Use of conditioned culture of umbilical cord interlining mesenchymal stem cells for healing and / or preventing formation and / or recurrence of traumatic or injured skin areas

Abstract

The invention also relates to a method of inducing, stimulating and / or promoting healing of a wounded or injured skin area wherein the method comprises topically treating the skin of a subject, which is the skin around the wounded or injured skin area, with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells. Likewise, the present invention relates to a method of preventing formation and / or recurrence of a wounded or injured skin area in a subject at risk of developing a wounded or injured skin area wherein the method comprises topically treating the skin of the subject with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells. Furthermore, the present invention relates to a method of treating rosacea, psoriasis, eczema, dermatitis, local steroid withdrawal syndrome, epidermis bullosa or skin injury caused by fragile skin, wherein the method comprises topically treating the skin of a subject around a wound or injured skin area to be treated with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells. The invention also relates to conditioned media, uses thereof, compositions comprising the same, methods for their production and uses of these compositions.

Description

Technical Field

[0001] The present invention relates to a method for inducing, stimulating, and / or promoting healing of a wound or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area. The present invention also relates to a method for preventing the formation and / or recurrence of a wound or damaged skin area in a subject who is at risk of developing a wound or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject. In addition, the present invention relates to a method for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin. The present invention also relates to conditioned medium, uses thereof, compositions comprising the same, methods for producing conditioned medium, and compositions containing the conditioned medium. These methods and compositions can be therapeutic methods, but can also be non-therapeutic or cosmetic methods or compositions. Background Art

[0002] Wounded and damaged skin, its healing complications, and the inability of patients at risk for developing wounded or damaged skin areas to effectively prevent their progression are major problems in both hospital and home settings. The ability of an organism to effectively and rapidly activate the healing process of wounded and damaged skin is essential not only for repairing skin that is constantly exposed to the external environment, but also because the skin serves as a primary defense system or protective barrier against the environment. Therefore, following an injury that results in wounded and damaged skin, the loss of extensive skin integrity can lead to significant disability or even death due to increased susceptibility to fungal, bacterial, and viral infections.

[0003] In order to understand the healing (i.e., repair and regeneration) process of damaged skin and wounds, it is necessary to understand the anatomy and physiological functions of normal skin. Histologically, the skin is divided into two functional layers, the epidermis and the dermis, which are separated by a basement membrane. The epidermis is multicellular, and new cells are continuously produced in the deepest layer (basal layer) of the epidermis and move outward toward the skin surface. The dermis is responsible for the mechanical properties of the skin and is composed of fibers and connective tissue, wherein the fibers are composed of elastin and collagen. Collagen fibers provide strength to the skin, and elastin fibers provide rebound strength. In normal skin, the dermis and epidermis form a protective barrier against the external environment and assist in numerous protective functions, including mechanical and light protection, immune surveillance, nutritional metabolism and repair. Because the skin plays such an important role in the body, the damaged skin needs to be replaced rapidly. When the protective barrier of the dermis and epidermis is destroyed, the structural integrity is impaired, and the vascular system below is destroyed, the physiological process of wound healing is initiated.

[0004] Wound healing involves the processes of hemostasis, inflammation, and proliferation. Skin repair begins with blood clot formation and platelet degranulation, which initiates hemostasis (homeostasis) by releasing growth factors required for wound repair and promoting the regeneration of damaged epidermis. In the early inflammatory phase, neutrophils are located at the site of the wound and are primarily responsible for clearing bacteria and foreign matter. In the late inflammatory phase, monocytes migrate to the site of the wound and transform into macrophages. The subsequent proliferative phase is characterized by increased granulation tissue formation, in which fibroblasts lay a collagen layer and produce new capillaries, ultimately leading to the formation of a new, complete epidermis in the affected area.

[0005] However, there are many types of wounds and diseases and conditions associated with damaged skin with different complications, in which the formation and / or recurrence of wounds or damaged skin cannot be effectively prevented and / or in which the healing process is insufficient or even inhibited. As an illustrative example, the development of pressure ulcers (also known as pressure sores or bedsores) is generally difficult to prevent. Pressure ulcers are injuries to the skin and underlying tissues, primarily caused by prolonged pressure on the skin, and may develop gradually or form within a few hours, affecting any part of the body that is subject to pressure. For example, pressure ulcers may particularly occur on bony parts of the body, such as the heels, elbows, hips, and the base of the spine. Typically, pressure ulcers affect, for example, bedridden patients, patients who sit in chairs or wheelchairs for long periods of time, elderly subjects or patients with mobility problems, subjects with skin that is easily damaged by dehydration, obese subjects or patients, subjects or patients with medical conditions that affect blood supply and make the skin more fragile or cause mobility problems, such as, but not limited to, diabetes, peripheral arterial disease, renal failure, heart failure, multiple sclerosis (MS), and Parkinson's disease. Strategies aimed at reducing the risk of developing pressure ulcers may include regular changes in body position and monitoring skin properties for signs and symptoms of pressure ulcers. Thus, there remains a need for effective and reliable compositions and methods for preventing the formation and / or recurrence of wounded or damaged skin, such as those resulting from pressure ulcers.

[0006] For chronic wounds, other problems of healing complications are known. Chronic wounds are usually accompanied by serious complications, because they comprise inflammatory cells and proteases that increase levels, and they suppress the healing process by degrading the extracellular matrix (ECM) that is mainly composed of collagen, and collagen plays an important role in the healing reaction, particularly in the above-mentioned proliferation stage. The example of chronic wounds is diabetic chronic wounds, such as diabetic foot ulcers, which are caused by poor blood flow and inflammation, and heal slowly, or may never heal if not treated. Out of other problems, this can lead to serious infection. In this case, the natural wound healing of chronic diabetic wounds is difficult, because the proinflammatory cytokines of elevated levels are secreted, and these proinflammatory cytokines hinder the natural wound healing that needs anti-inflammatory signals, and epithelial and endothelial migration activity is hindered, and this is then necessary for the process of closed wounds.

[0007] The typical procedure for treating wounds, particularly chronic wounds such as diabetic ulcers, venous ulcers and pressure ulcers, involves the use of dressings applied to the wound.

[0008] An example of an approved medical dressing is Apligraf, which is an active, bilayer skin substitute containing bovine type I collagen extracted and purified from bovine tendon and viable allogeneic human fibroblasts and keratinocytes isolated from human infant foreskin. This wound dressing / cover is particularly suitable for standard diabetic foot ulcer care and is used to treat full-thickness neuropathic diabetic foot ulcers of greater than 3 weeks duration that have not responded adequately to conventional ulcer therapy and that penetrate the dermis without exposing tendon, muscle, joint capsule, or bone. Another wound dressing / cover is It is a living, bilayer skin substitute containing bovine type I collagen extracted and purified from bovine tendon and living allogeneic human fibroblasts and keratinocytes isolated from human infant foreskin. It is indicated for the treatment of full-thickness diabetic foot ulcers of greater than 6 weeks' duration that penetrate the dermis without exposed tendon, muscle, joint capsule, or bone. It should be used in conjunction with standard wound care protocols and in patients who have an adequate blood supply to the involved foot.

[0009] However, these wound coverings are quite complex to manufacture and therefore relatively expensive, thereby adding considerable cost to the treatment / management of diabetic foot ulcers. Therefore, there is a need for new compositions that are easy to prepare and effective in treating ulcers such as diabetic foot ulcers. It is therefore an object of the present invention to provide such compositions for their subsequent use in wound healing. Summary of the Invention

[0010] The objects of the invention are achieved by a method, a conditioned medium and the use thereof having the features stated in the independent claims.

[0011] In a first aspect, the present invention provides a method for inducing, stimulating and / or promoting healing of a wound or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area.

[0012] In a second aspect, the present invention provides a method of preventing the formation and / or recurrence of a wound or damaged area of ​​skin in a subject who is at risk of developing a wound or damaged area of ​​skin, wherein the method comprises topically administering to the skin of the subject conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0013] In a third aspect, the present invention provides a method for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically treating the skin of a subject surrounding a wound or damaged skin area to be treated with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0014] In a fourth aspect, the present invention provides a method for producing a conditioned medium, the method comprising: (a) culturing umbilical cord mesenchymal stem cells in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 medium), M171 (Medium 171) and FBS (fetal bovine serum); (b) removing the umbilical cord mesenchymal stem cells from the culture medium; and (c) optionally culturing the umbilical cord mesenchymal stem cells in an additional culture medium, optionally, wherein the additional cell culture medium comprises a water-soluble antioxidant, wherein the conditioned medium is obtained by collecting the cell culture medium.

[0015] In a fifth aspect, the present invention provides a conditioned medium derived from umbilical cord mesenchymal stem cells, which is obtained or obtainable by the method according to the present invention.

[0016] In a sixth aspect, the present invention provides a composition comprising the conditioned medium of the present invention. Such a composition may be a pharmaceutical composition or a cosmetic composition (eg, in the form of an ointment, lotion, cream or gel) for treating a wound or skin condition as described herein.

[0017] In an eighth aspect, the present invention provides use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture in the preparation of a pharmaceutical composition for inducing, stimulating and / or promoting healing of wounded or damaged skin areas, and / or for preventing the formation and / or recurrence of wounded or damaged skin areas, and / or for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, or epidermolysis bullosa.

[0018] In a ninth aspect, the present invention provides a conditioned medium derived from umbilical cord mesenchymal stem cell culture for inducing, stimulating and / or promoting healing of wounded or damaged skin areas, and / or for preventing the formation and / or recurrence of wounded or damaged skin areas, and / or for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, and epidermolysis bullosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0020] Figure 1Shown is a technical information sheet for Lonza's Dulbecco's modified eagle medium, which includes the catalog number of DMEM used to prepare an exemplary example of culture medium (PTT-6) in the experimental section.

[0021] Figure 2 A technical information sheet of Lonza's Ham's F12 medium is shown.

[0022] Figure 3 Shown is a technical information sheet of Lonza's DMEM:F12 (1:1) medium, which includes the product catalog number of the DMEM:F12 (1:1) medium used to prepare an exemplary example (PTT-6) of the culture medium in the experimental section.

[0023] Figure 4 A technical information sheet for M171 medium from Life Technologies Corporation is shown, which includes the catalog number of the M171 medium used to prepare an illustrative example of the culture medium (PTT-6) in the experimental section.

[0024] Figure 5 Shown is an ingredient list including commercial suppliers and catalog numbers for each ingredient used to prepare PTT-6 medium in the experimental section.

[0025] Figure 6 The lesion size (cm) of 10 patients with chronic diabetic foot ulcers (DFU) treated with the conditioned medium of the present invention is shown. 2 ) decreased over time (week 1 to week 16).

[0026] Figure 7 AB shows the lesion size (cm) of 2 patients with chronic diabetic foot ulcers (DFU) (lower image is untreated, upper image is treated) with twice weekly administration of the conditioned medium of the present invention over a total duration of 16 weeks. 2 ) Image sequence of wound reduction and wound healing.

[0027] Figure 8 Shown are the lesion sizes (cm) of a patient with chronic diabetic foot ulcer (DFU) (lower image untreated, upper image treated) with three weekly administrations of the conditioned medium of the invention over a total duration of 8 weeks. 2 ) Image sequence of wound reduction and wound healing.

[0028] Figure 9AB shows the lesion size (cm) of patients with chronic diabetic foot ulcers (DFU) (left side - untreated, right side - treated) with twice weekly administration of the conditioned medium of the invention over a total duration of 5 weeks. 2 ) reduction and wound healing.

