Application of conditioned medium derived from culture of umbilical cord mesenchymal stem cells to induction, stimulation and promotion of hair growth and regeneration

Abstract

The present invention relates to a method of inducing, stimulating and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating the hair of a subject with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells. The invention also relates to a method of alleviating and / or reducing hair loss and / or hair sparseness, wherein the method comprises treating the hair of a subject with a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells. The present invention also relates to a method of producing a conditioned medium comprising a) culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco Modified Eagle Medium), F12 (Hamm F12 Medium), M171 (Medium 171) and FBS (fetal calf serum); b) removing the umbilical cord mesenchymal stem cells from the culture medium; wherein the conditioned medium is obtained by collecting the cell culture medium. The invention also relates to conditioned media obtained or obtainable by said production method, compositions thereof and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to International Patent Application No. PCT / SG2022 / 050834 filed on November 17, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention relates to a method for inducing, stimulating, and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. Similarly, the present invention relates to a method for alleviating and / or reducing hair loss and / or hair thinning, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. These methods can be non-therapeutic and cosmetic and can be used to treat hair loss and / or hair thinning in both men and women.

[0004] The present invention also relates to conditioned medium, its use, compositions comprising the same, and methods for producing the same, wherein the conditioned medium is derived from the culture of umbilical cord mesenchymal stem cells. In particular, a method for producing a conditioned medium derived from umbilical cord mesenchymal stem cells, the method comprising 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 (culture medium 171), and FBS (fetal bovine serum); b) removing the umbilical cord mesenchymal stem cells from the culture medium; wherein the conditioned medium is obtained by collecting the cell culture medium. After step b) of the method for producing the conditioned medium, the method may include one or more additional steps of culturing the umbilical cord mesenchymal stem cells in another culture medium, optionally wherein the second (additional) cell culture medium comprises a water-soluble antioxidant. The second cell culture medium may not contain growth factors and / or serum. In addition, in the second cell culture medium, cells may be cultured at a concentration of approximately 1 million cells per 1 ml. The umbilical cord mesenchymal stem cells of step c) can be cultured in the same medium as step a), i.e., a medium containing DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171) and FBS (fetal bovine serum), or in a cell culture medium different from that of step (a). The present invention also relates to a conditioned medium, its use, and a composition comprising the conditioned medium, wherein the conditioned medium is derived from the culture of umbilical cord mesenchymal stem cells and is obtained or obtainable by the production method of the present invention.

[0005] The present invention also relates to the use of the conditioned medium and its composition for inducing, stimulating and / or promoting hair growth and / or hair regeneration. The present invention also relates to the use of the conditioned medium and its composition for alleviating and / or reducing hair loss and / or hair thinning in a subject. Background Art

[0006] Hair consists of a shaft and a root. The shaft is the visible part of the skin, while the root lies just below the surface. At the base of the hair follicle is the dermal papilla, a pear-shaped structure composed of a group of fibroblasts. This structure supplies blood to the hair root, carrying nutrients for new hair production and controlling the regeneration of the hair follicle throughout its life cycle.

[0007] Normal hair follicles undergo a regeneration cycle defined by a growth phase (anagen), a regression phase (catagen), a resting phase (telogen), and a shedding phase (shedding). The growth phase is the phase in which the hair follicle grows and involves the periodic regrowth of the hair follicle. During the growth phase, dermal papilla fibroblasts secrete a variety of growth factors that maintain the active proliferation and differentiation of keratinocytes that form the proximal bulb of the hair fiber. At the end of the growth phase, the dermal papilla separates from the hair follicle, interrupting the blood supply and allowing the hair fiber to be pulled out, resulting in hair shedding.

[0008] According to the American Hair Loss Association, two-thirds of men over the age of 35 show signs of hair loss. Although it is more pronounced in men, hair loss also typically affects women from the age of 50 to 60. Consequently, recently, not only has male pattern baldness been increasing, but hair loss has also been increasing in women and young adults. For example, hair loss can be caused by various factors, including, for example, intrinsic factors such as genetic traits and the mental stress of daily life. In this context, with the progress of industrial development, environmental pollution, stress, and aging, hair loss symptoms will become more severe, and with the advent of the well-being era, people are increasingly concerned about their quality of life and appearance. Furthermore, hair loss may also be related to other factors, such as illness, hormonal changes, or side effects of medications. These can affect and inhibit hair production by causing physical damage to the hair follicles through abnormal hair follicle circulation and changes in hair follicle morphology.

