal stem cells (XSTEM) were stimulated with the pro-inflammatory cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) for 72 h. Culture supernatants were collected and analyzed for indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) by enzyme-linked immunosorbent assay (ELISA). The concentrations of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in culture supernatants released by XSTEM at steady state and with different cell numbers were also measured by Luminex multiplex assay.

[0134] result: PGE2 was significantly expressed in unstimulated XSTEM, and expression was further increased upon stimulation with IFNγ and TNFα (Figure 5A). IFNγ and TNFα stimulation of XSTEM also induced the expression of IDO (Figure 5B). PGE2 and IDO are anti-inflammatory molecules known to play an immunomodulatory role by suppressing T cell proliferation and promoting macrophage M2 polarization. In addition, XSTEM also secrete regenerative growth factors, including HGF and VEGF, which are known to promote skin wound healing (Figure 5C) (Guillamat-Prats et al. 2021). Based on previous work showing that integrin alpha10-selected MSCs secrete significantly more PGE2 than unselected MSCs (Uvebrant et al. 2019), secretion of other anti-inflammatory and immunomodulatory factors, such as IDO, HGF and VEGF, is also expected to be greater in integrin alpha10-selected MSCs (and compositions containing a majority of these MSCs) compared to MSCs not selected for integrin alpha10 expression.

[0135] conclusion Integrin alpha 10 selected MSCs secrete important cytokines and growth factors known to promote wound healing processes and tissue regeneration. Therefore, integrin alpha 10 selected MSCs have been successfully used to treat skin defect wounds, including chronic or difficult to heal wounds, such as those characterized by inflammation, due to their excellent ability to secrete factors that promote wound healing and the fact that they can regenerate skin, as shown in Example 3.

[0136] Example 3: In vivo treatment of skin defect wounds.

[0137] the purpose The aim of these experiments was to demonstrate and evaluate the therapeutic efficacy of integrin alpha 10-selected MSCs for the healing of skin defect wounds in a validated porcine model. Pig skin approximates human skin in terms of skin adherence, hair coverage, epidermis and dermis thickness, and healing mechanisms. Thus, pigs are considered to be a suitable species for wound healing studies (Sullivan et al. 2001). Furthermore, the size and depth of the full-thickness wounds created in our study resemble chronic wounds, as the large wound size does not allow for natural healing by contracture (Jeschke et al. 2017). The healing mechanisms of chronic wounds are in principle the same as non-chronic wounds. However, chronic wounds are often difficult to heal due to inflammation and / or the presence of infection or underlying disease at the wound site.

[0138] Materials and Methods Skin wound in vivo studies were performed in two pigs according to the following workflow: - On day 0, full thickness biopsies of skin including the underlying fat were surgically removed. The subcutaneous fat layer was mechanically dissociated. Adipose sections were minced and incubated with shaking in collagenase I for 90 min at 37°C to isolate adipose derived stem cells (ADSCs). Skin sections were cut into small pieces, which were incubated overnight in dispase at 4°C. The epidermis was then mechanically peeled off from the dermis and incubated with trypsin for 30 min at 37°C. After appropriate washing with phosphate buffered saline, cells were counted and resuspended in 2.5% human serum albumin (clinical grade) in saline. The volume was adjusted to 1 ml for each group depending on the target cell number. - On the following day (day 1), six critical size (at least 3x3 cm area) full thickness skin wounds were surgically incised on the back of each pig. Also on day 1, isolated autologous cells and integrin alpha 10 selected MSCs prepared as described in example 1 were applied in different concentrations: autologous keratinocytes (7×10 5 cells / wound), autologous stem cells (adipose-derived stem cells (ADSCs), 5 × 105 cells / wound), integrin alpha 10-selected MSCs (5 × 10 5 cells / wound), integrin alpha 10-selected MSCs + autologous keratinocytes (5 × 10 5 +5×10 5 cells / wound), integrin alpha 10-selected MSCs (1 × 10 6 After proper wound coverage and application of a non-absorbent dressing according to surgical guidelines, the cells were injected into the wound. - Day 7: After one week, the healing of the skin defect wound (wound size and epithelialization) was monitored. - After 2 weeks on day 14, the healing of the skin defect wound (wound size and epithelialization) was followed. Furthermore, the experiment was terminated and the wound was excised for histological evaluation. The wound was clinically evaluated and biopsies were collected, fixed and prepared for histological evaluation. Sections were stained with hematoxylin and eosin and Masson's trichrome according to local protocols.