[0029] Figure 10 Shown are the wound sizes (cm) of patients with chronic diabetic foot ulcers (DFU) (left side - untreated, right side - treated) with the administration of the conditioned medium of the invention three times a week for a total duration of 3 weeks. 2 ) reduction and wound healing. DETAILED DESCRIPTION

[0030] The present invention is directed to methods and compositions that are all suitable for inducing, stimulating, and / or promoting the healing of wounds or damaged skin areas. Furthermore, the methods and compositions of the present invention are all suitable for preventing the formation and / or recurrence of wounds or damaged skin areas in subjects who are at risk of developing wounds or damaged skin areas. Furthermore, the methods and compositions of the present invention are all suitable for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin.

[0031] The present invention has surprisingly found that conditioned medium derived from umbilical cord mesenchymal stem cell culture, especially conditioned medium derived from umbilical cord amniotic membrane mesenchymal stem cell culture, has the effect of inducing, stimulating and / or promoting the healing of wounds or skin injuries, without the need to apply the conditioned medium directly to or on the wound or damaged skin area itself, but by only applying the conditioned medium to the skin surrounding the wound or damaged skin area, that is, applying to an unwounded or damaged skin area. Particularly surprising is that, through this route of administration, the conditioned medium described herein can even heal / close wounds that are difficult to heal (e.g., chronic diabetic foot ulcers). Referring to Example 3, it is shown that 12 of 21 patients with chronic diabetic foot ulcers (DFUs) showed complete DFU closure. This discovery of the present invention allows conditioned medium to be formulated into preparations such as creams, gels, or lotions, which are easy to produce (at a much lower cost than, for example, wound coverings (e.g., Apligraf or Dermagraft)), are stable at room temperature, and therefore can be easily distributed to hospitals and treatment centers, and can also be easily applied, even by non-medically trained personnel or even the patient himself. These advantages make it possible to provide treatment for patients with chronic diabetic foot ulcers, for example, in less developed regions, who have not been able to afford treatment with wound coverings such as Apligraf or Dermagraft. In addition, by being able to close such chronic wounds, the present invention provides patients with the opportunity to, for example, return to work, thereby also improving the socioeconomic status of affected patients. Finally, by providing an affordable treatment option for chronic wounds (such as diabetic foot ulcers), the present invention provides an opportunity to reduce the overall cost of treating diabetic foot ulcers, particularly in countries with a high prevalence of diabetes. Without wishing to be bound by theory, it is believed that the induction, stimulation, promotion and / or prevention of healing of wounded or damaged skin is due to the ability of the conditioned medium to contain high concentrations of one or more biological factors, such as exosomes, growth factors, proteins, peptides and cytokines, which, when applied to the skin surrounding the wounded or damaged skin area or to the intact skin of a subject at risk of developing a wounded or damaged skin area, help to establish a robust extracellular matrix and support skin repair. In this way, the conditioned medium of the present invention provides excellent properties for inducing, stimulating and / or promoting the healing of wounded or damaged skin areas, and / or for preventing the formation and / or recurrence of wounded or damaged skin areas in subjects at risk of developing wounded or damaged skin areas, and / or for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin.

[0032] As demonstrated in the experimental section, patients with chronic and refractory ulcers were treated with the conditioned medium of the present invention, wherein the conditioned medium was applied only to the skin surrounding the wound, not to the wound itself. Patients experienced significant improvements in wound reduction that could be observed within 16, 8, 5, or even 3 weeks. In particular, wound healing was promoted by, for example, increasing skin cell turnover, promoting the formation of extracellular matrix, promoting regenerative healing through potent anti-inflammatory cytokines, and promoting healing through angiogenesis, which can be attributed to the fact that the conditioned medium is particularly rich in growth factors and other biological factors that are positively correlated with skin repair and regeneration, such as, but not limited to, PDGF-AA, PDGF-BB, transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), interleukin-10 (IL-10), angiopoietin 1 (Ang-1), and / or hepatocyte growth factor (HGF).

[0033] The conditioned medium of the present invention may comprise at least one of the biological factors produced by umbilical cord mesenchymal stem cells, more typically, a combination thereof (e.g., comprising at least one of the biological factors listed above, more typically, a combination thereof). For example, the conditioned medium of the present invention may comprise one or more (at least one) angiogenic cytokines and / or growth factors. For example, the conditioned medium of the present invention may comprise one or more selected from the following: PDGF-AA, PDGF-BB, transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), interleukin-10 (IL-10), angiopoietin 1 (Ang-1) and / or hepatocyte growth factor (HGF).

[0034] In this regard, it is generally known that growth factors can stimulate cell growth and / or proliferation and / or cell differentiation, and the positive effects of growth factors on the skin, in particular on wound healing, are known to those skilled in the art. For example, regarding the involvement of angiopoietin-1 in wound healing, see, for example, Li et al., Stem Cell Research & Therapy 2013, 4:113 “Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing” or Bitto et al., “Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression”, Clinical Science May 14, 2008, 114(12):707-718. For the involvement of hepatocyte growth factor (HGF) in wound healing, in particular the healing of chronic / non-healing wounds, see, for example, Yoshida et al., “Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation” J. Invest. Dermatol. 120: 335-343, 2003, Li, Jin-Feng et al., “HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin / ILK Pathway.” BioMed Research International 2013 (2013): 470418 or Conway et al., “Hepatocyte growth factor regulation: An integral part of why wounds become chronic”. Wound Rep Reg (2007) 15 683-692.For the involvement of vascular endothelial growth factor (VEGF) in wound healing, particularly the healing of chronic / non-healing wounds, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60-64 or Bao et al., “The Role of Vascular Endothelial Growth Factor in Wound Healing” J Surg Res. 2009 May 15; 153(2): 347-358. For the involvement of transforming growth factor β (including TGF-β1, TGF-β2 and TGF-β3) in wound healing, in particular the healing of chronic / non-healing wounds, see, for example, Ramirez et al., “The Role of TGFb Signaling in Wound Epithelialization” Advances In Wound Care, Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing, Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

[0035] Furthermore, the well-recognized positive effects of growth factors on the skin, and in particular on wound healing, are demonstrated, for example, by their ability to enhance the expression of elastin and hyaluronic acid in human dermal fibroblasts ( VM, et al., Transforming growth factor-beta up-regulates elastin gene expression in humanskin fibroblasts. Evidence for post-transcriptional modulation. Lab Invest, 1992.66(5):p.580-8; and Takami, Y., et al., Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid. Biochimica et al. Biophysica Acta (BBA)-Molecular Cell Research, 2005.1743(1):p.49-56). For example, umbilical cord mesenchymal stem cells can produce one or more factors involved in, for example, cell proliferation and migration (e.g., vascular endothelial growth factor (VEGF); platelet-derived growth factor AA (PDGF-AA); basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF1)); promoting angiogenesis (e.g., vascular endothelial growth factor (VEGF); anti-inflammation (e.g., transforming growth factor beta-1 (TGF-β1)); promoting elastin and / or hyaluronic acid expression (e.g., transforming growth factor beta-1 (TGF-β1); basic fibroblast growth factor (bFGF); insulin-like growth factor 1 (IGF1)), as well as other factors.

[0036] The conditioned medium described herein may contain exosomes. Exosomes are known to technicians, for example, by imparting anti-inflammatory properties, promoting angiogenesis and stimulating new tissue formation and having a positive effect on the wound healing process; see Danyang Li, Na Wu, Mechanism and application of exosomes in the wound healing process in diabetes mellitus, Diabetes Research and Clinical Practice, Volume 187, 2022; Li, X., Xie, X., Lian, W. et al., Exosomes from adipose-derived stem cells overexpressing Nrf2 accelerate cutaneous wound healing by promoting vascularization in a diabetic foot ulcer rat model. Exp Mol Med 50, 1–14 (2018). In this regard, the application of the conditioned medium of the present invention, for example, has a beneficial effect on all stages of wound healing by promoting the repair of wounds or damaged skin and / or preventing the formation and / or recurrence of wounds or damaged skin.

[0037] The present invention relates to the discovery that conditioned medium derived from a culture of umbilical cord mesenchymal cells, wherein the cells secrete, inter alia, growth factors and other biological factors into the conditioned medium, induces, stimulates and / or promotes the healing of wounds or damaged skin, prevents the formation and / or recurrence of wounds or damaged skin areas in subjects at risk of developing wounds or damaged skin areas, and / or is useful for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin lesions caused by fragile skin. In this manner, it is believed that the conditioned medium of the present invention derives high concentrations of growth factors and / or other biological factors that can effectively aid in the healing of wounds or damaged skin areas and / or prevent the formation and / or recurrence of such wounds or damaged skin areas and / or is useful for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin lesions caused by fragile skin.

[0038] In this context, it should be noted that culturing umbilical cord mesenchymal stem cells in a culture medium as described herein provides for the isolation of an extremely homogeneous and well-defined population of umbilical cord tissue mesenchymal stem cells (e.g., a population of mesenchymal stem cells from the umbilical cord Wharton's jelly or the umbilical cord amniotic membrane), as disclosed in WO 2019 / 199234A1 or WO 2018 / 067071A1. In particular, the isolation of the umbilical cord mesenchymal stem cell population results in greater than 90% or even 99% or more cells being positive for the three mesenchymal stem cell markers CD73, CD90, and at the same time these stem cells lack expression of CD34, CD45, and HLA-DR (see, e.g., WO 2019 / 199234A1 or WO 2018 / 067071A1), meaning that 99% or even more of the cells in the population express the stem cell markers CD73, CD90, and CD105 but do not express the markers CD34, CD45, and HLA-DR. In the present context, such a population of umbilical cord (tissue) mesenchymal stem cells fully meets the generally accepted criteria for human mesenchymal stem cells for cell therapy, as defined, for example, by Dominici et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., “Production of mesenchymalstromal / stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy 2013, 4: 66), Vonk et al., Stem Cell Research & Therapy (2015) 6: 94 or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75–79. This in turn means that conditioned medium derived from the culture of such an extremely homogeneous and well-defined cell population is ideal for producing highly homogeneous and well-defined components in the conditioned medium, such as highly concentrated growth factors.Furthermore, the use of the conditioned medium of the present invention in wound healing or healing damaged skin areas and / or preventing the formation and / or recurrence of such wounds or damaged skin areas has several other advantages over stem cell-based applications, as donor-recipient matching issues associated with stem cell-based treatments or transplants can be avoided, and conditioned medium is easier to prepare and less expensive than stem cell-based skin therapies.

[0039] Conditioned medium derived from umbilical cord mesenchymal stem cell culture that is considered to be suitable for healing and / or treating wounds or damaged skin areas and preventing the formation and / or recurrence of such wounds or damaged skin areas can be derived from the culture of any suitable umbilical cord (tissue) mesenchymal stem cell group known in the art. For example, umbilical cord mesenchymal stem cells can be derived from any mammalian tissue or compartment / body part known to contain umbilical cord mesenchymal stem cells. In an illustrative example, the mesenchymal stem cells can be mesenchymal stem cells of the umbilical cord amniotic membrane. The effect of promoting healing, treating and / or preventing wounds or damaged skin areas is achieved, for example, because these stem cells secrete growth factors and other biological factors that actively act on wound healing. For example, the biological factors that can be in the conditioned medium include but are not limited to growth factors, nucleic acids such as mRNA or DNA, exosomes, hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors and particulate matter. Such conditioned medium and any combination of growth factors and / or biological factors contained therein can be used in the method of the present invention.