[0009] For example, alopecia areata is considered an organ-specific autoimmune disease caused by a loss of immune privilege in hair follicles (HF), and therefore treatment is primarily immunosuppressive. As another example, androgenic alopecia (AGA), also known as male / female pattern baldness, is the most common cause of hair loss in the world today. It can affect up to 50% of Caucasian men. The causes of AGA are complex, and it is widely believed to be a multifactorial disease influenced by genetics, environment, diet, and hormones. The essential manifestation is the gradual shortening of the hair follicle growth cycle, and the hair produced within the cycle becomes shorter and thinner. Over time, the hair follicles in the scalp become completely inactive. In addition, hair loss can be induced after viral infection such as COVID-19 infection or the recently reported dengue virus infection, as described in Hussain N, Agarwala P, Iqbal K, Omar HMS et al., A systematic review of acute telogeneffluvium, a harrowing post-COVID-19 manifestation. J Med Virol. 2022 Apr; 94(4): 1391–1401. doi: 10.1002 / jmv.27534 or Chu & Yang, Dengue-associated telogeneffluvium: A report of 14 patients. Dermatologica Sinica, Volume 35, Issue 3, September 2017, Pages 124-126.

[0010] To alleviate the aforementioned hair loss phenomenon, a wide range of hair growth agents are available on the market. Pharmacological therapies specifically approved by the Food and Drug Administration (FDA) are limited to minoxidil and finasteride, both of which can have numerous side effects. Furthermore, while these two compounds prevent hair loss, their effects on hair growth are negligible. Therefore, there is a need to develop cost-effective and safe hair loss prevention technologies that not only prevent hair loss but also effectively promote hair growth and regeneration.

[0011] Therefore, there is still a need to discover new compounds and methods for promoting hair growth and regeneration. Therefore, it is an object of the present invention to provide such compounds and methods. Summary of the Invention

[0012] The above objects are achieved by a method, a conditioned medium and the use thereof having the features of the independent claims.

[0013] In a first aspect, the present invention provides a method for inducing, stimulating and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. The method may be non-therapeutic or cosmetic.

[0014] In a second aspect, the present invention provides a method for alleviating and / or reducing hair loss and / or hair thinning, wherein the method comprises treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. Similarly, the method can be non-therapeutic or cosmetic.

[0015] In a third aspect, the present invention provides a method for producing a conditioned medium, the method comprising (a) culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum); (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 is preferably serum-free and wherein optionally the additional culture medium contains a water-soluble antioxidant, wherein the conditioned medium is obtained by collecting the cell culture medium. In step (a) of the method, the umbilical cord mesenchymal stem cells can be cultured to about 70%, about 80%, about 90%, about 95% confluence, or cultured to even 100% confluence (complete confluence). In the optional step (c) of the method, the mesenchymal stem cells can be further cultured for any suitable period of time. Such a suitable time period may be, for example, a time period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

[0016] In a fourth aspect, the present invention provides a conditioned medium derived from umbilical cord mesenchymal stem cells obtained or obtainable by the method of the present invention.

[0017] In a fifth aspect, the present invention provides a composition comprising the conditioned medium of the present invention.

[0018] In a sixth aspect, the present invention provides the use of conditioned medium derived from umbilical cord mesenchymal stem cell culture for inducing, stimulating and / or promoting hair growth and / or hair regeneration; and / or for alleviating and / or reducing hair loss and / or hair thinning in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 1 A and Figure 1B shows representative images of immunohistochemical staining for elastin in human dermal fibroblasts (HDFs) after 48 hours of culture in DMEM / 10% FCS (control) versus red deer umbilical cord lining mesenchymal stem cell-conditioned medium (RD-CLMSC-CM). To compare the effects of DMEM / 10% FCS and RD-CLMSC-CM on elastin expression, HDFs (n=14) with varying donor characteristics (subjects ranged from 23 to 73 years old, from skin of different sites: forehead, eyelids, cheeks, neck, and temples) were cultured in DMEM / 10% FCS (control) and RD-CLMSC-CM for 48 hours, followed by immunocytochemical staining for elastin. Optical density was measured using ImageJ. RD-CLMSC-CM upregulated elastin expression in HDFs by 56% compared to HDFs cultured in DMEM / 10% FCS (control).

