Use of conditioned medium from umbilical cord lining mesenchymal stem cells for healing wounds and damaged skin areas and / or preventing their formation and / or recurrence - Patent Application 20070122999

Abstract

The present invention relates to a method for inducing, stimulating, and / or promoting the healing of a wound or damaged skin area, comprising topically treating a subject's skin surrounding the wound or damaged skin area with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. Similarly, the present invention relates to a method for preventing the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing wounds or damaged skin areas, comprising topically treating the subject's skin with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. Furthermore, the present invention relates to a method for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, comprising topically treating a subject's skin surrounding the wound or damaged skin area to be treated with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. The present invention is also directed to the conditioned medium, uses thereof, compositions comprising it, methods for producing the conditioned medium, and uses of these compositions.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for inducing, stimulating, and / or promoting healing of a wound or damaged skin area, comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject 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 at risk of developing a wound or damaged skin area, comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject. Furthermore, the present invention relates to a method for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, comprising topically administering 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 the conditioned medium, and compositions containing the conditioned medium. These methods and compositions may be therapeutic, but may also be non-therapeutic or cosmetic methods or compositions. [Background technology]

[0002] Background of the Invention Wounds and damaged skin, the complications associated with their healing, and the inability of patients at risk of developing wounds or damaged skin to effectively prevent their occurrence are major problems both in hospitals and at home. The body's ability to effectively and quickly activate the wound or damaged skin healing process is essential not only for restoring skin that is constantly exposed to the external environment, but also for the skin's function as a primary defense system or protective barrier against the environment. As a result, the loss of significant skin integrity following an injury resulting in a wound or damaged skin can increase susceptibility to fungal, bacterial, and viral infections, leading to severe disability or even death.

[0003] To understand the healing process of damaged skin and wounds, i.e., skin repair and regeneration, it is essential to understand the anatomical and physiological functions of normal skin. Histologically, skin is divided into two functional layers, the epidermis and dermis, separated by a basement membrane. The epidermis is multicellular, and new cells are constantly produced in the deepest layer of the epidermis, the stratum basalis, and migrate outward toward the skin's surface. The dermis, responsible for the skin's mechanical properties, is composed of fibers and connective tissue, with fibers composed of elastin and collagen. Collagen fibers provide skin strength, while elastin fibers provide recoil strength. In normal skin, the dermis and epidermis form a protective barrier against the external environment and serve various protective functions, including mechanical protection, photoprotection, immune surveillance, nutrient metabolism, and repair. Because skin plays such an important role in the body, damaged skin must be quickly replaced. The physiological process of wound healing is initiated when the protective barrier of the dermis and epidermis is disrupted, compromising its structural integrity and causing rupture of the underlying vasculature.

[0004] Wound healing involves hemostatic, inflammatory, and proliferative processes. Skin repair begins with the formation of a blood clot and platelet degranulation, which initiates homeostasis by releasing growth factors necessary for wound repair and promoting the regeneration of damaged epidermis. Early in the inflammatory phase, neutrophils are present at the wound site and are primarily responsible for removing bacteria and foreign bodies. Late in the inflammatory phase, monocytes migrate to the wound site and become macrophages. The subsequent proliferative phase is characterized by the increased formation of granulation tissue, in which fibroblasts lay down a layer of collagen and generate new capillaries; ultimately, a new, intact epidermis forms around the lesion.

[0005] However, there are many types of wounds and diseases and conditions associated with damaged skin, which have various complications, such as the formation and / or recurrence of wounds or damaged skin not being effectively prevented, and / or the healing process being insufficient or even inhibited.As a specific example, the occurrence of pressure sores, also known as pressure sores or bedsores, is generally difficult to prevent.A pressure sore is an injury to the skin and underlying tissues, mainly caused by prolonged pressure on the skin, and can develop gradually or form within hours, affecting any part of the body that is subjected to pressure; for example, pressure sores are particularly likely to occur in bony parts of the body, such as the heels, elbows, lower back, and base of the spine. Typically, pressure ulcers affect, for example, bedridden patients, patients who sit in a chair or wheelchair for long periods of time, elderly people or patients with mobility problems, subjects whose skin is easily damaged by dehydration, obese subjects or patients, and subjects or patients with medical conditions that affect blood supply and cause skin fragility or impaired mobility (such as, but not limited to, diabetes, peripheral arterial disease, renal failure, heart failure, multiple sclerosis (MS), Parkinson's disease, etc.). Measures to reduce the risk of developing pressure ulcers include regularly repositioning the patient and monitoring the condition of the skin for signs and symptoms of pressure ulcers. Therefore, there remains a need for effective and reliable compositions and methods for preventing the formation and / or recurrence of wounds or damaged skin, such as those caused by pressure ulcers.

[0006] Chronic wounds present other known complications related to healing. Chronic wounds often have serious complications because elevated levels of inflammatory cells and proteases inhibit the healing process by degrading the extracellular matrix (ECM), which is largely composed of collagen and plays a key role in the healing response, particularly the proliferative phase described above. An example of a chronic wound is a diabetic foot ulcer, which is caused by reduced blood flow and inflammation and may heal slowly or, if left untreated, never heal. This can lead, among other things, to severe infections. In such cases, increased levels of proinflammatory cytokines, which inhibit natural wound healing and require anti-inflammatory signals, disrupt the migratory activity of epithelial and endothelial cells, which are essential for wound closure, making natural wound healing difficult.

[0007] A common procedure for treating wounds, particularly chronic wounds such as diabetic ulcers, venous ulcers, and pressure ulcers, involves the use of a dressing that is applied to the wound.

[0008] One example of an approved medical dressing is Apligraf, a living, bilayered skin substitute containing type I bovine collagen extracted and purified from bovine tendon and viable allogeneic human fibroblast and keratinocyte cells isolated from human infant foreskin. This wound dressing / dressing is recommended for use in conjunction with standard diabetic foot ulcer treatment, particularly for the treatment of full-thickness, neuropathic diabetic foot ulcers that have not responded adequately to conventional ulcer treatments and that extend through the dermis but do not expose tendon, muscle, capsule, or bone, lasting more than three weeks. Yet another wound dressing / dressing is Dermagraft®, a living, bilayered skin substitute containing type I bovine collagen extracted and purified from bovine tendon and viable allogeneic human fibroblast and keratinocyte cells isolated from human infant foreskin. Dermagraft® is recommended for the treatment of full-thickness diabetic foot ulcers that extend through the dermis but do not expose tendons, muscles, joint capsules, or bone and last more than six weeks. Dermagraft® should be used in conjunction with a standard wound care regimen in patients with an adequate blood supply to the involved foot.

[0009] However, these wound dressings are relatively expensive because they are quite complex to manufacture, resulting in a significant cost for the treatment / management of diabetic foot ulcers. Therefore, there is a need for new compositions that are easy to manufacture yet effective in treating ulcers, such as diabetic foot ulcers. It is therefore an object of the present invention to provide such compositions for subsequent use in wound healing. Summary of the Invention

[0010] This object is achieved by a method, a conditioned medium and uses thereof having the features of 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, the method comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject surrounding the wound or damaged skin area.

[0012] In a second aspect, the present invention provides a method for preventing the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing such wounds or damaged skin areas, the method comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject.

[0013] In a third aspect, the present invention provides a method for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, the method comprising topically treating the skin of a subject surrounding the 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 the steps of: (a) culturing umbilical cord mesenchymal stem cells in a culture medium comprising 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 a further culture medium, optionally comprising a water-soluble antioxidant, wherein the further cell culture medium comprises a water-soluble antioxidant, and wherein the conditioned medium is obtained by recovering the cell culture medium.

[0015] In a fifth aspect, 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.

[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 or cosmetic composition, for example in the form of an ointment, lotion, cream, or gel, and may be used to treat a wound or skin condition as described herein.

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

[0018] In a ninth aspect, the present invention provides use of conditioned medium derived from a culture of umbilical cord mesenchymal stem cells for inducing, stimulating and / or promoting healing of wounds or damaged skin areas, and / or for preventing the formation and / or recurrence of wounds or damaged skin areas, and / or for treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa. [Brief explanation of the drawings]

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

[0020] [Figure 1-1] FIG. 1 shows the Lonza technical information sheet for Dulbecco's Modified Eagle's Medium (including the catalog number for DMEM) used to prepare the specific example medium (PTT-6) in the experimental section. [Figure 1-2] See description of Figure 1-1. [Figure 2] 1 shows Lonza's technical information sheet for Ham's F12 medium. [Figure 3]The experimental section provides the Lonza technical information sheet for DMEM:F12 (1:1) medium (including the catalog number for DMEM:F12 (1:1) medium) used to prepare the specific example medium (PTT-6). [Figure 4-1] FIG. 4 shows the Life Technologies Corporation technical information sheet for M171 medium (including the M171 medium catalog number) used to prepare the specific example medium (PTT-6) in the experimental section. [Figure 4-2] See description of Figure 4-1. [Figure 5] A list of the ingredients (including their commercial suppliers and catalog numbers) used in the experimental section to prepare medium PTT-6 is provided. [Figure 6] 1 shows the reduction in wound size (cm2) over time (week 1 to week 16) in 10 patients with chronic diabetic foot ulcers (DFUs) treated with conditioned medium of the present invention. [Figure 7A] 7A-B show image sequences of wound size (cm) reduction and wound healing over a total of 16 weeks in two patients with chronic diabetic foot ulcers (DFUs) (bottom image: untreated, top image: treated), in which the conditioned medium of the present invention was applied twice weekly. [Figure 7B] See legend to Figure 7A. [Figure 8] Image sequences of wound size (cm2) reduction and wound healing over a total of 8 weeks in a patient with a chronic diabetic foot ulcer (DFU) (bottom image: untreated, top image: treated) where the conditioned medium of the present invention was applied three times per week. [Figure 9] 9A-B show the reduction in wound size (cm2) and wound healing over a total of 5 weeks in a patient with a chronic diabetic foot ulcer (DFU) (left: untreated, right: treated), in which the conditioned medium of the present invention was applied twice weekly. [Figure 10]Reduction in wound size (cm2) and wound healing over a total of 3 weeks in a patient with a chronic diabetic foot ulcer (DFU) (left: untreated, right: treated) where the conditioned medium of the present invention was applied 3 times per week. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description The present invention is directed to a method and composition suitable for inducing, stimulating, and / or promoting the healing of wounds or damaged skin areas.In addition, the method and composition of the present invention are 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.In addition, the method and composition of the present invention are suitable for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin.