[0040] In a first aspect, the present invention relates to a method for inducing, stimulating and / or promoting the healing of a wound or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area. As used herein and in the context of the present invention, wound or damaged skin refers to a destruction or damage to the integrity of the skin (particularly the surface of the skin), and may also include destruction or damage to the subcutaneous tissue. For example, the wound or damaged skin is caused by a burn, bite, trauma, surgery or disease (such as a skin disease or condition or metabolic disorder). The skin disease or condition or metabolic disorder can be any disease, condition or disorder associated with the occurrence of wound or damaged skin, particularly those in which wound or damaged skin healing is needed / required. Non-limiting examples of such skin diseases, skin conditions, or metabolic disorders are type I or type II diabetes with chronic ulcers (e.g., diabetic foot ulcers (DFUs), pressure ulcers, limb ischemia, and leg venous ulcers), rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin. In some embodiments, the wound or damaged skin is a chronic wound or chronically damaged skin. In some embodiments, the damaged skin area is a wound. In some embodiments, the wound or damaged skin area is a wound, wherein the wound is selected from diabetic foot ulcers (DFUs), pressure ulcers, wounds caused by chronic limb ischemia, wounds caused by chronic limb-threatening limb ischemia, leg venous ulcers, surgical wounds, puncture wounds, burns, and bite wounds.

[0041] As used herein, "induce, stimulate and / or promote healing of wounded or damaged skin areas" refers to the ability of conditioned medium to accelerate and / or initiate (induce) the healing process by repairing and / or regenerating the skin, particularly in patients with wounds or damaged skin areas associated with healing complications. This ability to accelerate and / or initiate wound healing can be attributed to the presence of growth factors and other biological factors (such as exosomes). The secretion of growth factors and other biological factors into the culture medium (and therefore the growth factors and biological factors contained in the conditioned medium of the present invention) can be measured / determined by any suitable method, for example, by measuring the amount of growth factors by means of commercially available antibodies / immunoassays (see Experimental Section). Such measurements can be performed in an automated manner using, for example, a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).

[0042] In a second aspect, the present invention also relates to a method for preventing the formation and / or recurrence of a traumatic or damaged skin area in a subject who is at risk of developing a traumatic or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject. As used herein, "preventing the formation and / or recurrence of traumatic or damaged skin" means that the conditioned medium of the present invention is capable of avoiding and / or reducing the risk of the occurrence of traumatic or damaged skin in a subject who is at risk of developing a traumatic or damaged skin injury, wherein the risk may be due to a previous skin disease, skin disorder, or metabolic disease, among others. In order to prevent the formation and / or recurrence of traumatic or damaged skin, the conditioned medium can be applied to a functionally and physiologically intact skin area of ​​the subject.

[0043] Conditioned medium of the present invention for the treatment of can be, for example, administered with a final amount of, for example, about 5% or 10% to about 80%, and more preferably administered with a final amount of about 10% to about 50%, more preferably about 10% to about 30%, more preferably about 20%, wherein the % can refer to the % (v / v) of the total volume of the composition comprising the conditioned medium or the % (w / w) of the total weight of the composition comprising the conditioned medium. In this regard, referring to the illustrative examples of specific formulations described herein. For this purpose, the conditioned medium of the present invention can be diluted in any suitable diluent / carrier medium (prepared with any suitable diluent / carrier medium). Carrier medium can be a liquid, gel or cream-like preparation. For example, conditioned medium can be diluted in a carrier medium, such as PBS, water or basal medium, to name a few suitable culture media. Basal medium used herein refers to a mixture containing carbohydrates, amino acids, water etc. required for cell survival, including commercially prepared culture medium, such as but not limited to Dulbecco's modified Eagle's medium (DMEM), endothelial differentiation medium (EDM), minimum essential medium (MEM), basal medium Eagle (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (G-MEM) and Iscove's modified Dulbecco's medium. In some instances, the conditioned medium of the present invention may be included in a solution containing DMEM. The culture medium may further include an antioxidant, such as a soluble antioxidant. Water-soluble antioxidants can be added to the culture medium to prevent oxidative damage. The example of suitable water-soluble antioxidants includes curcumin, glutathione, uric acid, Trolox or Allicidin, only mentioning a few.

[0044] In a third aspect, the present invention relates to a method for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically applying to the skin of a subject a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0045] In order to treat the experimenter, the conditioned medium of the present invention is applied by topical administration in this article. In principle, this article means any topical administration mode. For example, before conditioned medium is applied to the experimenter's skin, conditioned medium can be mixed with emulsifiable paste, ointment, gel, lotion, suspension or any other suitable preparation. Conditioned medium can also or alternatively remain in place, and this can be realized (referring to embodiment 3) by dressing or by soaking conditioned medium in gauze, then this gauze is applied to the experimenter's skin.

[0046] Conditioned medium or a composition comprising a conditioned medium as described herein and according to the present invention is topically applied to the skin surrounding a wound or damaged skin area of ​​a subject. Thus, the method does not involve applying the conditioned medium to the wound or damaged skin area. In some examples, the skin surrounding a wound or damaged skin area is the skin directly surrounding the wound or damaged skin. In some instances, periwound skin refers to the peri-wound area, i.e., skin beyond the wound margins but close to or adjacent to the wound margins. In some embodiments, the skin to be treated is intact skin. In particular, periwound skin can refer to intact periwound skin that can perform the normal functions of skin (e.g., absorption, excretion, protection, secretion, temperature regulation, and immunity, to name a few) and that maintains the physical properties of skin (e.g., elasticity and surface integrity). In some embodiments, the skin to be treated according to the present invention is thin / fragile skin. As used herein, fragile skin preferably refers to particularly vulnerable and / or fragile skin that has been impacted by the wound or damaged skin but remains intact, i.e., can perform the normal functions of skin and maintains its physical properties as described above. The term "thin skin" is used herein with its conventional meaning to refer to skin that is easy to tear, easy to bruise or easy to break. Thin skin is sometimes referred to as thinning skin or fragile skin. Thin skin is a common condition in the elderly and is most obvious on the face, arms and hands. People with thin skin may find that they can see veins, tendons, bones and capillaries under the skin of their hands and arms. However, thin skin is not only relevant to aging, but also to disease factors (such as diabetes), the use of some drugs (such as steroids), or may also be caused by, for example, ultraviolet exposure, heredity or lifestyle. The thick fibrous tissue of the dermis is made of collagen and elastin. The dermis provides strength, flexibility and elasticity to the skin. Thin skin is the result of the thinning of the dermis.

[0047] In some embodiments, the conditioned medium is applied in the form of a gel. In particular, the present invention describes a gel formulation comprising a conditioned medium for topical application, wherein the formulation comprises a conditioned medium for inducing, stimulating and / or promoting the healing of wound healing or damaged skin areas, and / or for preventing the formation and / or recurrence of such wound or damaged skin areas, and / or for treating an effective amount of a disease associated with a wound or skin injury as described herein. The gel formulation used herein refers to a gel containing a certain amount of the conditioned medium of the present invention, the certain amount being an amount that effectively heals wounds or heals damaged skin areas as described herein, and / or prevents the formation and / or recurrence of such wound or damaged skin areas, and / or treats a disease associated with skin injury or wounds, and in addition, the gel can contain a water-soluble, pharmaceutically and / or cosmetically acceptable polymer, such as polyacrylic acid (carbomer). Such a gelling agent can provide a suitable viscosity of the gel, for example, within the range of topical application, such as 1000-200,000 cps at room temperature. The gel formulation has the advantage of spreading evenly over the surrounding area of ​​the wound or damaged skin area, which increases the contact time of the skin with the active components of the formulation (such active components include, for example, growth factors and / or other biological factors) and delivers the active components of the formulation over time. The conditioned medium of the present invention in gel form can be particularly useful in a hospital setting, i.e., for treating a wound or damaged skin area of ​​a patient in need of treatment for a wound or damaged skin area. Thus, the conditioned medium in gel form can be particularly useful for treating patients in a hospital with a wound or damaged skin area, where the wound or damaged skin area developed before or during hospitalization. In an illustrative example, such a wound can be a diabetic foot ulcer or a pressure ulcer, where a pressure ulcer develops due to prolonged pressure applied to a specific area of ​​the body.

[0048] In some embodiments, the conditioned medium is applied in a liquid form such as a lotion or ointment. Liquid formulations used herein refer to solutions or liquid formulations containing a certain amount of the conditioned medium of the present invention, wherein the certain amount is an amount that can effectively heal a wound or heal an injured skin area, and / or prevent the formation and / or recurrence of such wounds or injured skin areas, and / or treat a skin disease associated with the wound or injured skin. The conditioned medium of the present invention in liquid form can be particularly useful for application in a home environment, that is, a liquid formulation comprising the conditioned medium of the present invention can be applied without the need for a doctor. For example, the conditioned medium in liquid form can be applied by a subject who is at risk of developing a wound or injured skin area but is not hospitalized or does not need to be hospitalized. For example, the conditioned medium in liquid form can be applied directly by a subject who is at risk of developing a diabetic ulcer as a "daily skin conditioner," wherein the subject is at home and not treated in a hospital or wound care center.

[0049] In a fourth aspect, the present invention relates to a method of producing a conditioned medium. This medium can then be used for cosmetic or therapeutic applications as described herein. The method may comprise:

[0050] (a) Umbilical cord mesenchymal stem cells were cultured in a medium containing DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum);

[0051] (b) removing the umbilical cord mesenchymal stem cells from the culture medium; and

[0052] (c) optionally culturing the umbilical cord mesenchymal stem cells in an additional culture medium, optionally wherein the additional cell culture medium comprises a water-soluble antioxidant, wherein the conditioned medium is obtained by collecting the cell culture medium.

[0053] Conditioned medium can be obtained by culturing umbilical cord mesenchymal stem cells in the above-mentioned other culture medium after mesenchymal stem cells are cultured in the culture medium comprising DMEM, F12, M171 and FBS. This other culture medium can be any (other) basal medium suitable for animal cell growth, particularly basal medium suitable for mesenchymal stem cell culture. In some instances, this other cell culture medium does not include growth factors and / or is a serum-free culture medium and / or wherein the cells are cultured at a concentration of about 1 million cells per 1ml. In some instances, this other culture medium can be a serum-free culture medium. Non-limiting examples of such serum-free culture medium include minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Losvi Parker Memorial Institute culture medium (RPMI), keratinocyte culture medium (KM), KBM (keratinocyte basal culture medium), EpiLife KM (keratinocyte-EpiLife culture medium) etc. Other culture medium can also contain serum. As an example of such a serum-containing medium, the conditioned medium used can be a conditioned medium described and commonly used for isolating and culturing a population of mesenchymal stem cells from the umbilical cord amniotic membrane, such as medium PTT-4. This medium PTT-4 is composed of 90% (v / v) CMRL1066 and 10% (v / v) FBS and has been used in U.S. patent application US 2008 / 0248005 and the corresponding international patent application WO 2007 / 046775 for isolating and culturing a population of mesenchymal stem cells from the umbilical cord amniotic membrane. In these U.S. patent application US 2008 / 0248005 and the international patent application WO 2007 / 046775, this medium was shown to have excellent wound healing properties. In some instances, a cell culture medium as described herein, such as a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 medium), M171 (culture medium 171), and FBS (fetal bovine serum), can be used to culture umbilical cord mesenchymal stem cells, wherein after culture, for example, once the cells reach complete confluence, the cell culture medium is removed and replaced with another culture medium / solution, such as a culture medium solution comprising, for example, DMEM, which may contain an antioxidant component. Typically, during the optional culture step in the additional culture medium of the method for preparing a conditioned medium as described herein, the mesenchymal stem cells can be cultured for any suitable period of time. Such a suitable period of time can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or even longer, for example, if desired, for a period of 10 days or 14 days.In the present context, for the sake of clarity, it is noted that both the conditioned medium obtained in the first culturing step and the conditioned medium obtained in the second (additional) culturing step can be used as described herein to induce, stimulate and / or promote healing of wounded or damaged skin areas, and / or for preventing the formation and / or recurrence of wounded or damaged skin, and / or for treating diseases associated with wounded or damaged skin.