[0021] Figure 2 A and Figure 2 B shows representative images of immunohistochemical staining of hyaluronic acid (HA) in HDFs after 48 hours of culture in DMEM / 10% FCS (control) vs. RD-CLMSC-CM. To compare the effects of DMEM / 10% FCS and RD-CLMSC-CM on HA expression, HDFs (n=14) with different donor characteristics (subjects aged 23 to 73 years, from skin of different sites: forehead, eyelids, cheeks, neck, and temporal) were cultured in DMEM / 10% FCS (control) and RD-CLMSC-CM for 48 hours, followed by immunocytochemical staining of HA. Optical density was measured using ImageJ. Compared to HDFs cultured in DMEM / 10% FCS (control), RD-CLMSC-CM upregulated hyaluronic acid expression in HDFs by 83%.

[0022] Figure 3 Depicted are total cell counts, which were performed to compare the proliferative effects of DMEM / 10% FCS (control), human foreskin fibroblast-conditioned medium (FSF-CM), human umbilical cord lining mesenchymal stem cell-conditioned medium (H-CLMSC-CM), and red deer umbilical cord lining mesenchymal stem cell-conditioned medium (RD-CLMSC-CM) on aged skin HDFs. Starting with the same seeding density, the average total cell count for each group was calculated after 5 days of culture in the test medium. Compared to the control group, the highest increase in average total cell count was observed in the RD-CLMSC-CM group (113%), followed by the H-CLMSC-CM group (112%), and then the FSF-CM group (16%).

[0023] Figure 4An in vitro "scratch" wound assay is shown to compare the pro-migratory effects of red deer umbilical cord lining mesenchymal stem cell-conditioned medium (RD-CLMSC-CM) and human foreskin fibroblast-conditioned medium (FSF-CM). A "scratch" wound was created on a confluent human dermal fibroblast (HDF) monolayer using a p200 pipette tip, and images were taken on days 0 and 5 to document the progression of migration. On day 5, HDFs cultured in RD-CLMSC-CM completely filled the "scratch" wound, compared to 80% in FSF-CM.

[0024] Figure 5 Shown are top views of the scalp before (left) and 6 weeks after treatment (after 6 treatments) with a composition comprising conditioned medium derived from umbilical cord mesenchymal stem cell culture (right).

[0025] Figure 6A -G shows the results of the treatment with a composition comprising a conditioned medium cultured from mesenchymal stem cells derived from the amniotic membrane of the umbilical cord of red deer (conditioned medium of mesenchymal stem cells derived from the umbilical cord of red deer) before and after treatment ( Figure 6B -G) or weekly ( Figure 6A , B) Top view of the scalp with interval representation.

[0026] Figure 7 Shown are top views of the scalp of a patient with COVID-19-induced hair loss, with visible scalp, brittle hair, and low hair density, with the top photo representing the scalp before treatment. The middle photo shows a top view of the patient's scalp 3 months after treatment, with reduced scalp visibility and the beginning of hairline regrowth. The bottom photo shows the results 6 months after the start of 5 monthly stem cell serum applications, with minimal scalp visibility and restored hair density.

[0027] Figure 8 Shown are human hair follicle dermal papilla cells (HFDPCs) before treatment, after treatment with 2 μg / mL minoxidil, 0.2 μg / mL minoxidil concentrations, after treatment with 160-fold diluted (legend "PC 160-fold diluted") and 320-fold diluted (legend "PC 320-fold diluted") conditioned medium, and compared to human hair follicle dermal papilla cells (HFDPCs) after treatment with 160-fold diluted and 320-fold diluted (legend "NC 320-fold diluted" and "PC 320-fold diluted") negative control (NC).