[0022] Surprisingly, it has been found herein that the conditioned medium derived from the culture of mesenchymal stem cells from umbilical cord, particularly the conditioned medium derived from the culture of mesenchymal stem cells from the amniotic membrane of umbilical cord, can induce, stimulate, and / or promote the healing of wounds or skin injuries by administering the conditioned medium only to the skin surrounding the wound or injured skin area, i.e., only to the unwounded or uninjured skin area, rather than directly applying the conditioned medium to the wound or injured skin area itself.It is particularly surprising that, by this administration route, the conditioned medium described herein can even heal / close difficult-to-heal wounds, such as chronic diabetic foot ulcers.See Example 3, which shows that 12 out of 21 patients with chronic diabetic foot ulcers (DFUs) achieved complete closure of the DFUs. This finding of the present invention allows for the formulation of conditioned medium into preparations such as creams, gels, or lotions; these preparations are easily manufactured (e.g., at a much lower cost than wound dressings such as Apligraf and Dermagraft), are stable at room temperature, and can therefore be easily distributed to hospitals and treatment centers and applied by untrained medical personnel or even by patients themselves. These advantages make it possible to provide treatment to patients with chronic diabetic foot ulcers, for example in developing countries, who previously could not receive treatment with wound dressings such as Apligraf and Dermagraf. Furthermore, by enabling closure of such chronic wounds, the present invention provides patients with opportunities, for example, to return to work, thereby improving their socioeconomic situation. Finally, by providing an affordable treatment option for chronic wounds such as diabetic foot ulcers, the present invention offers an opportunity to reduce the overall cost of diabetic foot ulcer treatment, especially 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 wound or damaged skin healing is caused by the ability of the conditioned medium, which contains high concentrations of one or more biological factors, such as exosomes, growth factors, proteins, peptides, and cytokines, that contribute to building a strong extracellular matrix and supporting skin repair when administered to the skin surrounding a wound or damaged skin area or to the intact skin of a subject at risk of developing a wound or damaged skin area. This allows the conditioned medium of the present invention to provide excellent properties for inducing, stimulating, and / or promoting the healing of wounds or damaged skin areas, and / or for preventing the formation and / or recurrence of wounds or damaged skin areas in subjects at risk of developing wounds or damaged skin areas, and / or for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin.

[0023] As demonstrated in the experimental section, patients with chronic and refractory ulcers are treated with the conditioned medium of the present invention, but in this case the conditioned medium is administered only to the skin surrounding the wound, not to the wound itself, and the patients experience significant improvements in wound relief, observed within 16 weeks, 8 weeks, 5 weeks, or even 3 weeks. The promotion of wound healing, particularly through increased skin cell turnover, enhanced extracellular matrix formation, enhanced regenerative healing through potent anti-inflammatory cytokines, and enhanced angiogenesis, is thought to be due to the fact that conditioned medium is particularly rich in growth factors and other biological factors that are actively involved in skin repair and regeneration; such growth factors and biological factors include, but are 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).

[0024] The conditioned medium of the present invention may contain at least one, more typically a combination, of biological factors produced by umbilical cord mesenchymal stem cells (e.g., at least one, more typically a combination of the above biological factors). For example, they may contain one or more (at least one) angiogenic cytokines and / or growth factors. For example, they may contain one or more selected from the group consisting of 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).

[0025] In this context, growth factors are generally known to be capable of stimulating cell growth and / or proliferation and / or cell differentiation, and the active involvement of growth factors in skin and, in particular, wound healing is known to those skilled in the art. For example, for 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, particularly in 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 the 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 in 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 beta (including TGF-β1, TGF-β2, and TGF-β3) in wound healing, particularly in the healing of chronic / non-healing wounds, see, for example, Ramirez et al. "The Role of TGFβ 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.

[0026] Furthermore, the generally known positive involvement of growth factors in the skin and in particular wound healing is demonstrated, for example, by their ability to increase the expression of elastin and hyaluronic acid in human skin fibroblasts (see Kaehaeri, VM, et al., Transforming growth factor-beta upregulates elastin gene expression in human skin fibroblasts. Evidence for post-transcriptional modulation. Lab Invest, 1992. 66(5): pp. 580-8; and Takami, Y., et al., Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2005. 1743(1): pp. 49-56). For example, umbilical cord mesenchymal stem cells can produce one or more factors involved in 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-inflammatory (e.g., transforming growth factor β1 (TGF-β1)); promoting the expression of elastin and / or hyaluronic acid (e.g., transforming growth factor β1 (TGF-β1), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF1)), as well as other factors.

[0027] The conditioned medium described herein may contain exosomes. Those skilled in the art will recognize that exosomes actively participate in the wound healing process, for example, by conferring anti-inflammatory effects, promoting angiogenesis, and stimulating new tissue formation; see, for example, 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, application of the conditioned medium of the present invention has beneficial effects at all stages of wound healing, for example, by promoting the repair of wounds or damaged skin and / or preventing the formation and / or recurrence of wounds or damaged skin.

[0028] The present invention has discovered that a conditioned medium derived from a culture of umbilical cord mesenchymal cells, the cells secreting, inter alia, growth factors and other biological factors into the conditioned medium, can induce, stimulate, and / or promote healing of wounds or damaged skin, prevent the formation and / or recurrence of wounds or damaged skin areas in subjects at risk of developing wounds or damaged skin areas, and / or treat skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin. Thus, it is believed that the conditioned medium of the present invention can deliver high concentrations of growth factors and / or other biological factors that 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 treat skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin.

[0029] In this regard, it is noted that culturing umbilical cord mesenchymal stem cells in the culture media described herein results in the isolation of highly homogeneous and well-defined mesenchymal stem cell populations of umbilical cord tissue (e.g., mesenchymal stem cell populations of umbilical cord Wharton's jelly or amniotic membrane), as disclosed in WO 2019 / 199234 A1 or WO 2018 / 067071 A1. In particular, isolation of umbilical cord mesenchymal stem cell populations yields greater than 90%, or even 99% or more, of cells that are positive for the three mesenchymal stem cell markers CD73, CD90, while at the same time these stem cells lack expression of CD34, CD45 and HLA-DR (see, e.g., WO 2019 / 199234 A1 or WO 2018 / 067071 A1); that is, for example, 99% or more of the cells in this population express the stem cell markers CD73, CD90 and CD105, but do not express the markers CD34, CD45 and HLA-DR. In this regard, such mesenchymal stem cell populations from umbilical cord tissue fully meet the generally accepted criteria for human mesenchymal stem cells to be used in 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, pp. 315-317; Sensebe et al., "Production of mesenchymal stromal / 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, pp. 75-79.This, in turn, means that conditioned medium derived from the culture of such highly homogeneous and well-defined cell populations is ideal for inducing highly homogeneous and well-defined components, such as high concentrations of growth factors, into the conditioned medium. Furthermore, the use of the conditioned medium of the present invention in wound healing or healing of damaged skin areas and / or preventing the formation and / or recurrence of such wounds or damaged skin areas has several additional advantages over stem cell-based applications, as the conditioned medium can avoid donor-recipient compatibility issues with stem cell-based treatments or transplants, and is easy and inexpensive to prepare compared to stem cell-based skin treatments.

[0030] Conditioned medium derived from the culture of umbilical cord mesenchymal stem cells, 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 mesenchymal stem cell population from the umbilical cord (tissue) 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 a specific example, the mesenchymal stem cells can be mesenchymal stem cells from the amniotic membrane of the umbilical cord. For example, these stem cells secrete growth factors and / or other biological factors that are actively involved in wound healing, thereby achieving the effect of promoting, treating, and / or preventing the healing of wounds or damaged skin. For example, biological factors that may be present 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 granules. Any combination of such conditioned media and the growth factors and / or biological factors contained therein can be used in the methods of the present invention.