[0054] Umbilical cord (tissue) mesenchymal stem cell for preparing conditioned medium herein can be from (derived from) any compartment of the umbilical cord tissue containing mesenchymal stem cell.Mesenchymal stem cell group can be the mesenchymal stem cell group (AM) of amniotic membrane, perivascular (PV) mesenchymal stem cell group, the mesenchymal stem cell group of Wharton's jelly (WJ), the mesenchymal stem cell group (also referred to as umbilical cord lining mesenchymal cell) of umbilical cord amniotic membrane and the mixed property (MC) mesenchymal stem cell group of umbilical cord, mixed property (MC) mesenchymal stem cell group means the mesenchymal stem cell group of two or more stem cells including these compartments.In some instances, the mesenchymal stem cell is the mesenchymal stem cell of umbilical cord amniotic membrane.In some instances, umbilical cord mesenchymal stem cell includes the mesenchymal stem cell of umbilical cord amniotic membrane. Mesenchymal stem cells from these compartments and their isolation are known to those skilled in the art and are described, for example, by Subramanian et al., “Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells”, PLoS ONE 10(6):e0127992, 2015 and references cited therein, Van Pham et al., “Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications”, Cell Tissue Bank (2016) 17:289-302, 2016. A mixed population of mesenchymal stem cells from the umbilical cord can be obtained, for example, by dissecting the arteries and veins from the umbilical cord tissue, mincing the remaining tissue and Wharton's jelly into pieces, and culturing the umbilical cord tissue in a culture medium such as PTT-6 (by the tissue fragment method).Mixed umbilical cord mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue with intact umbilical cord vasculature by the tissue dissociation block method under conditions as described by Schugar et al. “High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009: 789526 (cultured in DMEM supplemented with serum with 10% fetal bovine serum, 10% horse serum and 1% penicillin / streptomycin). In this context, it should be noted that mesenchymal stem cell populations from the umbilical cord-placenta junction can be isolated as described by Beeravolu et al. “Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224.

[0055] According to the above, it is noted here that the conditioned medium is derived from the culture of mesenchymal stem cells of umbilical cord tissue, wherein the stem cells can be cultured in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 medium), M171 (culture medium 171) and FBS (fetal bovine serum), and before being cultured in a culture medium as described herein, the stem cells can be separated from their natural environment. This method is particularly useful for mesenchymal stem cell populations of the umbilical cord. Such stem cell populations (e.g., mesenchymal stem cell populations of Wharton's jelly) can first be isolated as described above in Subramanian et al., 2015, PLoS ONE, cited above, or in International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord", and then the isolated mesenchymal stem cell population is cultured in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 medium), M171 (culture medium 171) and FBS (fetal bovine serum). Placental mesenchymal stem cell populations can also be isolated from the placenta, for example, as described in European patent application EP1288293, Talwadekar et al., “Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood ABPlasma” Biomed Res J 2014; 1(2): 126-136, Talwadekar et al., “Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts - a paired sample study” Scientific Reports 5: 15784 (2015), or Beeravolu et al., “Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224, and subsequently cultured in the culture medium described herein.

[0056] In this context, it should be noted that the culture medium described herein also allows for the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from their natural environment. Thus, the culture medium described herein also allows for the isolation of a population of mesenchymal stem cells under conditions that allow the proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. In some instances, the cell culture medium used to isolate and / or culture umbilical cord mesenchymal stem cells can be the culture medium used to culture cells to produce the conditioned medium of the present invention.

[0057] In light of the above disclosure, it is noted that the mesenchymal stem cell populations described herein can be isolated and cultured from any umbilical cord tissue (i.e., can be derived from any umbilical cord tissue) as long as the umbilical cord tissue contains the amniotic membrane (also known as the "cord lining"). Thus, as described in the experimental section of this application, the mesenchymal stem cell populations can be isolated from (a portion of) the entire umbilical cord. Thus, in addition to the amniotic membrane, the umbilical cord tissue can comprise any other tissue / component of the umbilical cord. For example, as shown in FIG16 of U.S. Patent Application No. 2006 / 0078993 or International Patent Application No. WO2006 / 019357, the amniotic membrane of the umbilical cord is the outermost portion of the umbilical cord, covering the umbilical cord. In addition, the umbilical cord contains a vein (which delivers oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's jelly, a jelly-like substance primarily made of mucopolysaccharides. Therefore, the umbilical cord tissue used in this article may also include this one vein, two arteries and Wharton's jelly. Using this entire (complete) part of the umbilical cord has the following advantages: the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the separation step, and therefore makes the method of the present invention simpler, faster, more error-prone and more economical. Therefore, the separation of mesenchymal stem cells can be started by the tissue separation block method (tissue explant), and if a larger amount of mesenchymal stem cells are needed, the mesenchymal stem cells separated by subsequent passage culture (cultivation) can be carried out subsequently. Alternatively, the amniotic membrane can also be separated from the other components of the umbilical cord, and then the mesenchymal umbilical cord lining stem cells are separated from the amniotic membrane by culturing the amniotic membrane in the culture medium as described herein. The culture can also be carried out by the tissue separation block method, optionally followed by passage culture of the mesenchymal stem cells separated.

[0058] In this context, the term "tissue separation block method" or "tissue separation block method" is used with its conventional meaning in the art and refers to a method in which, once tissue (e.g., umbilical cord tissue) is harvested, the tissue or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, and over time, stem cells migrate from the tissue to the surface of the culture dish. These primary stem cells can then be further amplified and transferred to a fresh culture dish by micropropagation (passage culture) as described herein. In this context, it is noted that in the first step of separating / obtaining a population of umbilical cord mesenchymal stem cells (such as amniotic membrane or Wharton's jelly mesenchymal stem cells), a master cell bank of isolated mesenchymal stem cells is obtained, and subsequent passage culture can obtain a working cell bank. If the conditioned medium derived from umbilical cord mesenchymal stem cell culture of the present invention is used in an application such as promoting wound healing, the cell population of the working cell bank is typically used for this purpose. The stem cell population of the separation step (which can constitute the master cell bank) and the stem cell population of the passage culture step (which can constitute the working cell bank) can both be stored, for example, in the form of cryopreservation.

[0059] "DMEM" refers to Dulbecco's Modified Eagle's Medium, a modification of Basal Eagle's Medium (BME) developed in 1969 (see Figure 1 , which shows a data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for culturing embryonic mouse cells. Since then, DMEM has become the standard culture medium for cell culture and is commercially available from a variety of sources, such as ThermoFisher Scientific (Cat. No. 11965-084), Sigma Aldrich (Cat. No. D5546), or Lonza, to name just a few. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is the DMEM culture medium available from Lonza as Catalog No. 12-604F. This culture medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is the DMEM culture medium from Sigma Aldrich as Catalog No. D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0060] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed for culturing a variety of mammalian and hybridoma cells when used with serum and in combination with hormones and transferrin (see Figure 2, which shows a data sheet for Ham's F12 medium from Lonza. Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (Cat. No. 11765-054), Sigma Aldrich (Cat. No. N4888), or Lonza, to name a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0061] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium (see Figure 3 , which shows a data sheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is also a widely used basal medium for supporting the growth of many different mammalian cells and is commercially available from multiple suppliers such as ThermoFisherScientific (catalog number 11330057), Sigma Aldrich (catalog number D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium available from Lonza as catalog number 12-719F (which is DMEM:F12 with L-glutamine, 15mM HEPES, and 3.151g / L glucose).

[0062] "M171" refers to Medium 171, which has been developed as a basal medium for growing normal human mammary epithelial cells (see Figure 4 , which shows a data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely used and can be commercially obtained from, for example, ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalog number M171500.

[0063] " FBS " means fetal bovine serum (also referred to as " fetal bovine (calf) serum "), i.e., the portion of blood retained after the blood naturally coagulates and is then centrifuged to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement in eukaryotic cell in vitro cell culture because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. Preferably, FBS is obtained from members of the International Serum Industry Association (ISIA), which focuses on ensuring the safety and safe use of serum and animal-derived products by proper traceability of origin, authenticity of labels, and proper standardization and supervision. Suppliers of FBS as members of ISIA include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare with catalog number A15-151.

[0064] The culture medium for deriving a conditioned medium from umbilical cord mesenchymal stem cell culture as described herein may include DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v). The numerical values ​​of "% (v / v)" used herein refer to the volume of a single component relative to the final volume of the culture medium. This means, for example, if DMEM is present in the culture medium at a final concentration of about 55 to 65% (v / v), then 1 liter of culture medium contains about 550 to 650 ml of DMEM. In other embodiments, the culture medium may comprise DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In further embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0065] In addition to the above components, the culture medium may include supplements advantageous for cultivating the umbilical cord mesenchymal stem cells and for deriving the conditioned medium for inducing, stimulating and / or promoting the healing of wounds or damaged skin and / or for preventing wounds or skin damage from forming and / or recurring and / or for treating wounds or damaged skin. For example, culture medium as described herein may include epidermal growth factor (EGF). If present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some such embodiments, the culture medium may include EGF at a final concentration of about 10 ng / ml.

[0066] The culture medium described herein may also contain insulin. If present, the insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some such embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0067] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may comprise adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0068] As described herein, umbilical cord tissue can be cultured until a suitable number of (primary) mesenchymal stem cells (such as umbilical cord lining stem cells, Wharton's jelly or placental stem cells) grow out of the tissue. Typically, umbilical cord tissue is cultured until the cell outgrowth of the mesenchymal stem cells of the corresponding tissue reaches about 70% to about 80% confluence. It should be noted here that the term "confluency" is used in its conventional sense in the field of cell culture and is intended to be an estimate / indicator of the number of adherent cells in a culture dish or culture flask, referring to the proportion of the surface covered by cells. For example, 50% confluence means that approximately half of the surface is covered and there is still room for cells to grow. 100% confluence means that the surface is completely covered with cells and there is no longer room for cell monolayer growth.

[0069] Once the primary cells (mesenchymal stem cells) of appropriate quantity have been obtained from the corresponding tissue by the tissue separation block method, the mesenchymal stem cells are removed from the culture vessel for cultivating. By doing this, it is possible to obtain the master cell bank containing (primary) separation of for example umbilical cord or placenta mesenchymal stem cells. Conventionally, because this type of mesenchymal stem cell is an adherent cell, standard enzyme treatment is used to harvest cells. For example, the enzyme treatment can include trypsinization as described in International U.S. Patent Application 2006 / 0078993, International Patent Application WO2006 / 019357 or International Patent Application WO2007 / 046775, meaning that the cells grown outward can be harvested for further amplification by trypsinization (0.125% trypsin / 0.05% EDTA). If the mesenchymal stem cells gathered in the crops are for example used to produce a master cell bank, then the cells can also be cryopreserved and stored for further use, as described below.