[0028] Figure 9TNF-α release from normal human epidermal keratinocytes (NHEK) after exposure to UVB (UV+) and not exposed to UVB (UV-) as a negative control is shown. Inhibition of TNF-α production in the presence of conditioned medium diluted with factors 2, 4, 8, 16, 32, and 64 and in the presence of vitamin D. Figure 9 It was shown that in this experiment, the conditioned medium of the present invention reduced the expression / production of the key inflammatory cytokine TNF-α by 30-fold, which is comparable to the effect of vitamin D.

[0029] Figure 10 Shown is a technical information sheet for Lonza's Dulbecco's modified eagle medium, which includes the catalog number of DMEM used to prepare an exemplary example of culture medium (PTT-6) in the experimental section.

[0030] Figure 11 A technical information sheet of Lonza's Ham's F12 medium is shown.

[0031] Figure 12 Shown is a technical information sheet of Lonza's DMEM:F12 (1:1) medium, which includes the product catalog number of the DMEM:F12 (1:1) medium used to prepare an exemplary example (PTT-6) of the culture medium in the experimental section.

[0032] Figure 13 A technical information sheet for M171 medium from Life Technologies Corporation is shown, which includes the catalog number of the M171 medium used to prepare an illustrative example of the culture medium (PTT-6) in the experimental section.

[0033] Figure 14 Shown is an ingredient list including commercial suppliers and catalog numbers for each ingredient used to prepare PTT-6 medium in the experimental section.

[0034] Figure 15 The Norwood classification of male pattern baldness is shown.

[0035] Figure 16 The Ludwig classification of female pattern hair loss is shown.

[0036] Figure 17 The objective evaluation of 10 patients suffering from alopecia who applied a composition comprising the conditioned medium of the present invention to their capillitium is shown. Results. Among them, Figure 17A shows follicular units per square centimeter (FU / sqcm) during 12 weeks of treatment. Figure 17 B shows the interfollicular distance (mm) during 12 weeks of treatment. Figure 17 C shows the total hair count per cm2 during the 12 weeks of treatment. Figure 17 D shows the sum of hair widths per square centimeter (millions / square centimeter) during the 12 weeks of treatment.

[0037] Figure 18 The subjective assessment of patients by a standardized questionnaire is shown, where Figure 18 A shows the patient's satisfaction with hair growth, Figure 18 B shows the possibility of the patient covering the hair, Figure 18 C shows the quality of life of patients affected by their hair loss.

[0038] FIG19 shows Table 6, which shows the objective evaluation by the main outcome parameter (mean and standard deviation (mean / SD) and P value of the linear regression model) during the 12-week treatment period The main outcome parameters shown in Table 6 are (from top to bottom): interfollicular distance (mm), T:V ratio, follicular units (FU) per cm², average number of hairs / FU, average hair width (micm), terminal hair count per cm², total hair count per cm² (total), sum of terminal hair width per cm² (micm), and sum of total hair width per cm² (micm).

[0039] Figure 20 shows Table 7, which shows the results of the subjective assessment of patients by standardized questionnaires. The main outcome parameters (mean and standard deviation (mean / SD) and P value of the linear regression model) within the 12-week treatment period are shown. The main outcome parameters shown in Table 7 are (from top to bottom): satisfaction with hair growth, satisfaction with hair density, satisfaction with hair thickness, satisfaction with hair quality, satisfaction with the results, comfort with hair, self-confidence, friends / family evaluation of hair quality, thoughts on hair appearance, covering hair, re-combing hair, and affected quality of life. DETAILED DESCRIPTION

[0040] The present invention relates to methods that are all suitable for inducing, stimulating and / or promoting hair growth and / or hair regeneration by using conditioned medium derived from umbilical cord amniotic mesenchymal stem cell cultures. In addition, the methods of the present invention are all suitable for alleviating and / or reducing hair loss and / or hair thinning by using conditioned medium derived from umbilical cord mesenchymal stem cell cultures. Therefore, the present invention is based on the discovery that conditioned medium derived from umbilical cord mesenchymal stem cell cultures secretes biological factors (e.g., proteins) that provide excellent hair loss prevention and hair growth effects for conditioned medium used to treat subject hair, particularly hairline skin. In this regard, the inventors were surprised to find that umbilical cord mesenchymal stem cells secrete biological factors that are effective for hair growth, and also confirmed that conditioned medium obtained by culturing umbilical cord mesenchymal stem cells exhibits efficient hair growth promoting effects.