[0139] result Autologous ADSCs and keratinocytes as well as human integrin alpha 10 selected MSCs (XSTEM) were applied to a full thickness, critical size skin wound model and followed for 2 weeks. Cells were administered at 5 × 10 5 5 × 10 with and without autologous keratinocytes 5 XSTEM cells, as well as 1 × 10 6 For the control, 5 × 10 5 Autologous ADSCs, 7 × 10 5 Autologous keratinocytes and vehicle were applied.

[0140] Macroscopically, no signs of inflammation, dehiscence or infection were detected in any of the wounds (Figure 1).

[0141] Hematoxylin and eosin staining showed that XSTEM epidermal structure of 0.5 million cells was comparable to normal skin (Figures 2E and A). When cells were combined with autologous keratinocytes, the epidermis was thinner (Figure 2F). XSTEM of 1 million cells showed that the cells were associated with a disorganized epidermis and hyperkeratosis (Figure 2G). Epidermal development was less efficient with autologous stem cells, keratinocytes or vehicle (Figures 2D, 2C, and 2B, respectively).

[0142] Masson's trichrome staining showed that the collagen fascicle cell pattern and staining intensity of XSTEM of 0.5 million cells was comparable to normal skin (Figure 3E and Figure 3A). When cells were combined with autologous keratinocytes or autologous stem cells, the intensity of collagen staining was higher than controls (Figure 3F and Figure 3D). XSTEM of 1 million cells was associated with less organization of the epidermis and less intense collagen staining (Figure 3G). Meanwhile, autologous keratinocytes were associated with less collagen production compared to vehicle (Figure 3C and Figure 3B).

[0143] conclusion 5×10 after 14 days 5 Topical treatment of skin defects with (500,000) integrin alpha 10-selected MSCs / wound resulted in regeneration of the basement membrane, keratinization of the skin, and formation of high levels of collagen that was structured into bundles and resembled collagen naturally occurring in skin. Topical treatment with 1 million integrin alpha 10-selected MSCs / wound also resulted in regeneration of the dermis and epidermis, but a less organized epidermis. These effects were not observed in skin defects treated with vehicle (placebo), autologous stem cells, or autologous keratinocytes.

[0144] References Hady Shahin,Moustafa Elmasry,Ingrid Steinvall,Katrin Markland,Pontus Blomberg,Folke Sjoberg,Ahmed El-Serafi(2020). Human Serum Albumin as a Clinically Accepted Cell Carrier Solution for Skin Regenerative Application. Scientific Reports 10:14486. Matilda Karlsson, Ingrid Steinvall, Pia Olofsson, Johan Thorfinn, Folke Sjоberg, Liselott Astrand, Fayiz S., Ahmad Khalaf, Divyasree Parambath, Ahmed T.El-Serafi, Moustafa Elmasry (2020). Sprayed Cultured Autologous Keratinocytes in the Treatment of Severe Burns:A Retrospective Matched Cohort Study. Annals of Burns and Fire Disasters 33:134-142. Anjali Raghuram,Roy Yu,Andrea Lo,Cynthia Sung,Melissa Bircan,Holly Thompson,Alex K Wong(2020). Role of stem cell therapies in treating chronic wounds:A systematic review. World Journal of Stem Cells 12,659-675. Nina Kosaric,Harriet Kiwanuka and Geoffrey C Gurtner(2019)Stem cell therapies for wound healing. Expert Opinion on Biological Therapy 19:575-585. Ahmed T El-Serafi,Moustafa Elmasry and Folke Sjоberg(2018). Cell Therapy,the Future Trend for Burn Management. Editorial in Clinics in Surgery 3:1896. Ahmed T.El-Serafi,Ibrahim El-Serafi,Moustafa Elmasry,Ingrid Steinvall,Folke Sjoberg(2017). Skin Regeneration in Three Dimensions,Current Status,Challenges and Opportunities. Differentiation 96:26-29. Dominici M et al.,Cytotherapy.8(4):315-7(2006) Garcia-Bernal,D.,et al.,The Current Status of Mesenchymal Stromal Cells:Controversies,Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy. Front Cell Dev Biol,2021.9:p.650664. Uvebrant K.,Reimer Rasmusson L.,Talts JF.,Alberton P.,Aszodi A.and Lundgren-Akerlund E.“Integrinα10β1-selected Equine MSCs have Improved Chondrogenic Differentiation,Immunomodulatory and Cartilage Adhesion Capacity.” Ann Stem Cell Res.2,001-009(2019).Sullivan TP,Eaglstein WH,Davis SC,Mertz P.The pig as a model for human wound healing. Wound Repair Regen.2001 Mar-Apr;9(2):66-76. Jeschke MG,Sadri A-R,Belo C,Amini-Nik S.A surgical device to study the efficacy of bioengineered skin substitutes in mice wound healing models. Tissue Eng Part C Methods.2017;23:237-4 Guillamat-Prats,R.,The Role of MSC in Wound Healing,Scarring and Regeneration. Cells,2021.10(7)