[0070] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculture. If a population of umbilical cord mesenchymal stem cells previously isolated from its natural environment is used, subculture or culture (the two terms are used interchangeably hereinafter) will also be performed. The subculture can also be started from frozen primary cells, i.e., from the master cell bank. For subculture, any suitable number of cells can be seeded in a culture vessel such as a cell culture plate. For this purpose, the mesenchymal stem cells can be grown at, for example, about 0.5 x 10 6 cells / ml to approximately 5.0 x 10 6 Cells are suspended in a suitable culture medium for subculture (most conveniently, the culture medium of the present invention) at a concentration of about 1.0 x 10 cells / ml. 6The cells are suspended at a concentration of 10 cells / ml for subculture. The subculture can be carried out by culturing in a simple culture bottle or in a multi-layer system that can be stacked in an incubator, such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (part of Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA). Alternatively, the subculture can also be carried out in a closed self-contained system (such as a bioreactor). Bioreactors of different designs are known to those skilled in the art, for example, parallel plates, hollow fibers or microfluidic bioreactors. See, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", cited above. Illustrative examples of commercially available hollow fiber bioreactors are Cell Expansion System (Terumo BCT, Inc), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048–1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell populations described herein is the Xuri Cell Expansion System available from GE Healthcare. If a working cell bank is to be produced under GMP conditions and a large number of cells are required, then, for example, It is particularly beneficial to culture mesenchymal stem cell populations in an automated system such as the Cell Expansion System.

[0071] The subculture of the mesenchymal stem cells of the present invention can be carried out in a culture medium as described herein. Therefore, the culture medium can be used to isolate a mesenchymal stem cell population from, for example, the amniotic membrane of the umbilical cord or Wharton's jelly and for subsequent cultivation of isolated primary cells by culture. Similarly, for cultivation, mesenchymal stem cells can be cultured until a suitable amount of cells are grown. In some embodiments, mesenchymal stem cells are cultured until the mesenchymal stem cells reach about 70% confluence, about 80% confluence, about 90% confluence, about 95% confluence, to about 96% or even 100% confluence.

[0072] The isolation / culturing of the mesenchymal stem cell population can be carried out under standard conditions for culturing mammalian cells. Typically, the method of isolating a mesenchymal stem cell population is typically carried out under the following conditions (temperature, atmosphere): the conditions are generally used to culture cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37° C. in a normal atmosphere containing 5% CO 2 , respectively. In this context, it is noted that the mesenchymal cell populations described herein can be derived from any mammalian species, such as humans, red deer, mice, rats, guinea pigs, pigs, rabbits, goats, horses, dogs, cats, sheep or monkeys. In an exemplary embodiment, the umbilical cord mesenchymal stem cells are red deer or human.

[0073] Once the desired / appropriate amount of mesenchymal stem cells are obtained from self-culture or passage culture, the mesenchymal stem cells are harvested by removing them from the culture container for cultivation. Usually, the mesenchymal stem cells are harvested again by enzyme treatment, including trypsin digestion of cells. The separated mesenchymal stem cells are subsequently collected and used directly or preserved for further use. Usually, they are preserved by cryopreservation. The term "cryopreservation" is used in this article with its conventional meaning to describe the process of preserving mesenchymal stem cells by cooling to a low subzero temperature (such as (usually) -80°C or -196°C (boiling point of liquid nitrogen)). Cryopreservation can be performed as known to those skilled in the art, and may include the use of a cryoprotectant such as dimethyl sulfoxide (DMSO) or glycerol, which slows down the formation of ice crystals in umbilical cord cells.

[0074] The isolated mesenchymal stem cell populations obtained by the culture and / or isolation methods described herein are highly defined and homogeneous. Such highly homogeneous mesenchymal stem cell populations derived from tissues such as umbilical cord amniotic membrane or Wharton's jelly are reported in WO 2018 / 067071A1 and WO 2019 / 199234A1. For example, as disclosed and shown in the experimental section of WO 2019 / 199234A1A1, at least about 90%, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in a population of umbilical cord mesenchymal stem cells (e.g., isolated from umbilical cord Wharton's jelly or amniotic membrane) express each of the following markers: CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR, meaning that this percentage of the isolated cell population expresses each of CD73, CD90, and CD105, and lacks expression of the following markers: CD34, CD45, and HLA-DR. This highly homogenous population of mesenchymal stem cells derived from the umbilical cord amniotic membrane meets the criteria for mesenchymal stem cells for cell therapy (see also the experimental section in WO 2019 / 199234A1, and, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra, cited above). In this context, it is noted that this population of mesenchymal stem cells can be obtained by the isolation method described in WO 2019 / 199234A1, or, if desired, by a different method (such as cell sorting).

[0075] In another aspect, the present invention relates to a method for inducing, stimulating and / or promoting wound healing, and / or preventing wound formation and / or recurrence, and / or treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises treating the skin surrounding a wound or damaged skin area of ​​a subject or the intact skin of a subject in a skin area where a wound or damage is developing with a conditioned medium derived from umbilical cord mesenchymal stem cells cultured in a medium as described herein, wherein the medium is mixed as follows to obtain a final volume of 500 mL of medium:

[0076] i. 250 ml of DMEM

[0077] ii.118ml M171

[0078] iii. 118ml DMEM / F12

[0079] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0080] As mentioned above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, when using this method of preparing the medium, the final concentrations (v / v) in a total volume of 500 ml are as follows:

[0081] DMEM: 250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v)

[0082] M171: 118 ml, corresponding to 118 / 500 = 23.6% (v / v)

[0083] F12: 59 ml, corresponding to 59 / 500=11.8% (v / v).

[0084] The culture medium as described herein further included the addition of: v. 1 ml of EGF stock solution (5 μg / ml) to give a final EGF concentration of 10 ng / ml, and vi. Insulin 0.175 ml of stock solution (14.28 mg / ml) to give a final insulin concentration of 5 μg / ml.

[0085] It should be noted here that, when mixing, the volume of these components i to vi above-mentioned obtains the substratum that final volume is 499.675ml.If no longer in substratum, add other components, then remaining 0.325ml (volume being added to 500ml) can be for example arbitrarily among component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of substratum is 500ml.In addition, also note that component i to iv need not add according to the order that they are listed, but certainly can use any order to make these components mix to obtain substratum as herein described. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to achieve final concentrations as described herein, i.e., a final concentration of about 55 to 65% (v / v) of DMEM, a final concentration of about 5 to 15% (v / v) of F12, a final concentration of about 15 to 30% (v / v) of M171, and a final concentration of about 1 to 8% (v / v) of FBS.

[0086] In other embodiments, the method further comprises adding one or more of the following supplements to the DMEM: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) in a volume of 0.325 ml, thereby obtaining a total volume of 500 ml of culture medium. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows:

[0087] about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine,

[0088] About 1 to 10 μg / ml hydrocortisone,

[0089] About 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0090] In some embodiments, umbilical cord mesenchymal stem cells are cultured in a cell culture medium described herein, for example, in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 medium), M171 (culture medium 171), and FBS (fetal bovine serum), and the conditioned medium of the present invention is produced by removing the umbilical cord mesenchymal stem cells from the culture medium, wherein the conditioned medium is obtained by collecting the cell culture medium. In addition, in some instances, the culture medium in which the stem cells have been cultured can be replaced by a second cell culture medium, and the cells can be cultured in the second cell culture medium, which can be a cell culture medium that is the same or different from the first cell culture medium. In some instances, the second cell culture medium can be a culture medium without growth factors and / or a serum-free culture medium. Alternatively, previously cultured umbilical cord mesenchymal stem cells (e.g., stored cultured stem cells) can be further cultured in a culture medium without growth factors and / or serum. In either case, the resulting culture medium is referred to as a conditioned medium. The resulting conditioned medium is harvested and can be further processed to produce a composition comprising a conditioned medium of the present invention.

[0091] Conditioned medium of the present invention or the composition comprising the conditioned medium of the present invention can be diluted in any suitable diluent / carrier culture medium, as long as when topically applied to the skin of the experimenter, the carrier culture medium does not affect the function and safety of the conditioned medium. For example, conditioned medium can be diluted in PBS, water, basal culture medium or cream formulation. Basal culture medium used herein refers to a mixture containing saccharides, amino acids, water etc. required for cell survival, including commercially prepared culture medium, such as but not limited to Dulbecco's modified Eagle's medium (DMEM), endothelial differentiation medium (EDM), minimum essential medium (MEM), basal culture medium Eagle (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (G-MEM) and Iscove's modified Dulbecco's medium. In addition, the conditioned medium to be used can be any basic medium suitable for growing animal cells, non-limiting examples of which include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Resveratrol Memorial Institute Medium (RPMI), Keratinocyte Medium (KM), KBM (Keratinocyte Basal Medium), EpiLife KM (Keratinocyte-EpiLife Medium), etc. In some examples, the conditioned medium comprises the cell culture medium described herein, such as a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).

[0092] On the other hand, the present invention provides a conditioned medium derived from umbilical cord mesenchymal stem cells obtained or obtainable by a method according to the present invention. In a related aspect, the present invention provides a composition comprising the conditioned medium of the present invention. In some embodiments, the composition described herein is a pharmaceutical or cosmetic composition for inducing, stimulating and / or promoting wound healing, and / or for preventing the formation and / or recurrence of wounds, and / or for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, or epidermolysis bullosa. The composition can be prepared in any dosage form conventionally prepared in the art and generally comprises the conditioned medium of the present invention and an acceptable carrier / receptor suitable for use in cosmetic products or pharmaceutically.

[0093] In some examples, the present invention provides a composition comprising conditioned medium derived from umbilical cord mesenchymal stem cell culture, wherein the conditioned medium is formulated for use in skin care or skin treatment. Examples of such formulations are described in more detail below.

[0094] In examples of such compositions, the composition may further comprise an anti-inflammatory compound. Any suitable anti-inflammatory compound may be used in such compositions. The anti-inflammatory compound may be a small molecule drug of natural origin or chemical synthesis. In illustrative examples, the anti-inflammatory compound may be curcumin, sulfacetamide, a non-steroidal anti-inflammatory compound (e.g., capsaicin or diclofenac), niacinamide (vitamin B3), sea buckthorn oil, calendula, chamomile (plant-based) extract, or Centella asiatica extract, to name a few. The anti-inflammatory properties of curcumin are reviewed in, for example, Peng et al., “Anti-Inflammatory Effects of Curcumin in Inflammatory Diseases: Status, Limitations and Countermeasures”, Drug Design, Development and Therapy 2021: 15 4503–4525. Calendula is a natural oil extracted from marigolds, which are native to the Mediterranean region. This sweet juice contains flavonoids with antibacterial properties and has anti-inflammatory abilities that can aid in wound healing and soothe eczema. Chamomile (botanical) extract is the powdered essence of German chamomile (also known as Hungarian chamomile or blue chamomile), whose scientific name is Matricaria recutita, and is used in traditional remedies for wound healing. Chamomile (botanical) extract is the powdered essence of German chamomile (also known as Hungarian chamomile or blue chamomile), whose scientific name is Matricaria recutita, and is used in traditional remedies for wound healing. In addition to its traditional use in wound healing, chamomile extract is often included in skincare products to address irritation and inflammation. The anti-inflammatory properties of Centella asiatica extract are discussed in Gohil et al., “Pharmacological Review on Centella asiatica: A Potential Herbal Cure-all,” Indian J Pharm Sci. 2010 Sep-Oct; 72(5): 546–556.

[0095] The composition may further include a wetting agent. The term "wetting agent" is used herein in its conventional sense in the field of skin care, and means a component that draws moisture to the skin. Any suitable wetting agent can be used in the corresponding compositions described herein. Illustrative examples of wetting agents include, but are not limited to, glycerol, propylene glycol, triethylene glycol, tripropylene glycol, sorbitol, hexylene glycol, butylene glycol, urea, collagen, butylene glycol, aloe vera gel, and mixtures thereof. In some examples of compositions described herein, the wetting agent includes glycerol, propylene glycol, or both glycerol and propylene glycol (also see below).