[0041] As shown in the experimental section, the present invention uses conditioned medium derived from umbilical cord mesenchymal stem cell culture, which has been shown to effectively and sustainably improve hair quality, density and coverage of inactive or dormant hair follicles in patients with hair thinning / hair loss. For example, as shown in the experimental section, conditioned medium derived from umbilical cord mesenchymal stem cell culture was used to After 6 weeks of treatment, patients with thinning hair / hair loss had objective improvements in hair density and coverage (Example 6; Figure 6A -K). As another example, conditioned medium derived from umbilical cord mesenchymal stem cell culture Stimulate hair growth in patients with androgenetic alopecia (AGA) (Example 7, Figure 5 ), androgenic alopecia is also known as male and female pattern hair loss and is the most common cause of hair loss in the world today. In addition, a patient who developed telogen effluvium (TE) (i.e., diffuse hair loss) after COVID-19 infection, after not responding to conventional hair loss treatment, received monthly treatment with conditioned medium derived from umbilical cord mesenchymal stem cells for a total of 5 times, and with the regrowth of the frontal hairline and villi in the prefrontal region, the hair density gradually increased (Example 8, Figure 7 Under these circumstances, the present invention has found that a conditioned medium derived from umbilical cord mesenchymal stem cell culture has an excellent effect on hair growth.

[0042] Therefore, the present invention relates to a culture medium conditioned by umbilical cord mesenchymal stem cell culture. Therefore, as described herein, by culturing umbilical cord mesenchymal stem cells in a cell culture medium, and separating the resulting culture medium from cells, the conditioned culture medium is obtained so that the conditioned culture medium of the present invention contains secreted stem cell products (referred to as biological factors). Therefore, the conditioned culture medium contains biological factors and is substantially free of or free of stem cells. The biological factors that can be in the conditioned culture medium include, but are not limited to, growth factors, exosomes, hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and particulate matter. Such conditioned culture medium and the combination of any biological factors therein (including but not limited to growth factors) can be used in the method of the present invention.

[0043] As shown in the experimental examples herein, conditioned medium derived from umbilical cord mesenchymal stem cell culture is particularly rich in growth factors, which are believed to help reverse the damaged hair follicle activity in conditions of hair thinning / hair loss (such as traditional hair loss and COVID-19-induced hair loss). For example, epidermal growth factor (EGF) is known to have a positive effect on hair growth because EGF promotes the growth phase and regulates the elongation of hair follicles while stimulating angiogenesis; see Mak KK, Kingston SY, Epidermal growth factor as a biologic switch in hair growth cycle. J. Invest Dermatol 2001: 117 (6): 1594-600). As another example, fibroblast growth factor 8FGF is known to have a positive effect on the development of hair follicles. As shown in the experimental section herein (Example 2), when conditioned medium - derived from culture of red deer umbilical cord mesenchymal stem cells (RD-CLMSCs) in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) - was applied to human dermal fibroblasts (HDFs) to evaluate the effects of the conditioned medium on human skin, the conditioned medium significantly upregulated the expression of elastin and hyaluronic acid compared to administration of DMEM / 10% FCS to HDFs (Example 3, Figure 1 and 2 Without wishing to be bound by theory, the upregulation of elastin and hyaluronan expression seen in RD-CLMSC-CM is due to the presence of growth factors including transforming growth factor (TGF) β1 and TGFβ2, insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and fibroblast growth factor 7 (FGF-7) present in RD-CLMSC-CM.