[0031] 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, comprising topically administering a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells to the skin of a subject surrounding the wound or damaged skin area. As used herein in the context of the present invention, the term wound or damaged skin refers to injury or damage to the integrity of the skin, particularly the integrity of the skin surface, and may also include injury or damage to the underlying tissue. For example, the wound or damaged skin may be caused by a burn, bite, trauma, surgery, or a disease, such as a skin disease, skin condition, or metabolic disorder. The skin disease, skin condition, or metabolic disorder may be any disease, condition, or disorder associated with the occurrence of a wound or damaged skin, particularly one in which healing of the wound or damaged skin is desired / required. Non-limiting examples of such skin diseases, skin conditions, or metabolic disorders include type I or type II diabetes mellitus suffering from chronic ulcers such as diabetic foot ulcers (DFUs), pressure ulcers, limb ischemia, and venous leg 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, and the wound is selected from the group consisting of diabetic foot ulcers (DFUs), pressure ulcers, wounds due to chronic limb ischemia, wounds due to chronic limb-threatening limb ischemia, venous leg ulcers, surgical wounds, puncture wounds, burns, or bite wounds.

[0032] As used herein, "inducing, stimulating, and / or promoting healing of wounds or damaged skin" refers to the ability of a conditioned medium to promote and / or initiate (induce) the healing process by skin repair and / or regeneration, particularly in patients suffering from wounds or damaged skin areas with healing complications. The ability to promote and / or initiate wound healing may be due 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 thus the growth factors and biological factors contained in the conditioned medium of the present invention) can be measured / quantified by any suitable method, for example, by measuring the amount of growth factors and biological factors using commercially available antibodies / immunoassays (see Experimental Section). Such measurements can be performed automatically, for example, using a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).

[0033] In a second aspect, the present invention also relates to a method for preventing the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing such wounds or damaged skin areas, the method comprising topically treating the subject's skin with conditioned medium derived from culture of umbilical cord mesenchymal stem cells. As used herein, "preventing the formation and / or recurrence of wounds or damaged skin" refers to the ability of the conditioned medium of the present invention to avoid and / or reduce the risk of developing wounds or damaged skin in a subject at risk of developing wounds or skin damage; wherein the risk may be due to a previous skin disease, skin condition, metabolic disease, or the like. To prevent the formation and / or recurrence of wounds or damaged skin, the conditioned medium can be administered to a functionally and physiologically normal skin area of ​​the subject.

[0034] The conditioned medium of the present invention used for treatment can be applied, for example, in a final amount of about 5% or about 10% to about 80%, more preferably about 10% to about 50%, more preferably about 10% to about 30%, more preferably about 20% to about 30%, and more preferably about 20%, where % refers to either the % (v / v) of the total volume of the composition containing the conditioned medium or the % (w / w) of the total weight of the composition. In this regard, please refer to the specific formulation examples described herein. For this purpose, the conditioned medium of the present invention can be diluted (formulated) with any suitable diluent / carrier medium. The carrier medium may be a liquid, gel, or cream formulation. For example, the conditioned medium can be diluted with a carrier medium such as PBS, water, or basal medium, to name just a few suitable media. As used herein, basal medium refers to a mixture containing sugars, amino acids, water, and other components necessary for cell survival. Examples include, but are not limited to, commercially available prepared media such as Dulbecco's Modified Eagle's Medium (DMEM), Endothelial Cell Differentiation Medium (EDM), Minimal Essential Medium (MEM), Basal Medium Eagle's Medium (BME), RPMI 1640, F-10, F-12, α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. In some examples, the conditioned medium of the present invention may be contained in a solution containing DMEM. The medium may further contain antioxidants, such as soluble antioxidants. Adding water-soluble antioxidants to the medium can prevent oxidative damage. Examples of suitable water-soluble antioxidants include curcumin, glutathione, uric acid, Trolox, or Allicidin, to name just a few.

[0035] In a third aspect, the present invention also relates to a method for treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, the method comprising topically administering to the skin of a subject a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0036] To treat a subject, the conditioned medium of the present invention is administered here by topical administration. In principle, any topical administration method is contemplated herein. For example, before applying the conditioned medium to the subject's skin, the conditioned medium can be formulated into a cream, ointment, gel, lotion, suspension, or other suitable formulation. The conditioned medium can also or alternatively be held in place by, for example, a dressing or by soaking the conditioned medium into gauze and then applying it to the subject's skin (see Example 3).

[0037] The conditioned medium according to the present invention or a composition comprising the conditioned medium described herein is topically administered to the subject's skin surrounding a wound or damaged skin area. Thus, this method does not include administering the conditioned medium onto the wound or damaged skin area. In some instances, the skin surrounding a wound or damaged skin area refers to the skin immediately surrounding the wound or damaged skin. In some instances, the skin surrounding a wound refers to the periwound area, which is the skin beyond the wound edge but close to or adjacent to the wound edge. In some embodiments, the treated skin is intact skin. In particular, the skin surrounding a wound refers to intact periwound skin that can perform normal skin functions such as absorption, excretion, protection, secretion, thermoregulation, and immunity, and maintains the physical properties of skin such as elasticity and surface integrity. In some embodiments, the skin treated according to the present invention is thin / weak skin. As used herein, fragile skin preferably refers to particularly vulnerable and / or fragile skin that has been affected by wounds or damaged skin but is still intact, i.e., capable of performing the normal functions of skin as described above and maintaining its physical properties. The term "thin skin" is used herein in its ordinary sense to refer to skin that easily tears, breaks, or tears. Thin skin is also sometimes referred to as thinning skin or fragile skin. Thin skin is a common condition among older people and is most noticeable on the face, arms, and hands. People with thin skin may notice visible veins, tendons, bones, and capillaries beneath the skin of their hands and arms. However, thin skin is not only associated with aging, but also with diseases such as diabetes, the use of certain medications such as steroids, and can also be caused by UV exposure, genetics, or lifestyle factors. The thick fibrous tissue of the dermis is made up of collagen and elastin. The dermis provides skin with strength, flexibility, and elasticity. Thin skin is the result of dermal thinning.

[0038] In some embodiments, the conditioned medium is administered in the form of a gel. In particular, the present invention describes a gel formulation containing a conditioned medium for topical application; the formulation contains an effective amount of conditioned medium as described herein for inducing, stimulating, and / or promoting the healing of wounds or damaged skin areas, and / or preventing the formation and / or recurrence of such wounds or damaged skin areas, and / or treating diseases associated with wounds or skin damage. As used herein, the term "gel formulation" refers to a gel formulation containing the conditioned medium of the present invention in an amount effective for wound healing or healing of damaged skin areas, and / or preventing the formation and / or recurrence of such wounds or damaged skin areas, and / or treating diseases associated with skin damage or wounds, as described herein, and may further include a pharmaceutically and / or cosmetically acceptable water-soluble polymer, such as polyacrylic acid (carbomer). Such a gelling agent can provide a suitable gel viscosity within the range of topical application, for example, 1,000 to 200,000 cps at room temperature. The gel formulation has the advantage of spreading evenly around the wound or damaged skin area, thereby increasing the contact time between the active ingredients of the formulation (such as growth factors and / or other biological factors) and the skin, and delivering the active ingredients of the formulation over time.The conditioned medium of the present invention in the form of a gel can be used, particularly in hospitals, to treat wounds or damaged skin areas of patients who need treatment for wounds or damaged skin areas.Therefore, the conditioned medium in the form of a gel can be particularly useful for treating hospitalized patients suffering from wounds or damaged skin areas that occurred before or during hospitalization.In a specific example, such wounds are diabetic foot ulcers or pressure ulcers, which develop due to prolonged pressure on certain parts of the body.

[0039] In some embodiments, the conditioned medium is administered in a liquid form, such as a lotion or ointment. As used herein, a liquid formulation refers to a solution or liquid preparation containing the conditioned medium of the present invention in an amount effective for healing wounds or damaged skin areas, and / or preventing the formation and / or recurrence of such wounds or damaged skin areas, and / or treating skin diseases associated with wounds or damaged skin. The conditioned medium of the present invention in liquid form is particularly useful for home administration, i.e., a liquid formulation containing the conditioned medium of the present invention can be administered without the need for a doctor. For example, the conditioned medium in liquid form can be administered by a subject who is at risk of developing a wound or damaged skin area but is not hospitalized or does not need to stay in a hospital. For example, the conditioned medium in liquid form can be directly applied as a "daily skin conditioner" to a subject at risk of developing a diabetic ulcer when they are not receiving treatment at a hospital or wound care center and are at home.

[0040] In a fourth aspect, the present invention relates to a method for producing a conditioned medium, which can then be used in a cosmetic or therapeutic application as described herein, comprising the steps of: (a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle 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; (c) optionally culturing the umbilical cord mesenchymal stem cells in an additional culture medium, wherein the additional cell culture medium optionally comprises a water-soluble antioxidant. wherein the conditioned medium is obtained by harvesting the cell culture medium.