[0096] The composition comprising conditioned medium, an anti-inflammatory compound and a wetting agent may comprise an amount of about 5 to 40% (v / v) of the total volume of the composition, an amount of the conditioned medium derived from umbilical cord mesenchymal stem cell culture, an amount of the wetting agent, and an amount of about 1.0 to about 3.0% (v / v) of the composition or an amount of 0.5 to 5.0% (w / w) of the total weight of the composition. In other examples, such a composition may comprise an amount of about 10 to 30% (v / v) of the total volume of the composition, an amount of the wetting agent, and an amount of about 15 to 25% (v / v) of the total volume of the composition, and an amount of the anti-inflammatory compound, or an amount of 0.75 to 3.0% (w / w) of the total weight of the composition.

[0097] Alternatively, the composition comprising a conditioned medium, an anti-inflammatory compound, and a wetting agent may comprise an amount of about 5% to 40% (w / w) of the conditioned medium derived from umbilical cord mesenchymal stem cell culture, an amount of about 10% to 30% (w / w) of the wetting agent, and an amount of about 0.5% to 5.0% (w / w) of the anti-inflammatory compound, based on the total weight of the composition. For the sake of clarity, it is noted that if a component is typically in liquid form at room temperature, it is preferred to use volume percentage (% v / v) to define the content of the component in the composition, and if a component is typically in solid form at room temperature, it is preferred to use weight percentage (% w / w) to define the content of the component in the composition.

[0098] In an illustrative example of such a composition, the composition comprises

[0099] -Conditioned medium derived from umbilical cord mesenchymal stem cell culture,

[0100] - glycerol, propylene glycol or both glycerol and propylene glycol, and

[0101] -Curcumin.

[0102] The compositions described herein may further comprise a solvent / carrier. Any cosmetically acceptable solvent / carrier may be used. In illustrative examples, the solvent / carrier may be water or an aqueous buffer solution.

[0103] The compositions described herein may further comprise a preservative. As used herein, a preservative is used in its conventional sense to refer to a natural or synthetic ingredient that protects the integrity of the composition (preparation). Preservatives can prevent the growth of bacteria or other microorganisms. Illustrative examples of suitable preservatives include, but are not limited to, 1,2-hexanediol, benzyl alcohol, benzyl benzoate, benzalkonium chloride, citric acid, parabens, formaldehyde-releasing preservatives (e.g., DMDMhydantoin diazolydinal urea or imidazolidine urea), anisic acid, sodium salicylate, chlorphenism, stearalkonium chloride, to name a few.

[0104] In an illustrative example, such a composition as described herein may comprise

[0105] - water in an amount of about 50% to about 65% (v / v), preferably about 55% to about 58% (v / v), most preferably 57% (v / v), based on the total volume of the composition,

[0106] - glycerol in an amount of about 10% to about 35% (v / v), preferably about 15% to about 25% (v / v), most preferably about 20% (v / v), of the total amount of the composition,

[0107] - conditioned medium in an amount of about 10% to about 35% (v / v), preferably about 15% to about 25% (v / v), and most preferably 20% (v / v) of the total amount of the composition,

[0108] -1,2-hexanediol in an amount of about 1% to about 55% (v / v), preferably about 1.5% to about 3.5% (v / v), and most preferably 2% (v / v) of the total amount of the composition,

[0109] - Curcumin in an amount of about 0.5% to about 5.0% (w / w), preferably about 0.75% to about 2.5% (w / w), and most preferably 1.0% (w / w), based on the total weight of the composition.

[0110] In other illustrative examples, the compositions described herein may include

[0111] - water in an amount of about 40% to about 65% (w / w), preferably about 45% to about 55% (w / w), most preferably about 52% (w / w), based on the total weight of the composition,

[0112] - conditioned medium in an amount of about 5% to about 40% (w / w), preferably about 10% (w / w) to about 30% (w / w) or about 12.5% ​​to about 25% (w / w), most preferably about 17.5% to 20.0% (w / w), based on the total weight of the composition,

[0113] - glycerol and / or propylene glycol in an amount of about 10% to 30% (w / w) of the total weight of the composition, preferably about 10% (w / w) to about 30% (w / w) or about 12.5% ​​to about 25% (w / w), most preferably about 17.5% to 20.0% (w / w), of the total weight of the composition, and

[0114] - Curcumin in an amount of about 0.5% to 5.0% (w / w) of the total weight of the composition, preferably about 0.75% to about 2.5% (w / w) or 0.8% to about 1.5% (w / w), and most preferably about 0.9% to 1.0% (w / w) of the total weight of the composition.

[0115] The compositions described herein may also include one or more of a gelling agent, a buffer, and an emollient. Any suitable gelling agent may be used in such compositions. Illustrative examples of gelling agents include, but are not limited to, gelling agents selected from poly(acrylic acid), pectin, starch, alginates such as sodium alginate, gelatin, cellulose derivatives (e.g., hydroxypropyl methylcellulose or methylcellulose), and polyvinyl alcohol clays.

[0116] As used herein, "buffer" is used in its conventional sense to refer to a compound used to stabilize or adjust the pH of a cosmetic product. Similarly, the term "emollient" is used in its conventional sense in cosmetic product formulations to refer to a compound that softens the skin and is incorporated into cosmetic products to improve skin feel. Any suitable buffer may also be used in the compositions described herein. Illustrative examples of buffers include, but are not limited to, sodium phosphate salts, calcium carbonate, sodium bicarbonate, citric acid, triethanolamine, and Good's buffer. Examples of suitable Good's buffers are HEPES, Tris, BisTris, glycylglycine, MOPS (3-(N-morpholino)propanesulfonic acid), and Tricine, to name a few. Similarly, any suitable emollient may be used in the compositions described herein, illustrative examples of suitable emollients being olive oil PEG-7 esters, triglycerides, lanolin, polyols, and fatty acid esters (e.g., isopropyl myristate, dioctyl sebacate, and dioctyl maleate).

[0117] Further illustrative examples of compositions of the present invention are shown in the following table.

[0118] Table 1: Exemplary compositions of the present invention formulated as lotions

[0119] Ingredients (NCI name) content Function Water(Aqua / water) 57.00%,(v / v) Solvent / Carrier Glycerol (Glycerin) 20.00%,(v / v) Wetting agent Conditioned medium 20.00% (v / v) Active ingredients for skin care and wound healing 1,2-Hexanediol 2.00%,(v / v) preservative Curcumin 1.00%, (w / w) anti-inflammatory agents

[0120] It should be noted that since curcumin is a solid (powder), the amount of curcumin is expressed as a weight percentage of the total weight of the formulation, while the other components are liquid at room temperature, so the amounts of these components are given as volume percentages of the total volume of the formulation. In contrast, if the anti-inflammatory agent is, for example, calendula (an oil), its amount in the composition is usually defined as a volume percentage of the total volume of the formulation.

[0121] The lotions described in Table 1 can, for example, be advantageously used as daily skin conditioning agents for patients to use at home, for example, to prevent the formation and / or recurrence of wounds. To this end, patients, such as diabetic patients, can apply the lotion topically to the skin of the feet or ankles to improve the condition of diabetic skin, thereby preventing the formation of diabetic wounds (such as foot ulcers).

[0122] Table 2: Exemplary compositions of the invention formulated as gels

[0123]

[0124]

[0125] The gels described in Table 2 can, for example, be advantageously used to induce, stimulate and / or promote healing of wounded or damaged skin areas, wherein the method comprises topically applying conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a patient, the skin of the patient being the skin surrounding a wound (e.g., a pressure ulcer or a diabetic foot ulcer).

[0126] The composition of the present invention may also contain other conventional additives and adjuvants in cosmetic products, pharmaceuticals or dermatology, such as fragrances, fillers, antiseptics, odor absorbers and dyes or colorants. Typically, the composition can be used by applying it directly to the skin via topical / dermal application. Preferably, the composition comprising the conditioned medium of the present invention is a cosmetic product composition. In some embodiments, the composition is suitable for topical application. Compositions suitable for topical application can be in liquid or viscous form. Examples include gels, ointments, creams and lotions. The preparation of such compositions is within the knowledge of those skilled in the art and is described, for example, in Gennaro, AL and Gennaro, AR (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA. In this context, a composition comprising the conditioned medium of the present invention for topical application should be understood as a preparation that can be absorbed by the skin in a targeted manner, particularly wherein the active ingredients of the conditioned medium of the present invention or the composition comprising the conditioned medium of the present invention can penetrate the epidermis and dermis but cannot be absorbed into the systemic circulation. In some examples, the conditioned medium of the invention or a composition comprising a conditioned medium of the invention crosses the surface of the skin by transepidermal diffusion or by transdermal diffusion.

[0127] On the other hand, the present invention provides conditioned medium derived from umbilical cord mesenchymal stem cell culture for inducing, stimulating and / or promoting wound or damaged skin area healing, and / or for preventing the formation and / or recurrence of wound or damaged skin area, and / or for treating rosacea, psoriasis, eczema, dermatitis, local steroid withdrawal syndrome, bullous epidermolysis. On the other hand, the present invention provides conditioned medium derived from umbilical cord mesenchymal stem cell culture for preparing a drug for inducing, stimulating and / or promoting wound or damaged skin area healing, and / or for preventing the formation and / or recurrence of wound or damaged skin area, and / or for treating rosacea, psoriasis, eczema, dermatitis, local steroid withdrawal syndrome, bullous epidermolysis. Therefore, a method for treating a subject with a conditioned medium derived from umbilical cord mesenchymal stem cell culture is described. The method includes applying (treating) an effective amount of the conditioned medium of the present invention to the skin of the subject. The treatment process may include repeatedly applying the effective amount within any desired time period. As described herein, the composition / preparation containing conditioned medium can, for example, be applied once a day or twice or three times a week over any desired time period. Referring to Example 3, a preparation comprising conditioned medium as described herein is applied for up to 16 weeks, twice to three times a week, to achieve closure of chronic diabetic foot ulcers. It will be appreciated by those skilled in the art that the time of applying the preparation of the present invention depends on the patient's specific circumstances (age, health status, wound size, etc.), but treatment can be continued until the desired effect is achieved. As also shown in the Examples section (see again Example 3), an "effective amount of conditioned medium" can be any amount of a preparation containing conditioned medium (e.g., an amount of about 20% (w / w) of the gross weight of the preparation) and applied to the skin of the subject to cover the target skin area for each treatment.

[0128] In principle, any subject is suitable for treatment with the conditioned medium of the present invention. Finally, the present invention also provides a method of treating a non-human mammal or human subject, the method comprising topically treating the skin surrounding a wound or damaged skin area of ​​the subject or the intact skin of a subject at risk of developing a wound or damaged skin area with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0129] The present invention is further illustrated by the following non-limiting experimental examples.

[0130] The sequences used herein are depicted in Table 3 below.

[0131] Table 3. Amino acid sequences used herein.

[0132]

[0133]

[0134]

[0135]

[0136] Experimental Examples

[0137] Example 1: Isolation and culture of umbilical cord mesenchymal stem cells (CLMSCs)

[0138] Culture medium preparation

[0139] a. Prepare 500 ml of PTT-6 (culture / growth medium) by adding the following substances in the order listed:

[0140] i.DMEM, 250ml

[0141] ii.M171 118ml

[0142] iii. DMEM F12 118ml

[0143] iv. FBS 12.5ml (final concentration 2.5%)

[0144] v. EGF 1ml (final concentration 10ng / ml)

[0145] vi. Insulin 0.175 ml (final concentration 5 μg / ml)

[0146] The volume of above-mentioned components i to vi obtains the culture medium that final volume is 499.675ml.If no longer add other components in culture medium, then remaining 0.325ml (volume added to 500ml) can be any one among for example component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of culture medium is 500ml.Alternatively, can add the storing solution of antibiotic (such as penicillin-streptomycin-amphotericin (Amphotericin)) to obtain final volume is 500ml.Can also add one or more in the following fill-in of 0.325ml volume in described culture medium: adenine, hydrocortisone and 3,3 ', 5-triiodo-L-thyronine sodium salt (T3), obtain thus the culture medium that cumulative volume is 500ml.