Claims

1. A pharmaceutical composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in the treatment and / or regeneration of skin defect wounds in a mammal, wherein at least 60% of the MSCs in the composition express integrin alpha 10.

2. A pharmaceutical composition comprising integrin alpha 10-selected mesenchymal stem cells (MSCs) for use in preventing fibrosis in mammalian skin, wherein at least 60% of the MSCs in the composition express integrin alpha 10.

3. A pharmaceutical composition for reducing scar formation resulting from healing of a skin defect wound in a mammal, wherein at least 60% of MSCs in said composition express integrin alpha 10.

4. The pharmaceutical composition according to claim 1 , wherein the skin defect wound is a wound that is difficult to heal, such as a chronic wound.

5. 5. The pharmaceutical composition of claim 4, wherein the difficult-to-heal wound is a difficult-to-heal wound associated with venous insufficiency, arterial insufficiency and diabetic complications, rare diseases such as sickle cell anemia or bedsores, and / or the difficult-to-heal wound is a skin ulcer, wound or skin defect wound caused by vascular insufficiency or diabetic disorders of blood vessels.

6. The pharmaceutical composition according to claim 1, wherein the skin defect wound is a skin disorder induced by an external factor, such as a bedsore, a skin disease caused by friction or mechanical stress, a skin disease caused by a foreign body, a skin disease induced or aggravated by exposure to cold air, a skin disease induced by heat or electricity, a skin disease caused by light or ultraviolet light, a skin disease caused by ionizing radiation, allergic contact dermatitis, photoallergic contact dermatitis, irritant contact dermatitis, allergic contact urticaria, protein contact dermatitis, allergic contact sensitization, a phototoxic reaction to skin contact with a photoactive agent, a skin reaction to a poisonous organism or animal, a burn, a bedsore caused by prolonged bed rest, a skin defect wound induced by trauma, or an amputation.

7. The pharmaceutical composition according to claim 1, wherein the skin defect wound is an inflammatory skin disease, such as dermatitis, eczema, atopic dermatitis, papillary squamous cell disease, urticaria, angioedema or other scleral disorders, inflammatory erythema and other reactive inflammatory skin diseases, immune blistering diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory skin diseases.

8. The pharmaceutical composition of claim 1, wherein the skin defect is a post-treatment disorder of the skin, such as an insufficient surgical scar of the skin, skin flap necrosis, myocutaneous flap necrosis, skin graft failure, composite graft failure, or surgical incision.

9. The pharmaceutical composition of claim 1 , wherein the skin defect wound is a genetic and / or developmental disorder affecting the skin, such as epidermolysis bullosa and / or a genetic skin disease.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is administered locally to the skin defect wound, by injection, or by any combination thereof.

11. The composition is applied to the skin defect wound to be treated. ２ The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is administered to contain 20,000 to 150,000 MSCs per administration.

12. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is administered in the form of a cell suspension in a pharma- ceutically acceptable liquid medium, for example a liquid medium containing human serum albumin.

13. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100% of the MSCs express integrin alpha 10 subunit.

14. the MSCs are MHCII-negative, CD45-negative, CD34-negative, CD11b-negative, CD19-negative and / or CD79alpha-negative; and / or The pharmaceutical composition according to any one of claims 1 to 3, wherein the MSCs express CD73, CD90 and / or CD105.

15. The pharmaceutical composition of any one of claims 1 to 3, wherein the MSCs are allogeneic or autologous.

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