[0041] The conditioned medium may be derived from culturing umbilical cord mesenchymal stem cells in the additional culture medium described above after culturing mesenchymal stem cells in a medium containing DMEM, F12, M171, and FBS. This additional culture medium may be any (additional) basal culture medium suitable for the growth of animal cells, particularly a basal medium suitable for culturing mesenchymal stem cells. In some examples, this additional cell culture medium does not contain growth factors and / or is serum-free, and / or cells are cultured at a concentration of about 1 million cells per ml. In some examples, this additional medium may be serum-free. Non-limiting examples of such serum-free media include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), and keratinocyte medium (KM), KBM (keratinocyte basal medium), EpiLife KM (keratinocyte-EpiLife medium), etc. Alternatively, this additional culture medium may contain serum. As an example of such a serum-containing medium, the medium used may be one that has been described and commonly used for the isolation and culture of mesenchymal stem cell populations from umbilical cord amniotic membranes, such as medium PTT-4, which consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS and was used in U.S. Patent Application No. US2008 / 0248005 and corresponding International Patent Application No. WO2007 / 046775 for the isolation and culture of mesenchymal stem cell populations from umbilical cord amniotic membranes, and was shown to have excellent wound healing properties in U.S. Patent Application No. US2008 / 0248005 and corresponding International Patent Application No. WO2007 / 046775. In some examples, the cell culture medium described herein, such as a medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), can be used to culture umbilical cord mesenchymal stem cells, where the cell culture medium is removed after culturing, for example, after the cells reach full confluency, and replaced with another culture medium / solution, such as a culture medium / solution containing DMEM (which may contain antioxidant components). Generally, during any culture step in the additional medium of the conditioned medium preparation method described herein, the mesenchymal stem cells can be cultured for an appropriate 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 up to 10 or 14 days, if desired. In this regard, it should be noted for clarity that the conditioned medium obtained in the first culture step and the conditioned medium obtained in the second (further) culture step can both be used to induce, stimulate and / or promote healing of wounds or damaged skin areas, and / or to prevent the formation and / or recurrence of wounds or damaged skin, and / or to treat diseases associated with wounds or damaged skin, as described herein.

[0042] The umbilical cord (tissue) mesenchymal stem cells used herein to prepare conditioned medium may be from any compartment of umbilical cord tissue that contains mesenchymal stem cells (or may be derived from any compartment). The mesenchymal stem cell population may be an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, a mesenchymal stem cell population from the amniotic membrane of the umbilical cord (also referred to as umbilical cord lining mesenchymal cells), or even a mixed umbilical cord mesenchymal stem cell population (MC), i.e., a mesenchymal stem cell population containing stem cells from two or more of these compartments. In some examples, the mesenchymal stem cells are from the amniotic membrane of the umbilical cord. In some examples, the umbilical cord mesenchymal stem cells include amniotic membrane mesenchymal stem cells. Mesenchymal stem cells from these compartments and their isolation are known to those skilled in the art and are described, for example, in 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. Mixed mesenchymal stem cell populations from the umbilical cord can be obtained (by tissue explantation), for example, by removing arteries and veins from umbilical cord tissue, dissecting the remaining tissue and Wharton's jelly, and culturing the umbilical cord tissue in a culture medium such as PTT-6.Mixed umbilical cord mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue, including intact umbilical vessels, as tissue explants under conditions such as those described in 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 (culture in serum-supplemented DMEM containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin / streptomycin). In this regard, it should be noted that mesenchymal stem cell populations from the umbilical-placental junction can be isolated as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224.

[0043] In accordance with the above, it should be noted that the conditioned medium is derived from the culture of mesenchymal stem cells from umbilical cord tissue, and these stem cells can be cultured in culture media including DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (171 Medium), and FBS (fetal bovine serum), or can be isolated from their natural environment prior to culture in the culture media described herein. Such an approach is particularly useful for umbilical cord mesenchymal stem cell populations. Such stem cell populations, for example, mesenchymal stem cell populations from Wharton's jelly, can be first isolated as described in Subramanian et al., 2015, PLoS ONE, supra, or International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord", and the isolated mesenchymal stem cell population can then be cultured in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).Placental mesenchymal stem cell populations may also be isolated from the placenta as described, for example, in European Patent Application EP1 288 293; Talwadekar et al, "Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma" 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 then cultured in the culture medium described herein.

[0044] In this regard, it should be noted that the culture medium described herein therefore also allows for the isolation of a mesenchymal stem cell population (also referred to herein as "mesenchymal stem cells") from its natural environment. Thus, the culture medium described herein also allows for the isolation of a mesenchymal stem cell population under conditions that allow cell proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. In some examples, the cell culture medium used to isolate and / or culture umbilical cord mesenchymal stem cells can be the medium used to culture the cells to produce the conditioned medium of the present invention.

[0045] In accordance with the above disclosure, it should be noted that the mesenchymal stem cell populations described herein can be isolated and cultured (i.e., derived) from any umbilical cord tissue, so long as the umbilical cord tissue contains the amniotic membrane (also referred to as the "cord lining"). Thus, as described in the Experimental Section of this application, mesenchymal stem cell populations can be isolated from the entire umbilical cord (or a portion thereof). Therefore, the umbilical cord tissue may contain other tissues / components of the umbilical cord in addition to the amniotic membrane. For example, as shown in Figure 16 of U.S. Patent Application Publication 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 and covers it. Additionally, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-poor blood away from the fetus). These three blood vessels are embedded in Wharton's jelly, a gelatinous substance composed primarily of mucopolysaccharides, for protection and mechanical support. Therefore, umbilical cord tissue, as used herein, can also include this one vein, two arteries, and Wharton's jelly. Using such an entire (intact) portion of the umbilical cord has the advantage that it is not necessary to separate the amniotic membrane from other components of the umbilical cord. This reduces the isolation steps, thus making the method of the present invention simpler, faster, less error-prone, and more economical. Thus, mesenchymal stem cell isolation can begin with tissue explantation, and if larger quantities of mesenchymal stem cells are required, the isolated mesenchymal stem cells can be subsequently subcultured (cultured). Alternatively, mesenchymal umbilical cord lining stem cells can be isolated from the amniotic membrane by first separating the amniotic membrane from other components of the umbilical cord and culturing the amniotic membrane in the culture medium described herein. This culture can also be performed by tissue explantation, followed by optional subculture of the isolated mesenchymal stem cells.

[0046] In this context, the terms "tissue explant" or "tissue explant method" are used in their usual sense in the art to refer to a method in which, once harvested, a tissue (e.g., umbilical cord tissue) or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, allowing stem cells to migrate from the tissue to the surface of the culture dish over time. These primary stem cells can then be further expanded by micropropagation (subculture), as also described herein, and transferred to a new dish. In this context, it should be noted that the initial step of isolating / obtaining a mesenchymal stem cell population from the umbilical cord, such as amniotic membrane or Wharton's jelly mesenchymal stem cells, results in a master cell bank of the isolated mesenchymal stem cells, and subsequent subcultures result in a working cell bank. When the conditioned medium derived from the culture of umbilical cord mesenchymal stem cells of the present invention is used, for example, to promote wound healing, a cell population from the working cell bank is typically used for this purpose. Both the stem cell population from the isolation step (which may constitute a master cell bank) and the stem cell population from the subculture step (which may constitute a working cell bank) may be stored, for example, in a cryopreserved form.

[0047] "DMEM" refers to Dulbecco's Modified Eagle's Medium, developed in 1969 and modified from Basal Medium Eagle (BME) (see Figure 1, which shows the data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from various sources, including ThermoFisher Scientific (catalog number 11965-084), Sigma-Aldrich (catalog number D5546), and 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 DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich DMEM medium, catalog number D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0048] "FI2" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed to cultivate a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 2, which shows the data sheet for Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., the medium from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number N4888), or Lonza, to name just a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0049] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 culture medium (see FIG. 3, which shows the data sheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is a widely used basal medium for supporting the growth of various mammalian cells and is commercially available from various sources, such as ThermoFisher Scientific (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 under catalog number 12-719F (which is DMEM:F12 containing L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0050] "M171" refers to Culture Medium 171, which was developed as a basal medium for the culture or growth of normal human mammary epithelial cells (see Figure 4, which shows the data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely used and is commercially available from suppliers such as 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 M171 medium available from Life Technologies Corporation under catalog number M171500.

[0051] "FBS" refers to fetal bovine serum (also known as "fetal calf serum"), i.e., the blood fraction remaining after natural clotting of blood and subsequent centrifugation (to remove any remaining red blood cells). Fetal bovine serum is the most widely used serum supplement for in vitro eukaryotic cell culture because of its extremely low antibody levels, higher growth factor content, and versatility for a variety of cell culture applications. FBS is preferably obtained from members of the International Serum Industry Association (ISIA), whose primary focus is the safety and safe use of serum and animal-derived products through proper origin traceability, label authenticity, and appropriate standardization and oversight. Suppliers of FBS that are ISIA members include, to name just a few, Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, Inc. In a currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0052] The culture medium described herein may contain a final concentration of approximately 55-65% (v / v) DMEM, approximately 5-15% (v / v) F12, approximately 15-30% (v / v) M171, and approximately 1-8% (v / v) FBS to produce conditioned medium from the culture of umbilical cord mesenchymal stem cells. As used herein, the "% (v / v)" values ​​refer to the volume of each component relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium at a final concentration of approximately 55-65% (v / v), for example, one liter of culture medium will contain approximately 550-650 ml of DMEM. In another embodiment, the culture medium may contain DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v). In a further embodiment, the culture medium may contain 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).