[0147] vii. Label the bottle with "PTT-6," the date the medium was prepared, the operator's initials, and the expiration date followed by the phrase "Expiration." The expiration date is the earliest expiration date of any component or one month from the preparation date, whichever comes first.

[0148] b. In a 50 ml centrifuge tube, add 2.5 ml FBS to 47.5 ml HBSS to prepare rinse medium (Hank's buffered saline solution (HBSS) with 5% FBS without calcium or magnesium). Label the tube "Rinse Medium," the operator's initials, and the date the medium was prepared.

[0149] c. All media were sterile tested using Bactec Lytic / 10 (Dickinson & Company) and Bactec Pluc+Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared culture medium was injected into each bottle.

[0150] Collection and isolation of umbilical cord tissue from red deer (RD)

[0151] Red deer (RD) umbilical cords were obtained from a farm in New Zealand that raises these animals for horn velvet, according to specific operating instructions. RD-CLMSCs were isolated from the umbilical cords according to the protocol described in International Patent Application WO 2006 / 019357A1.

[0152] Briefly, the umbilical cord of red deer was rinsed clean and immediately transferred to a 500 ml sterile glass bottle containing culture transport medium (L-15 medium supplemented with 50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml fungizone, 50 μg / ml gentamicin; all reagents were purchased from Invitrogen) and then transferred to the laboratory. In the laboratory, stem cell extraction was performed under sterile conditions in a laminar flow hood. The umbilical cord was first transferred to a sterile stainless steel tray. All residual blood in the umbilical cord vessels was removed by multiple syringe washes using warm phosphate-buffered saline (PBS) supplemented with 5 IU / ml heparin (from Sigma). Pure PBS without heparin was used in the final wash. The umbilical cord tissue samples were then cut into 2 cm long segments and transferred to 10 cm diameter cell culture dishes, where they were further washed and sterilized with 70% ethanol and then washed several times with PBS containing an antibiotic cocktail (50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml amphotericin, 50 μg / ml gentamicin; all purchased from Invitrogen) until the solution became clear.

[0153] Isolation and culture of RD-CLMSCs

[0154] First, the umbilical cord tissue is dissected to separate the amniotic membrane from Wharton's jelly (i.e., the umbilical cord stroma) and other internal components. The separated amniotic membrane is then cut into small segments (0.5 cm x 0.5 cm) for cell isolation. These small segments are then placed on tissue culture dishes under different cell culture conditions for tissue explant isolation to isolate mesenchymal stem cells.

[0155] To isolate / culture mesenchymal cells, tissue explants were immersed in PTT-6 medium (CellResearch Corp, Singapore) and maintained in a CO2 cell culture incubator at 37°C. The medium was changed every 2 or 3 days. Cell outgrowth was monitored under a light microscope. At approximately 70% confluence, cells were treated with trypsin (0.05% trypsin / 0.02% EDTA) for further expansion or cryopreservation. H-CLMSCs (#CLMC43) were also obtained from a CRC tissue bank.

[0156] Example 2: Conditioned medium derived from red deer umbilical cord lining mesenchymal stem cells (RD-CLMSCs)

[0157] Cryovials containing RD-CLMSCs were retrieved from storage and rapidly thawed in a water bath at 37° C. FSF were donated to CRC (Cell Research Corporation, Singapore) by the Stem Cell and Wound Healing Research Group, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore.

[0158] RD-CLMSCs were cultured using PTT-6 culture medium (CellResearch Corporation, Singapore) at 37°C and 5% CO2. The cell culture medium was replaced every 2 to 3 days. When 80% to 100% confluent, the cell culture medium was removed, the cells were optionally rinsed in PBS, and then cultured in DMEM basal medium, wherein the culture medium optionally contained a water-soluble antioxidant, but did not contain growth factors or serum. The culture dish was incubated for another 48 hours, and the spent culture medium was collected into a centrifuge tube and centrifuged at 1800 rpm for 10 minutes. The supernatant was collected into a labeled test tube as conditioned medium (CM). The conditioned medium was stored at -80°C until use.

[0159] Example 3: Evaluation of Conditioned Medium in Healing Persistent Chronic Diabetic Foot Ulcer (DFU) Wounds

[0160] Experimental setup

[0161] Conditioned medium was derived from red deer umbilical cord lining stem cell conditioned medium as described in Examples 1 and 2 above and formulated into a liquid solution or gel as described in Table 1 above, the formulation containing approximately 20% (w / w) conditioned medium.

[0162] Patients with chronic diabetic foot ulcers (duration> 3 months) were recruited into this case series through the hospital DFU registry. In all cases, the DFU persisted after standard care (including regular debridement by a podiatrist, local dressings, and decompression shoe wear). Patients with active infection were excluded from this case series. Conditioned medium was applied to the intact skin (ISSW) surrounding the clean, debrided wound by a liquid solution soaked in gauze or by directly applying to the gel formulation described in Table 1 of the ISSW (which contains approximately 20% (w / w) conditioned medium). After 5 minutes, the gauze or excess gel was removed and the wound was bandaged according to routine care. Treatment was twice or three times a week.

[0163] A total of 21 patients were enrolled: 15 men and 6 women. The mean age of the patients was 56.7 years (range, 40-72 years), and the mean HbA1c was 7.9% (SD = 1.99). The DFUs treated were located in various locations on the foot, with the majority being plantar ulcers. No significant systemic complications, such as allergic reactions or sepsis, were observed during the application period.

[0164] method

[0165] The patient's trauma is managed as follows:

[0166] i) Routinely debride and clean the wound;

[0167] ii) soaking 5 ml of the conditioned medium preparation in gauze;

[0168] iii) applying gauze to the intact skin surrounding the wound (ISSW) for 5 minutes, or applying gel to the ISSW;

[0169] iv) conditioned medium was not applied directly to any open skin or wound;

[0170] v) remove gauze or excess gel; and

[0171] vi) Use topical dressings as prescribed for previous treatment

[0172] result

[0173] At the time of results collection, 12 of the 21 patients achieved complete DFU closure, with ulcer closure occurring more rapidly in those treated with the gel formulation. Of the remaining patients, 8 (5 of whom are still receiving treatment) experienced a significant reduction in lesion size. One patient showed no improvement.

[0174] Figure 6 shows a reduction in wound size in a subgroup of 10 patients treated with gauze soaked with conditioned medium, Figure 7-10 Exemplary photographs of patients with healing wounds are shown. In this patient group were 8 male patients and 2 female patients. The mean age of these patients was 54.7 (SD=9.4) years and the mean HbA1c was 8.2% (SD=2.5). 8 (80%) patients had at least 1 palpable distal foot pulse. During treatment, 6 patients (60%) experienced wound healing, 3 patients (30%) showed a decrease in wound size, and 1 (10%) patient had stable wound size. Of those patients who healed, patients (n=2) who applied conditioned medium twice a week healed within 16 weeks, and patients (n=4) who applied conditioned medium three times a week healed within 4 to 7 weeks. In this case, the patients showed a significant reduction in their wound size and wound closure. In particular, as Figure 7-10 As exemplified in Figure 1, application of conditioned medium to intact skin surrounding the wound (ISSW) promoted the healing of chronic persistent wounds (DFUs), thereby regenerating the skin and restoring healthy skin. No patient experienced any side effects from treatment with the conditioned medium, and no systemic or local safety issues were observed.

[0175] In this context, reference is made to the results of a pivotal, single-blind phase 3 clinical trial of Dermagraft for the treatment of DFU. In this clinical trial, stratification was based on ulcer size (≥1 to ≤2 cm). 2 and >2 to ≤20cm 2 ) and key inclusion criteria were: chronic DFU, >6 weeks of wound duration; ulcer size 1-20 cm 2 , allowing extension through the dermis (without exposing muscle, tendon, bone, or joint capsule). The trial included 130 patients (n=130), with Dermagraft applied on day 0, followed by up to 7 additional applications at weekly intervals in combination with standard care. The results of this trial were as follows: the incidence of complete wound closure at 12 weeks was 30.0% (39 of 130 patients), compared to 18.3% (21 of 115 patients) in the control group (P=0.023). Although the results of this study (using 21 patients and no control group) cannot be directly compared with the results of the clinical trial using Dermagraft, the wound closure rate of approximately 57% (12 of 21 patients showed complete DFU closure, see above) suggests that treatment of chronic DFUs using the conditioned medium described herein should be at least as effective as or even more effective than treatment with Dermagraft (a tissue-engineered active human dermis substitute approved by the US FDA for the treatment of DFUs).

[0176] Therefore, the results of the present application indicate that applying the conditioned culture medium of mesenchymal stem cells from umbilical cord tissue (such as umbilical cord lining) to the skin around wounds (such as chronic diabetic foot ulcers) achieves effective wound closure, thereby providing a new, inexpensive and easily applicable method for treating such intractable wounds.

[0177] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0178] All patents and publications mentioned in this specification represent the levels of ordinary skill in the art to which the invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually incorporated by reference.

[0179] The invention described herein in an illustrative manner can be practiced in the absence of any element or elements, or any limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprise," "include," and "contain" are to be construed broadly and without limitation. Furthermore, the terms and expressions employed herein have been used as words of description, not limitation, and their use is not intended to exclude any equivalents of the features shown and described, or portions thereof, but rather to recognize that various modifications may be made within the scope of the claimed invention. Thus, it should be understood that while the invention has been specifically disclosed with reference to preferred embodiments and optional features, modifications and variations of the invention presented herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the claimed invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This generic description of the invention is encompassed, without precondition or negative limitation, by removing any subject matter from the genus, whether or not the deleted matter is specifically described herein. Furthermore, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will be apparent from the following claims.

Claims

1. A method for inducing, stimulating and / or promoting healing of a wound or damaged skin area, wherein the method comprises topically applying a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area.

2. A method of preventing the formation and / or recurrence of a wound or damaged skin area in a subject who is at risk of developing a wound or damaged skin area, wherein the method comprises topically administering to the skin of the subject conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

3. A method for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically administering to the skin of a subject a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

4. The method of claim 1, wherein the method does not comprise applying the conditioned medium to the wound or the damaged skin area.

5. The method according to any one of claims 1 to 4, wherein the conditioned medium is administered in the form of a gel, a cream or a liquid.

6. The method according to any one of claims 1 to 5, wherein the skin to be treated is intact skin.

7. The method according to claims 1 to 6, wherein the skin to be treated is diabetic skin or thin (weak) skin.

8. The method of any one of claims 1 to 7, wherein the wounded or damaged skin area is a chronic wound or chronically damaged skin.

9. The method of any one of claims 1-8, wherein the damaged skin area is a wound.

10. The method according to any one of claims 1 to 9, wherein the wound or injured skin area is selected from the group consisting of diabetic foot ulcers (DFUs), pressure ulcers, wounds caused by chronic limb ischemia, wounds caused by chronic limb-threatening limb ischemia, venous leg ulcers, surgical wounds, puncture wounds, burns and bite wounds.