[0044] In this regard, the presence of growth factors involved in inducing enhanced elastin and hyaluronic acid expression is known to those skilled in the art. In humans, transforming growth factor 1 (TGF-β1) plays a central role in the expression of tropoelastin (TE), the soluble form of elastin; see Kuang, PP, et al., Activation of elastin transcription by transforming growth factor-beta in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol, 2007. 292(4): p. L944-52. TGF-β1 also stabilizes tropoelastin mRNA transcripts; see VM, et al., Transforming growth factor-beta up-regulates elastin gene expression in human skin fibroblasts. Evidence for post-transcriptional modulation. Lab Invest, 1992. 66(5): p. 580-8; and Kucich, U., et al., Stabilization of elastin mRNA by TGF-beta: initial characterization of signaling pathway. Am J Respir Cell Mol Biol, 1997. 17(1): p. 10-6. Interestingly, the combination of TGF-β1 and hyaluronic acid oligomers synergistically enhances the level of elastin in the extracellular matrix of cultured vascular smooth muscle cells; see Joddar, B. and A. Ramamurthi, Elastogenic effects of exogenous hyaluronan oligosaccharides on vascular smooth muscle cells. Biomaterials, 2006. 27(33): p. 5698-707. TGF-β1 also reduces the degradation of elastin by reducing the levels and activity of elastin hydrolytic proteases, including matrix metalloproteinase (MMP)-2 and matrix metalloproteinase (MMP)-9; see Dai, J., et al., Overexpression of transforming growth factor-beta1 stabilizes already-formed aorticaneurysms: a first approach to induction of functional healing by endovascular gene therapy. Circulation, 2005. 112(7): p. 1008-15. Treatment of HDFs with various concentrations of TGF-β1 or TGF-β2 for 24 hours resulted in a dose-dependent increase in steady-state levels of elastin mRNA, with a maximum 30-fold increase observed at a dose of 1 ng / ml; see VM, et al., Transforming growth factor-beta up-regulateselastin gene expression in human skin fibroblasts. Evidence for post-transcriptional modulation. Lab Invest, 1992.66(5):p.580-8. In mammals, three hyaluronan synthases (HAS): HAS-1, 2, and 3 synthesize HA chains of various lengths; see Weigel, PH, V.C. Hascall and M. Tammi, Hyaluronan synthases. J Biol Chem, 1997. 272(22): p. 13997-4000; Itano, N., et al., Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J Biol Chem, 1999. 274(35): p. 25085-92; and Itano, N. and K. Kimata, Mammalian hyaluronan synthases. IUBMB Life, 2002. 54(4): p. 195-9. TGF-β1 differentially upregulates gene expression of HAS-1 and HAS-2 in the dermis and epidermis; see Stuhlmeier, KM and C. Pollashek, Differential effect of transforming growth factor beta (TGF-beta) on the genes encoding hyaluronan synthases and utilization of the p38 MAPK pathway in TGF-beta-induced hyaluronan synthase 1 activation. J Biol Chem, 2004. 279(10): p.8753-60; and Stern, R. and HIMaibach, Hyaluronan in skin: aspects of aging and its pharmacologic modulation. Clin Dermatol, 2008. 26(2): p.106-22.Fibroblast growth factor (FGF) -7 can stimulate the mRNA expression of HAS-2 and HAS-3, which activates keratinocyte migration and stimulates wound healing, resulting in the accumulation of intermediate molecular weight HA in the culture medium and in keratinocytes. TGF-β2 and platelet-derived growth factor (PDGF) -BB have been shown to induce osteoblasts to express HAS-2 and hyaluronan synthase; see Nikitovic, D., et al., Transforming Growth Factor-βas a key molecule triggering the expression ofversican isoforms v0 and v1, Hyaluronan Synthase-2and synthesis of Hyaluronanin Malignant Osteosarcoma cells. IUBMB Life, 2006. 58 (1): p. 47-53.

[0045] Therefore, the significant upregulation of elastin and hyaluronic acid expression observed in all exposed HDFs can be attributed to the presence of TGF-β, PDGF-BB, and FGF-7 in RD-CLMSC-CM. In addition to the known involvement of growth factors in enhancing elastin and hyaluronic acid expression, a positive role for growth factors in hair growth was also found herein, including but not limited to transforming growth factor (TGF) β1 and TGFβ2, insulin-like growth factor 1 (IGF-1), and platelet-derived growth factor (PDGF, including, for example, PDGFaa, PDGFbb, and / or PDGFab).