[0053] In addition to the above components, the culture medium may contain supplements advantageous for culturing umbilical cord mesenchymal stem cells, for inducing, stimulating, and / or promoting healing of wounds or damaged skin, and / or for preventing the formation and / or recurrence of wounds or skin damage, and / or for generating a conditioned medium for treating wounds or damaged skin. The culture medium described herein may contain, for example, epidermal growth factor (EGF). When 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 of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0054] The culture medium described herein may also contain insulin. When present, insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0055] 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 contain all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0056] As described herein, umbilical cord tissue can be cultured until an appropriate number of (primary) mesenchymal stem cells, such as umbilical cord lining stem cells, Wharton's jelly, or placental stem cells, outgrow the tissue. Typically, umbilical cord tissue is cultured until cell outgrowth of the mesenchymal stem cells from each tissue reaches approximately 70% to 80% confluency. Note that, as used herein, the terms "confluency" and "confluence" are used in their usual sense in the cell culture art, as an estimate / indicator of the number of adherent cells in a culture dish or flask, and refer to the percentage of the surface covered by cells. For example, 50% confluence means that approximately half of the surface is covered, with additional room for cell growth. 100% confluence means that the surface is completely covered with cells, with no room left for cells to grow as a monolayer.

[0057] Once an appropriate number of primary cells (mesenchymal stem cells) are obtained from each tissue by tissue explantation, the mesenchymal stem cells are removed from the culture vessel used for culturing. This allows for the creation of a master cell bank containing isolated (primary) mesenchymal stem cells, for example, from the umbilical cord or placenta. Generally, because such mesenchymal stem cells are adherent cells, the cells are harvested using standard enzyme treatments. For example, enzyme treatments can include trypsin treatment as described in International Patent Application No. 2006 / 0078993, International Patent Application No. WO2006 / 019357, or International Patent Application No. WO2007 / 046775. That is, growing cells can be harvested by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further expansion. When the harvested mesenchymal stem cells are used, for example, to create a master cell bank, they can also be cryopreserved as described below and stored for further use.

[0058] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculturing. Subculturing or culturing (hereafter, both terms are used interchangeably) may also be performed when using a population of mesenchymal stem cells from the umbilical cord that have previously been isolated from their natural environment. Subculturing can also be initiated from frozen primary cells, i.e., from a master cell bank. For subculturing, an appropriate amount of cells is seeded into a culture vessel, such as a cell culture plate. For this purpose, the mesenchymal cells are cultured in an appropriate medium for subculturing (most conveniently, the culture medium described herein), at a concentration of, for example, about 0.5 × 10 6 pieces / ml ~ approx. 5.0×10 6 For example, the cells can be suspended at a cell concentration of about 1.0 x 10 cells / ml for subculture. 6The cells are suspended at a concentration of 10 ... An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT), 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 Heathcare. Culturing mesenchymal stem cell populations in an automated system such as the Quantum® Cell Expansion System is particularly beneficial when working cell banks are to be generated under GMP conditions and large numbers of cells are desired.

[0059] The subculture of the mesenchymal stem cells of the present invention can be carried out in the culture medium described herein.Therefore, this culture medium can be used for both the isolation of mesenchymal stem cell populations, for example, from the amniotic membrane of the umbilical cord or Wharton's jelly, and the subsequent culture of the isolated primary cells.In addition, in culture, mesenchymal stem cells can be cultured until they proliferate to an appropriate amount.In some examples, mesenchymal stem cells are cultured until they reach about 70% confluency, about 80% confluency, about 90% confluency, about 95% confluency, about 96% confluency, or 100% confluency.

[0060] Isolation / culturing of mesenchymal stem cell populations can be performed under standard mammalian cell culture conditions. Generally, methods for isolating mesenchymal stem cell populations are performed under conditions (temperature, atmosphere) typically used for culturing 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 standard atmosphere of 5% CO2, respectively. In this regard, it should be 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 exemplary embodiments, umbilical cord mesenchymal stem cells are red deer or human.

[0061] Once a desired / appropriate number of mesenchymal stem cells have been obtained from culture or subculture, they are harvested by removing them from the culture vessel used for culturing. Harvesting of mesenchymal stem cells is typically performed by enzymatic treatment, such as trypsinization of the cells. The isolated mesenchymal stem cells are then recovered and used directly or preserved for further use. Preservation is typically achieved by cryopreservation. The term "cryopreservation" is used herein in its ordinary sense to refer to the process of preserving mesenchymal stem cells by cooling them to subzero temperatures, such as -80°C (typically) or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be performed as known to those skilled in the art and can include the use of cryoprotectants, such as dimethyl sulfoxide (DMSO) or glycerol, which retard the formation of ice crystals within the umbilical cord cells.

[0062] The isolated populations of mesenchymal stem cells obtained by the culture and / or isolation methods described herein are highly defined and homogeneous. Such highly homogeneous populations of mesenchymal stem cells derived from tissues such as the amniotic membrane of the umbilical cord or Wharton's jelly are reported in WO 2018 / 067071 A1 and WO 2019 / 199234 A1. For example, as disclosed and shown in the experimental section of WO 2019 / 199234 A1, 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; that is, 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. Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord meets the criteria for mesenchymal stem cells to be used in cell therapy (see the experimental section of WO 2019 / 199234 A1 and, e.g., Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," supra). In this regard, it should be noted that this mesenchymal stem cell population can be obtained not only by the isolation method described in WO 2019 / 199234 A1, but also by different methods, such as cell sorting, if desired.

[0063] In a further aspect, the present invention is directed to a method of inducing, stimulating, and / or promoting wound healing and / or preventing the formation and / or recurrence of wounds and / or treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, comprising treating the skin of a subject surrounding a wound or damaged skin area or the intact skin of a subject at risk of developing a wound or damaged skin area with conditioned medium derived from culturing umbilical cord mesenchymal stem cells in a culture medium as described herein; wherein the culture medium is mixed as follows to obtain a final volume of 500 ml of culture medium: i. 250 ml of DMEM ii. 118ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) (to a final concentration of 2.5% (v / v)).

[0064] As explained 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, if you use this method to make culture medium, the final concentrations (v / v) for a total volume of 500 ml will be as follows: DMEM: 250ml+59ml=309ml, equivalent to 309 / 500=61.8%(v / v); M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v); F12: 59ml, equivalent to 59 / 500=11.8% (v / v).

[0065] The culture media described herein may further comprise the addition of the following components: v. 1 ml of EGF stock solution (5 μg / ml) (resulting in a final EGF concentration of 10 ng / ml), and vi. 0.175 ml of insulin stock solution (14.28 mg / ml) (to give a final insulin concentration of 5 μg / ml).

[0066] It should be noted that the above volumes of components i-vi, when mixed, result in a final culture medium volume of 499.675 ml. If no further components are added to the culture medium, the remaining 0.325 ml (added to a volume of 500 ml) can be, for example, any of components i-iv, i.e., DMEM, M171, DMEM / F12, or FBS. Alternatively, it is possible to adjust the concentration of the EGF or insulin stock solution so that the total volume of the culture medium is 500 ml. Furthermore, it should be understood that components i-iv do not necessarily have to be added in the order listed; they can be mixed in any order to form the culture medium described herein. That is, for example, M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to reach the final concentrations described herein, i.e., a final DMEM concentration of about 55-65% (v / v), a final F12 concentration of about 5-15% (v / v), a final M171 concentration of about 15-30% (v / v), and a final FBS concentration of about 1-8% (v / v).

[0067] In other embodiments, the method further comprises adding 0.325 ml of one or more of the following supplements to the DMEM: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine sodium salt (T3), thereby bringing the total volume of the culture medium to 500 ml. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows: about 0.05-0.1 μg / ml adenine, for example about 0.025 μg / ml adenine; hydrocortisone at approximately 1–10 μg / ml; About 0.5-5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0068] In some embodiments, the conditioned medium of the present invention is produced by culturing umbilical cord mesenchymal stem cells in a cell culture medium described herein, such as a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), and then removing the umbilical cord mesenchymal stem cells from the culture medium. In this case, the conditioned medium is obtained by recovering the cell culture medium. Furthermore, in some instances, the culture medium in which the stem cells were cultured can be replaced with a second cell culture medium, and the cells can be cultured in the second medium; the second medium can be the same as or different from the first medium. In some instances, the second cell culture medium can be growth factor-free and / or serum-free. Alternatively, previously cultured umbilical cord mesenchymal stem cells (e.g., stored cultured stem cells) can be further cultured in growth factor-free and / or serum-free medium. In either case, the resulting medium is referred to as conditioned medium. The resulting conditioned medium can be collected and further processed to prepare a composition comprising the conditioned medium of the present invention.

[0069] The conditioned medium of the present invention or a composition containing the conditioned medium of the present invention can be diluted with any suitable diluent / carrier medium, as long as the carrier medium does not affect the function and safety of the conditioned medium when topically administered to the skin of a subject. For example, the conditioned medium can be diluted with PBS, water, a basal medium, or a cream formulation. As used herein, a basal medium refers to a mixture containing sugars, amino acids, water, and other components necessary for cell survival, and includes commercially available prepared media such as Dulbecco's Modified Eagle's Medium (DMEM), endothelial cell differentiation medium (EDM), minimal essential medium (MEM), basal medium Eagle's (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow minimal essential medium (G-MEM), and Iscove's Modified Dulbecco's Medium. Furthermore, the conditioned medium used can be any basal culture medium suitable for growing animal cells, non-limiting examples of which include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), and Keratinocyte Medium (KM), KBM (Keratinocyte Basal Medium), EpiLife KM (Keratinocyte-EpiLife Medium), etc. In some examples, the conditioned medium comprises a cell culture medium described herein, such as DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and a culture medium containing FBS (fetal bovine serum).