11. The method according to any one of the preceding claims, wherein the conditioned medium is derived from a culture of an umbilical cord amniotic membrane mesenchymal stem cell population, a culture of an umbilical cord Wharton's jelly (WJ) mesenchymal stem cell population, a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of an umbilical cord mixed (MC) mesenchymal stem cell population.

12. The method according to any one of the preceding claims, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

13. The method of claim 12, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

14. The method of claim 12 or 13, wherein the culture medium comprises DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v).

15. The method of any one of claims 12-14, wherein the culture medium comprises DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

16. The method according to any one of claims 12 to 15, wherein the culture medium further comprises (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), Optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml. 17 . The method according to claim 12 , wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

18. A method for producing conditioned medium, the method comprising (a) Umbilical cord mesenchymal stem cells were cultured in a medium containing DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum); (b) removing the umbilical cord mesenchymal stem cells from the culture medium; and (c) optionally culturing the umbilical cord mesenchymal stem cells in an additional culture medium, optionally wherein the additional cell culture medium comprises a water-soluble antioxidant, The conditioned medium is obtained by collecting the cell culture medium.

19. The method according to claim 18, wherein the additional cell culture medium does not comprise growth factors and / or is a serum-free medium and / or wherein the cells are cultured at a concentration of about 1 million cells / 1 ml. 20 . The method according to claim 18 or 19 , wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

21. The method according to any one of claims 18 to 20, wherein the additional culture medium is a basal medium suitable for culturing mesenchymal stem cells, preferably a serum-free medium.

22. The method of any one of claims 18-21, wherein the additional culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), DMEM-F12, RPMI medium, EpiLIfe medium, and Medium 171.

23. The method of any one of claims 18-22, wherein the conditioned medium is diluted in carrier medium.

24. The method of any one of claims 1-17, wherein the conditioned medium is obtained or obtainable by the method of any one of claims 18-23. 25 . A conditioned medium derived from umbilical cord mesenchymal stem cells, obtained or obtainable by the method of any one of claims 18 to 23 .

26. A composition comprising the conditioned medium of claim 25.

27. The composition of claim 26, wherein the composition further comprises an anti-inflammatory compound.

28. The composition of claim 27, wherein the anti-inflammatory compound is selected from the group consisting of curcumin, sulfacetamide, nonsteroidal anti-inflammatory compounds, niacinamide (vitamin B3), calendula, sea buckthorn oil, chamomile extract, centella asiatica extract, and mixtures thereof.

29. The composition of claim 27 or 28, wherein the composition further comprises a wetting agent.

30. The composition of claim 29, wherein the humectant is selected from the group consisting of glycerin, propylene glycol, triethylene glycol, tripropylene glycol, sorbitol, hexylene glycol, butylene glycol, urea, collagen, and mixtures thereof.

31. The composition of claim 30, wherein the humectant comprises glycerin, propylene glycol, or both glycerin and propylene glycol.

32. The composition of any one of claims 26 to 31, wherein the composition comprises conditioned medium derived from umbilical cord mesenchymal stem cell culture in an amount of about 5 to 40% (v / v) of the total volume of the composition, a wetting agent in an amount of about 10 to 30% (v / v) of the total volume of the composition, and an anti-inflammatory compound in an amount of about 1.0 to about 3.0% (v / v) of the composition or in an amount of 0.5 to 5.0% (w / w) of the total weight of the composition.

33. The composition of claim 32, wherein the composition comprises a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells in an amount of about 10 to 30% (v / v) of the total volume of the composition, a wetting agent in an amount of about 15 to 25% (v / v) of the total volume of the composition, and an anti-inflammatory compound in an amount of about 1.0 to about 3.0% (v / v) of the composition or in an amount of 0.75 to 3.0% (w / w) of the total weight of the composition.

34. The composition of any one of claims 26 to 31, wherein the composition comprises conditioned medium derived from a culture of umbilical cord mesenchymal stem cells in an amount of about 5% to 40% (w / w), based on the total weight of the composition, a wetting agent in an amount of about 10% to 30% (w / w), based on the total weight of the composition, and an anti-inflammatory compound in an amount of about 0.5% to 5.0% (w / w), based on the total weight of the composition.

35. according to any one of claims 26 to 34, wherein said composition comprises -Conditioned medium derived from umbilical cord mesenchymal stem cell culture, - glycerol, propylene glycol or both glycerol and propylene glycol, and -Curcumin.

36. The composition of any one of claims 26 to 35, wherein the composition further comprises a solvent / carrier.

37. The composition of claim 36, wherein the solvent / carrier is water or an aqueous buffer solution.

38. The composition of any one of claims 26 to 37, wherein the composition further comprises a preservative.

39. The composition of claim 38, wherein the preservative is selected from 1,2-hexanediol, benzyl alcohol, benzyl benzoate, benzalkonium chloride, citric acid, parabens, formaldehyde-releasing preservatives, anisic acid, salicylic acid, sodium salicylate, chlorphenism, and stearyltrimonium chloride.

40. A composition according to any one of claims 36 to 39, wherein the composition comprises - water in an amount of about 50% to about 65% (v / v), preferably about 55% to about 58% (v / v), most preferably 57% (v / v), based on the total volume of the composition, - glycerol in an amount of about 10% to about 35% (v / v), preferably about 15% to about 25% (v / v), most preferably about 20% (v / v), of the total amount of the composition, - conditioned medium in an amount of about 10% to about 35% (v / v), preferably about 15% to about 25% (v / v), and most preferably 20% (v / v) of the total amount of the composition, -1,2-hexanediol in an amount of about 1% to about 55% (v / v), preferably about 1.5% to about 3.5% (v / v), and most preferably 2% (v / v) of the total amount of the composition, - Curcumin in an amount of about 0.5% to about 5.0% (w / w), preferably about 0.75% to about 2.5% (w / w), and most preferably 1.0% (w / w), based on the total weight of the composition.

41. according to the composition described in any one of claims 36 to 39, wherein said composition comprises - water in an amount of about 40% to about 65% (w / w), preferably about 45% to about 55% (w / w), most preferably about 52% (w / w), based on the total weight of the composition, - conditioned medium in an amount of about 5% to about 40% (w / w), preferably about 10% (w / w) to about 30% (w / w) or about 12.5% ​​to about 25% (w / w), most preferably about 17.5% to 20.0% (w / w), based on the total weight of the composition, - glycerol and / or propylene glycol in an amount of about 10% to 30% (w / w) of the total weight of the composition, preferably about 10% (w / w) to about 30% (w / w) or about 12.5% ​​to about 25% (w / w), most preferably about 17.5% to 20.0% (w / w), of the total weight of the composition, and - Curcumin in an amount of about 0.5% to 5.0% (w / w) of the total weight of the composition, preferably about 0.75% to about 2.5% (w / w) or 0.8% to about 1.5% (w / w) of the total weight of the composition, and most preferably about 0.9% to 1.0% (w / w) of the total weight of the composition.

42. A composition according to any one of claims 36 to 41, wherein the composition further comprises one or more of a gelling agent, a buffering agent and an emollient.

43. A composition according to claim 42, wherein the gelling agent is selected from poly (acrylic acid), pectin, starch, alginates such as sodium alginate, gelatin, cellulose derivatives and polyvinyl clay.

44. The composition of claim 42 or 43, wherein the buffer is selected from the group consisting of sodium phosphate salts, calcium carbonate, sodium bicarbonate, citric acid, triethanolamine, and Good's buffer.

45. The composition of claim 44, wherein the Good's buffer is selected from the group consisting of HEPES, Tris, BisTris, glycylglycine, MOPS (3-(N-morpholino)propanesulfonic acid) and Tricine.

46. ​​A composition according to any one of claims 42 to 45, wherein the emollient is selected from olive oil PEG-7 esters, triglycerides, lanolin, polyols and fatty acid esters (e.g. isopropyl myristate, dioctyl sebacate and dioctyl maleate).

47. Use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture in the preparation of a pharmaceutical composition for inducing, stimulating and / or promoting healing of a wound or damaged skin area, wherein the use comprises topically applying the conditioned medium derived from umbilical cord mesenchymal stem cell culture to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area.

48. Use of conditioned medium derived from a culture of umbilical cord mesenchymal stem cells in the preparation of a pharmaceutical composition for preventing the formation and / or recurrence of a wound or damaged skin area in a subject who is at risk of developing a wound or damaged skin area, wherein the method comprises topically administering the conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject.

49. Use of a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells in the preparation of a pharmaceutical composition for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically administering the conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject.

50. The use according to any one of claims 47 to 49, wherein the conditioned medium derived from umbilical cord mesenchymal stem cell culture is obtained or obtainable by the method according to any one of claims 19 to 24.

51. The use of claim 47, wherein the method does not comprise applying the conditioned medium to the wound or the damaged skin area.

52. The use according to any one of claims 47 to 51, wherein the conditioned medium is administered in the form of a gel, a cream or a liquid.

53. Use according to any one of claims 47 to 52, wherein the skin to be treated is intact skin.

54. Use according to any one of claims 47 to 53, wherein the skin to be treated is diabetic skin or thin (weak) skin.

55. The use according to any one of claims 47 to 54, wherein the wounded or damaged skin area is a chronic wound or chronically damaged skin.

56. The use according to any one of claims 47 to 55, wherein the damaged area of ​​skin is a wound.

57. The use according to any one of claims 47 to 56, wherein the wound or injured skin area is selected from diabetic foot ulcers (DFUs), pressure ulcers, wounds caused by chronic limb ischemia, wounds caused by chronic limb-threatening limb ischemia, venous leg ulcers, surgical wounds, puncture wounds, burns and bite wounds.

58. The use according to any one of the preceding claims 47-57, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population of the umbilical cord amniotic membrane, a culture of a mesenchymal stem cell population of the umbilical cord Wharton's jelly (WJ), a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed (MC) mesenchymal stem cell population of the umbilical cord.

59. The use according to any one of the preceding claims 47-58, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

60. The use according to claim 59, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

61. The use according to claim 59 or 60, wherein the culture medium comprises DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v).

62. The method of claim 59, wherein the culture medium comprises DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

63. The use according to any one of claims 59-62, wherein the culture medium further comprises (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), Optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml. The use according to any one of claims 59 to 63 , wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

65. Use of a composition as defined in any one of claims 26 to 46 in: i) for inducing, stimulating and / or promoting healing of a wound or damaged skin area, wherein the use comprises topically administering a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject, wherein the skin of the subject is the skin surrounding the wound or damaged skin area, or ii) for preventing the formation and / or recurrence of a wound or damaged area of ​​skin in a subject who is at risk of developing a wound or damaged area of ​​skin, wherein the method comprises topically administering to the skin of the subject a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells, or iii) for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or skin damage caused by fragile skin, wherein the method comprises topically administering to the skin of the subject a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

66. The use according to claim 65i), wherein the method does not comprise applying the conditioned medium to the wound or the damaged skin area.

67. Use according to claim 65 or 66, wherein the skin to be treated is intact skin.

68. Use according to any one of claims 65 to 67, wherein the skin to be treated is diabetic skin or thin (weak) skin.

69. The use according to any one of claims 65 to 68, wherein the wounded or damaged skin area is a chronic wound or chronically damaged skin.

70. The use according to any one of claims 65-69, wherein the damaged skin area is a wound.

71. The use according to any one of claims 65 to 70, wherein the wound or injured skin area is selected from diabetic foot ulcers (DFUs), pressure ulcers, wounds caused by chronic limb ischemia, wounds caused by chronic limb-threatening limb ischemia, venous leg ulcers, surgical wounds, puncture wounds, burns and bite wounds.

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