[0070] In another aspect, 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 compositions described herein are pharmaceutical or cosmetic compositions 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, and epidermolysis bullosa. The compositions can be prepared as any formulation conventionally produced in the art, and typically comprise the conditioned medium of the present invention and a suitable cosmetically or pharmaceutically acceptable carrier / recipient.

[0071] In some examples, the present invention provides compositions comprising conditioned medium derived from the culture of umbilical cord mesenchymal stem cells formulated for skin care or skin treatment, examples of such formulations are described in more detail below.

[0072] In an example of such a composition, the composition may further comprise an anti-inflammatory compound. Any suitable anti-inflammatory compound can be used in such a composition. The anti-inflammatory compound can be naturally derived or a chemically synthesized small molecule drug. In specific examples, the anti-inflammatory compound can be curcumin, sulfacetamide, a nonsteroidal anti-inflammatory compound such as capsaicin or diclofenac, niacinamide (vitamin B3), sea buckthorn oil, calendula, chamomile (plant) extract, or Centella asiatica extract, to name just a few. The anti-inflammatory effects of curcumin are discussed, for example, in 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 marigold flowers, which are native to the Mediterranean region. This sweet liquid contains antiseptic flavonoids and has anti-inflammatory properties that aid in wound healing and eczema relief. Chamomile (botanical) extract is the powdered essence of German chamomile (also known as Hungarian chamomile or blue chamomile), scientifically known as Matricaria recutita, and is used in traditional wound healing remedies; similarly, chamomile botanical extract is the powdered essence of German chamomile (also known as Hungarian chamomile or blue chamomile), scientifically known as Matricaria recutita. In addition to its use in traditional wound healing remedies, chamomile extract is also frequently included in skin care 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.

[0073] The composition may further comprise a humectant. The term humectant is used herein in its usual sense in the field of skin care, and refers to a component that attracts moisture to the skin. Any suitable humectant can be used in each composition described herein. Specific examples of humectants include, but are not limited to, glycerin, 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 the compositions described herein, the humectant comprises glycerin, propylene glycol, or both glycerin and propylene glycol (see also below).

[0074] A composition comprising conditioned medium, an anti-inflammatory compound, and a humectant may comprise conditioned medium derived from the culture of umbilical cord mesenchymal stem cells in an amount of about 5-40% (v / v) of the total volume of the composition, a humectant in an amount of about 10-30% (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 0.5-5.0% (w / w) of the total weight of the composition. In another example, such a composition may comprise conditioned medium derived from the culture of umbilical cord mesenchymal stem cells in an amount of about 10-30% (v / v) of the total volume of the composition, a humectant in an amount of about 15-25% (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 0.75-3.0% (w / w) of the total weight of the composition.

[0075] Alternatively, a composition comprising conditioned medium, an anti-inflammatory compound, and a humectant may comprise conditioned medium derived from the culture of umbilical cord mesenchymal stem cells in an amount of about 5-40% (w / w) of the total weight of the composition, a humectant in an amount of about 10-30% (w / w) of the total weight of the composition, and an anti-inflammatory compound in an amount of about 0.5-5.0% (w / w) of the total weight of the composition. For clarity, it should be noted that, in this specification, when specifying the content of a certain component in a composition, it is preferable to use volume percentage (% v / v) if the component is generally in liquid form at room temperature, whereas it is preferable to use weight percentage (% w / w) if the component is generally in solid form at room temperature.

[0076] In a specific example of such a composition, the composition comprises: - conditioned medium derived from the culture of umbilical cord mesenchymal stem cells; - glycerin, propylene glycol, or both glycerin and propylene glycol; and - Curcumin.

[0077] The compositions described herein may further comprise a solvent / carrier. Any cosmetically acceptable solvent / carrier may be used. In a specific example, the solvent / carrier may be water or an aqueous buffer solution.

[0078] The compositions described herein may further comprise a preservative. The term "preservative" as used herein is used in its conventional sense to refer to a natural or synthetic ingredient that protects the integrity of the composition (formulation). Preservatives can prevent the growth of bacteria and other microorganisms. Specific 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., DMDM ​​hydantoin, diazolidinyl urea, or imidazolidinyl urea), anisic acid, salicylic acid, sodium salicylate, chlorphenism, and stearalkonium chloride, to name just a few.

[0079] In specific examples, such compositions described herein may include: - 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) of the total volume of the composition; glycerin 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), most preferably 20% (v / v) of the total volume 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), 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), most preferably 1.0% (w / w) of the total weight of the composition.

[0080] In other embodiments, such compositions described herein may include: 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) of 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)% of the total weight of the composition; glycerin 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 about 0.8 to about 1.5% (w / w), most preferably about 0.9 to 1.0% (w / w) of the total weight of the composition.

[0081] The compositions described herein may further comprise one or more of a gelling agent, a buffering agent, and a softening agent. Any suitable gelling agent can be used in such compositions. Specific examples of gelling agents include, but are not limited to, gelling agents selected from the group consisting of poly(acrylic acid), pectin, starch, alginates such as sodium alginate, gelatin, cellulose derivatives (e.g., hypromellose or methylcellulose), and polyvinyl alcohol clay.

[0082] The term "buffering agent" is used herein in its usual sense to stabilize or adjust the pH of a cosmetic product. The term "emollient" is also used in its usual sense in cosmetic formulations to refer to a compound that can soften the skin, and is incorporated into cosmetics to improve skin feel. Similarly, any suitable buffering agent can be used in the compositions described herein. Specific examples of buffering agents 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 just a few. Similarly, any suitable emollient may be used in the compositions described herein, and specific examples of suitable emollients are olive oil PEG-7 esters, triglycerides, lanolin, polyhydric alcohols, and esters of fatty acids (e.g., isopropyl myristate, dioctyl sebacate, and dioctyl maleate).

[0083] Further specific examples of compositions of the present invention are shown in the table below.

[0084] Table 1: Representative compositions of the present invention formulated as lotions TIFF2026500184000001.tif61128

[0085] It should be noted that since curcumin is a solid (powder), the amount of curcumin is expressed as a weight percent relative to the total weight of the formulation, while the amounts of other ingredients are expressed as volume percent relative to the total volume of the formulation because these ingredients are in a liquid state at room temperature. Alternatively, if the anti-inflammatory agent is, for example, calendula (oil), its amount in the composition is usually defined as a volume percent relative to the total volume of the formulation.

[0086] The lotions described in Table 1 can be advantageously used as daily skin conditioners for home use by patients, for example, to prevent wound formation and / or recurrence. To that end, patients, such as diabetics, can apply the lotions topically to the skin of their feet and ankles to improve the condition of their diabetic skin, thereby avoiding the formation of diabetic wounds, such as foot ulcers.

[0087] Table 2: Representative compositions of the present invention formulated as gels TIFF2026500184000002.tif94128

[0088] The gels described in Table 2 can be advantageously used, for example, to induce, stimulate, and / or promote healing of wounds or damaged skin areas, the method comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to a patient's skin surrounding a wound, such as a pressure ulcer or diabetic foot ulcer.

[0089] The compositions of the present invention may also contain excipients and adjuvants conventional in the cosmetic, pharmaceutical, or dermatological fields, such as fragrances, fillers, disinfectants, odor absorbers, dyes, or colorants. Generally, the compositions can be used topically or transdermally by applying them directly to the skin. Preferably, the compositions containing the conditioned medium of the present invention are cosmetic compositions. In some embodiments, the compositions are adapted for topical application. Compositions adapted 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 formulation that can be absorbed into the skin in a targeted manner, in particular, such that the active ingredients of the conditioned medium of the present invention or a composition comprising the conditioned medium of the present invention can penetrate the epidermal and dermal layers but are not absorbed into the systemic circulation. In some instances, the conditioned medium of the present invention or a composition comprising the conditioned medium of the present invention passes through the surface of the skin by transepidermal or transdermal diffusion.

[0090] In a further aspect, the present invention provides use of a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells as a culture medium for inducing, stimulating, and / or promoting healing of wounds or damaged skin areas, and / or preventing the formation and / or recurrence of wounds or damaged skin areas, and / or treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, and epidermolysis bullosa. In yet another aspect, the present invention provides use of a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells for the manufacture of a medicament for inducing, stimulating, and / or promoting healing of wounds or damaged skin areas, and / or preventing the formation and / or recurrence of wounds or damaged skin areas, and / or treating rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, and epidermolysis bullosa. Accordingly, a method of treating a subject with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells is described. The method comprises administering a (therapeutically) effective amount of the conditioned medium of the present invention to the skin of the subject. A treatment course may include repeated administration of such an effective amount over any desired period of time. As described herein, a composition / formulation containing conditioned medium can be applied, for example, once daily or two or three times per week for any desired period of time. See Example 3, in which a formulation containing conditioned medium described herein was applied two to three times per week for up to 16 weeks to achieve closure of a chronic diabetic foot ulcer. Those skilled in the art will recognize that the duration of application of the formulations of the present invention will vary depending on the patient's specific circumstances (e.g., age, health status, wound size, etc.), but treatment can be administered 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 formulation containing conditioned medium (e.g., in an amount of about 20% (w / w) based on the total weight of the formulation) that can be applied to a subject's skin per treatment to cover the desired skin area.

[0091] 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 a human subject, comprising topically treating the subject's skin surrounding a wound or damaged skin area, 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.

[0092] The present invention is further illustrated by the following non-limiting experimental examples.

[0093] The sequences used herein are shown in Table 3 below.

[0094] Table 3: Amino acid sequences described herein TIFF2026500184000003.tif150150TIFF2026500184000004.tif210150TIFF2026500184000005.tif221150 [Example]

[0095] Experimental example Example 1: Isolation and culture of umbilical cord mesenchymal stem cells (CLMSCs) Preparation of culture medium a. To prepare 500 ml of PTT-6 (culture / growth medium), add the following in the order listed: i. DMEM, 250ml ii. M171 118ml iii. 118ml of DMEM F12 iv. FBS 12.5ml (final concentration 2.5%) v. EGF 1ml (final concentration 10ng / ml) vi. Insulin 0.175ml (final concentration 5μg / ml)

[0096] The combined volumes of components i through vi above result in a final culture medium volume of 499.675 ml. If no additional components are added to this culture medium, the remaining 0.325 ml (to bring the total volume to 500 ml) can be, for example, any of components i through iv, i.e., DMEM, M171, DMEM / F12, or FBS. Alternatively, it is possible to adjust the concentration of an EGF or insulin stock solution to bring the total volume to 500 ml. Alternatively, a stock solution of an antibiotic, such as penicillin-streptomycin-amphotericin, can be added to bring the final volume to 500 ml. It is also possible to add 0.325 ml of one or more of the following supplements to the culture medium: adenine, hydrocortisone, or 3,3',5-triiodo-F thyronine sodium salt (T3), thereby bringing the total volume to 500 ml.

[0097] vii. Label the bottle "PTT-6" and write the date the medium was prepared, the operator's initials, and "expires on" followed by the expiration date, which should be the earliest expiration date of any of the ingredients or one month from the date of preparation, whichever comes first.

[0098] b. To prepare rinse medium (Hank's Buffered Salt Solution (HBSS) without calcium or magnesium and containing 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube "Rinse Medium" and include your initials and the date the medium was prepared.

[0099] c. All media / culture media are tested for sterility using Bactec Fytic / 10 (Dickinson & Company) and Bactec Pluc + Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared media / culture media is poured into each bottle.

[0100] Harvesting and isolation of red deer (RD) umbilical cord tissue Red deer (RD) umbilical cords are collected from farms in New Zealand where the animals are raised for horn velvet harvesting according to specific instructions, and RD-CLMSCs are isolated from the cords according to the protocol described in International Patent Application WO 2006 / 019357 A1.

[0101] Briefly, red deer umbilical cords are washed 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, and 50 μg / ml gentamicin; all reagents purchased from Invitrogen) before being transported to the laboratory. Once in the laboratory, stem cell extraction is performed under sterile conditions in a laminar flow hood. The cords are first transferred to a sterile stainless steel tray. Any blood remaining in the umbilical vessels is removed by multiple syringe washes with warm phosphate-buffered saline (PBS) supplemented with 5 IU / ml heparin (Sigma). The final wash is performed with pure PBS without heparin. The umbilical cord tissue specimens were then cut into 2 cm lengths and transferred to 10 cm diameter cell culture dishes, further washed and disinfected with 70% ethanol, followed by multiple washes with PBS containing an antibiotic mixture (50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml fungizone, 50 μg / ml gentamicin; all purchased from Invitrogen) until the solution became clear.

[0102] Isolation and culture of RD-CLMSCs First, umbilical cord tissue is dissected to separate the amnion from Wharton's jelly (i.e., the umbilical cord matrix) and other internal components. The isolated amnion is then cut into small pieces (0.5 cm x 0.5 cm) for cell isolation. For mesenchymal stem cell isolation, explantation is performed by placing the small pieces of amnion on tissue culture dishes under different cell culture conditions.

[0103] For mesenchymal cell isolation / culture, explants were submerged 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 trypsinized (0.05% trypsin / 0.02% EDTA) for further expansion or cryopreservation. H-CLMSCs (#CLMC43) were also obtained from the CRC tissue bank.

[0104] Example 2: Conditioned medium derived from red deer umbilical cord lining mesenchymal stem cells (RD-CLMSC) Cryovials containing RD-CLMSCs were removed from storage and rapidly thawed in a water bath at 37°C. FSFs were provided to CellResearch Corporation (CRC, Singapore) as a gift from the Stem Cell and Wound Healing Research Group, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore.

[0105] PTT-6 medium (Cell Research Corporation, Singapore) was used to culture RD-CLMSCs at 37°C and 5% CO2. This cell culture medium was changed every 2–3 days. At 80–100% confluency, the cell culture medium was removed, and the cells were optionally rinsed with PBS before being cultured in DMEM basal medium, which optionally contained water-soluble antioxidants but did not contain growth factors or serum. The culture dish was incubated for an additional 48 hours, and the spent medium was collected in a centrifuge tube and centrifuged at 1800 rpm for 10 minutes. The supernatant was collected in a labeled tube as conditioned medium (CM). The conditioned medium was stored at -80°C until use.

[0106] Example 3: Evaluation of conditioned medium in the healing of persistent chronic diabetic foot ulcer (DFU) wounds Experimental setup The conditioned medium was derived from conditioned medium of red deer umbilical cord lining stem cells as described above in Examples 1 and 2, and was formulated as a solution or gel containing approximately 20% (w / w) conditioned medium as shown in Table 1 above.

[0107] Patients with chronic diabetic foot ulcers (>3 months duration) were enrolled in this case series through the hospital's DFU registry. In all cases, the DFU persisted despite standard care, including regular debridement by a podiatrist, topical dressings, and offloading shoe wear. Patients with active infection were excluded from this case series. Conditioned medium was administered to clean, debridement-treated intact skin surrounding the wound (ISSW) via a solution soaked in gauze or via a gel formulation (containing approximately 20% (w / w) conditioned medium) described in Table 1 above, which was applied directly to the ISSW. After 5 minutes, the gauze or excess gel was removed, and the wound was dressed according to usual care. Treatments were performed twice or three times weekly.

[0108] Twenty-one 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 level was 7.9% (SD=1.99). The treated DFUs were located in various locations on the feet, but the majority were plantar ulcers. No serious systemic complications, such as allergic reactions or sepsis, were observed during the treatment period.

[0109] method The patient's wounds were treated as follows: i) Debride and cleanse the wound as per usual procedures; ii) soaking gauze with 5 ml of conditioned medium preparation; iii) applying gauze to the intact skin surrounding the wound (ISSW) for 5 minutes or applying a gel to the ISSW; iv) do not administer conditioned medium directly to open skin or wounds; v) Remove gauze or excess gel; and vi) Use of prescribed topical dressings is envisaged according to previous treatment.

[0110] result At the time of results collection, 12 of the 21 patients had complete closure of their DFUs, but ulcer closure appeared to be faster in patients treated with the gel formulation. Of the remaining patients, 8 (5 of whom are still undergoing treatment) experienced a significant reduction in wound size. One patient showed no improvement.

[0111] The reduction in wound size in a subgroup of 10 patients treated with conditioned medium-impregnated gauze is shown in Figure 6, and representative photographs of patients with healed wounds are shown in Figures 7–10. This patient group included eight male and two female patients. The mean age of these patients was 54.7 years (SD = 9.4), and the mean HbA1c was 8.2% (SD = 2.5). Eight patients (80%) had at least one palpable distal foot pulse. During the treatment period, six patients (60%) experienced wound healing, three patients (30%) showed a reduction in wound size, and one patient (10%) had a stable wound size. Of the healed patients, those receiving conditioned medium twice weekly (n = 2) healed in 16 weeks, while those receiving conditioned medium three times weekly (n = 4) healed in 4–7 weeks. Under these conditions, the patients demonstrated significant reduction in wound size and wound closure. Notably, as shown representatively in Figures 7-10, administration of conditioned medium to the intact skin (ISSW) surrounding the wound promoted healing of chronic persistent wounds in DFUs, thereby regenerating the skin and restoring healthy skin. Patients experienced no adverse reactions, and no systemic or local safety concerns were observed following treatment with conditioned medium.

[0112] In this context, we note the results of a pivotal, single-blind, phase 3 clinical trial of DFUs treated with Dermagraft, in which stratification was based on ulcer size (1 cm2 More than 2cm 2 Less than or equal to 2cm 2 super 20cm 2 The main inclusion criteria were as follows: chronic DFU, wound duration >6 weeks, ulcer size 1-20 cm 2 , with tolerated dermal spread (no exposure to muscle, tendon, bone, or joint capsule). Dermagraft was applied on day 0, followed by up to seven additional applications at 1-week intervals in conjunction with standard care; 130 patients participated in the study (n=130). Results of the study were as follows: At 12 weeks, the incidence of complete wound closure was 30.0% (39 of 130 patients) compared with 18.3% (21 of 115 patients) in the control group (P=0.023). Although the results of this study, which included 21 patients but did not have a control group, cannot be directly compared to the results of this clinical trial using Dermagraft, the wound closure rate was approximately 57% (12 of 21 patients demonstrated complete closure of their DFUs, see above), suggesting that treatment of chronic DFUs with the conditioned medium described herein should be at least as effective, if not more effective, than treatment with Dermagraft, a tissue-engineered living human dermal substitute approved by the U.S. FDA for the treatment of DFUs.

[0113] Thus, the results of the present application demonstrate that administration of conditioned medium from mesenchymal stem cells of umbilical cord tissue, such as the umbilical cord lining, to the skin surrounding a wound, such as a chronic diabetic foot ulcer, results in efficient wound closure, thereby providing a new, inexpensive, and easy-to-apply approach to treating such difficult-to-treat wounds.

[0114] It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0115] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0116] The invention illustratively described herein can suitably be practiced in the absence of one or more elements, or one or more limitations, not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be interpreted expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description, rather than terms of limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described, or portions thereof, recognizing that various modifications are possible within the scope of the claimed invention. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it will be understood that those skilled in the art can make modifications and variations of the invention disclosed and embodied therein, and that such modifications and variations are considered to be within the scope of the present invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings included in the generic disclosure also constitutes part of the invention. This includes the generic description of an invention with a proviso or negative limitation excluding any item from the genus, whether or not the excluded item is specifically described herein. Furthermore, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member of the Markush group or any subgroup thereof. Further aspects of the invention will be apparent from the appended claims.

Claims

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

2. A method for preventing the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing such wounds or damaged skin areas, comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the subject's skin.

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

4. 10. The method of claim 1, wherein the method does not include administering the conditioned medium onto the wound or the damaged skin area.

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

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

7. The method according to any one of 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 wound or the area of ​​damaged skin is a chronic wound or chronically damaged skin.

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

10. 10. The method of any one of claims 1 to 9, wherein the wound or damaged skin area is selected from the group consisting of a diabetic foot ulcer (DFU), a pressure ulcer, a wound due to chronic limb ischemia, a wound due to chronic limb-threatening limb ischemia, a venous leg ulcer, a surgical wound, a puncture wound, a burn, and a bite wound.

11. 10. The method of claim 1, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, a culture of a mesenchymal stem cell population from Wharton's jelly (WJ) of the umbilical cord, a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed mesenchymal stem cell population (MC) from the umbilical cord.

12. 10. The method of claim 1, 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 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).

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

14. 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. 15. The method of any one of claims 12 to 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 culture medium (i) Epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml; or (ii) the culture medium contains EGF at a final concentration of 10 ng / ml; or (iii) the culture medium contains insulin at a final concentration of 1 μg / ml to 10 μg / ml; or (iv) the culture medium contains insulin at a final concentration of 5 μg / ml; or (v) 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) the culture medium comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), and optionally the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml, hydrocortisone at a final concentration of 0.1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml; 16. The method according to any one of claims 12 to 15.

17. The method according to any one of claims 12 to 16, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

18. The following steps: (a) culturing umbilical cord mesenchymal stem cells in a culture 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, wherein the additional culture medium optionally comprises a water-soluble antioxidant. Including, The conditioned medium is obtained by collecting the cell culture medium. Methods for generating conditioned medium.

19. 19. The method of claim 18, wherein the additional cell culture medium does not contain growth factors and / or is serum-free medium, and / or the cells are cultured at a concentration of about 1 million cells per ml.

20. 20. The method of 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 further culture medium is a basal medium suitable for culturing mesenchymal stem cells, preferably a serum-free medium.

22. 22. The method of any one of claims 18 to 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. 23. The method of any one of claims 18 to 22, wherein the conditioned medium is diluted with a carrier medium.

24. The method of any one of claims 1 to 17, wherein the conditioned medium is obtained or obtainable by a method of any one of claims 18 to 23.

25. A conditioned medium derived from umbilical cord mesenchymal stem cells obtained or obtainable by the method according to any one of claims 18 to 23.

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

27. 27. The composition of claim 26, further comprising an anti-inflammatory compound.

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

29. 29. The composition of claim 27 or 28, further comprising a humectant.

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

32. 32. The composition of any one of claims 26 to 31, comprising a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells in an amount of about 5 to 40% (v / v) of the total volume of the composition, a humectant 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. 33. The composition of claim 32, comprising a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells in an amount of about 10-30% (v / v) of the total volume of the composition, a humectant in an amount of about 15-25% (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 0.75-3.0% (w / w) of the total weight of the composition.

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

35. below: - conditioned medium derived from the culture of umbilical cord mesenchymal stem cells; - glycerin, propylene glycol, or both glycerin and propylene glycol; and - Curcumin 35. The composition of any one of claims 26 to 34, comprising:

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

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

38. 38. The composition of any one of claims 26 to 37, further comprising a preservative.

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

40. below: - 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) of the total volume of the composition; glycerin 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 volume of the composition; - conditioned medium in an amount of about 10 to about 35% (v / v), preferably about 15 to about 25% (v / v), most preferably 20% (v / v) of the total volume of said 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), most preferably 2% (v / v) of the total volume 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) of the total weight of the composition; 40. The composition of any one of claims 36 to 39, comprising:

41. below: - 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) of the total weight of the composition; - about umbilical cord volume of conditioned medium in an amount of about 5 to about 40% (w / w), preferably about 10 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; - glycerin and / or propylene glycol in an amount of about 10 to about 30% (w / w) of the total weight of the composition, preferably about 10 to about 30% (w / w) or about 12.5 to about 25% (w / w), most preferably about 17.5 to about 20.0% (w / w) of the total weight of the composition; and curcumin in an amount of about 0.5 to about 5.0% (w / w) of the total weight of the composition, preferably about 0.75 to about 2.5% (w / w) or about 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; 40. The composition of any one of claims 36 to 39, comprising:

42. 42. The composition of any one of claims 36 to 41, further comprising one or more of a gelling agent, a buffering agent, and an emollient.

43. 43. The composition of claim 42, wherein the gelling agent is selected from the group consisting of poly(acrylic acid), pectin, starch, alginates such as sodium alginate, gelatin, cellulose derivatives, and polyvinyl alcohol clay.

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

45. 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. 46. ​​The composition of any one of claims 42-45, wherein the emollient is selected from the group consisting of olive oil PEG-7 esters, triglycerides, lanolin, polyhydric alcohols, and esters of fatty acids (e.g., isopropyl myristate, dioctyl sebacate, and dioctyl maleate).

47. Use of conditioned medium derived from culturing umbilical cord mesenchymal stem cells for the manufacture of a pharmaceutical composition for inducing, stimulating, and / or promoting healing of a wound or damaged skin area, comprising topically administering conditioned medium derived from culturing umbilical cord mesenchymal stem cells to the skin of a subject surrounding the wound or damaged skin area.

48. Use of conditioned medium derived from culturing umbilical cord mesenchymal stem cells for the manufacture of a pharmaceutical composition for preventing the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing wounds or damaged skin areas, the method comprising topically administering conditioned medium derived from culturing umbilical cord mesenchymal stem cells to the skin of the subject.

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

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

51. 48. The use of claim 47, wherein the method does not include administering the conditioned medium onto the wound or the damaged skin area.

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

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

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

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

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

57. 57. The use of any one of claims 47 to 56, wherein the wound or damaged skin area is selected from the group consisting of a diabetic foot ulcer (DFU), a pressure ulcer, a wound due to chronic limb ischemia, a wound due to global severe chronic limb ischemia, a venous leg ulcer, a surgical wound, a puncture wound, a burn, and a bite wound.

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

59. 59. The use according to any one of claims 47 to 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 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).

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

61. 61. The use of 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. 62. The use according to any one of claims 59 to 61, 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 culture medium (i) Epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml; or (ii) the culture medium contains EGF at a final concentration of 10 ng / ml; or (iii) the culture medium contains insulin at a final concentration of 1 μg / ml to 10 μg / ml; or (iv) the culture medium contains insulin at a final concentration of 5 μg / ml; or (v) 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) the culture medium comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), and optionally the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml, hydrocortisone at a final concentration of 0.1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml; 63. Use according to any one of claims 59 to 62.

64. 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 for: i) to induce, stimulate, and / or promote healing of a wound or damaged skin area, said use comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject surrounding said wound or damaged skin area; or ii) to prevent the formation and / or recurrence of wounds or damaged skin areas in a subject at risk of developing such wounds or damaged skin areas, the method comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of the subject; or iii) For treating skin damage caused by rosacea, psoriasis, eczema, dermatitis, topical steroid withdrawal syndrome, epidermolysis bullosa, or fragile skin, the method comprising topically administering conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to the skin of a subject.

66. 66. The use of claim 65 i), wherein the method does not include administering the conditioned medium onto the wound or the damaged skin area.

67. 67. The use of claim 65 or 66, wherein the skin to be treated is intact skin.

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

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

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

71. 71. The use of any one of claims 65 to 70, wherein the wound or damaged skin area is selected from the group consisting of a diabetic foot ulcer (DFU), a pressure ulcer, a wound due to chronic limb ischemia, a wound due to global severe chronic limb ischemia, a venous leg ulcer, a surgical wound, a puncture wound, a burn, and a bite wound.

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