[0046] Furthermore, the Examples show that conditioned medium of mesenchymal stem cells from red deer and human umbilical cord amniotic membrane applied to human dermal fibroblasts (HDF) significantly increased the proliferation capacity of HDF, which could not be achieved, for example, with DMEM / 10% FCS (control) or human foreskin fibroblasts (FSF) (Example 4, Figure 3 Furthermore, cell migration properties assessed by an in vitro “scratch” assay showed that conditioned medium derived from cultures of red deer umbilical cord amniotic membrane mesenchymal stem cells was more effective in promoting migration than conditioned medium derived from cultures of human foreskin fibroblasts (FSF) (Example 5, Figure 4The superior proliferative and pro-migratory properties of conditioned medium derived from umbilical cord amniotic membrane mesenchymal stem cells (red deer and human) compared to DMEM / 10% FCS (control) or human foreskin fibroblasts (FSF) suggest the presence of other proteins and / or growth factors in this conditioned medium that may have a positive impact on HDF proliferation. For example, VEGF significantly increases the proliferation capacity of HDFs and human lung fibroblasts (see, e.g., Bondarenko, N.A., et al., Effect of Vascular Endothelial Growth Factor and Erythropoietin on Functional Activity of Fibroblasts and Multipotent Mesenchymal Stromal Cells. Bulletin of Experimental Biology and Medicine, 2016. 160(4): p. 498-501.; Larsson-Callerfelt, A.-K., et al., VEGF induces ECM synthesis and fibroblast activity in human lung fibroblasts. European Respiratory Journal, 2017. 50(suppl 61): p. PA1045) and HGF was shown to induce proliferation of human keratinocytes with the same potency as FGF-7 (see, e.g., Takami, Y., et al., Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid. Biochimica et al., 2017. Biophysica Acta (BBA)-Molecular Cell Research, 2005.1743(1):p.49-56).

[0047] Therefore, the significantly enhanced proliferation and increased migration of HDFs cultured in RD-CLMSC-CM can be attributed to the proliferative and pro-migratory effects of VEGF, TGF-β1, and HGF. In addition to expressing pro-proliferative factors, conditioned medium derived from umbilical cord mesenchymal stem cell cultures can contain anti-apoptotic and maintenance factors to delay cell senescence, slow growth arrest, and reduce apoptosis. Furthermore, in this case, the stimulation of hair growth can also be attributed to VEGF and HGF, as these growth factors are known to those skilled in the art to also have positive effects on hair growth. In this regard, VEGF is known to be involved in hair follicle growth and circulation, wherein it promotes hair growth, an increase in the number of hair follicles and hair size by improving vascularization (especially during the growth phase of the hair cycle); see Yano et al., Control of hair growth and follicle size by VEGF. Journal of Clinical Investigation, 2001 Feb 15:107(4):409-417; Lachgar S, Moukadiri H, Jonca F et al., Vascular endothelial growth factor is an autocrine growth factor for hairdermal papilla cells. Journal of Investigative Dermatology. 1996; 106(1):17–23; Kozlowska U, Blume-Peytavi U, Kodelja V et al., Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle. Archives of Dermatological Research.1998;290(12):661–668; Rinaldi F. et al.: the role of up-stimulation of growth factors in hair transplantationimprove the revascularization of transplanted hair growth mediated byangiogenesis. Forum 2007:2.

[0048] A growth factor is an agent, such as a naturally occurring substance that is capable of stimulating cell growth and / or proliferation and / or cell differentiation. Typically, growth factors are proteins or steroid hormones. "Growth factor" and "factor" can be used interchangeably; however, as used herein, the term "biological factor" is not limited to growth factors. For example, umbilical cord mesenchymal stem cells can produce one or more factors involved in, for example, cell proliferation and migration (e.g., hepatocyte growth factor (HGF); vascular endothelial growth factor (VEGF); platelet-derived growth factor AA (PDGF-AA); basic fibroblast growth factor (bFGF); urokinase plasminogen activator receptor (UPAR); intercellular adhesion molecule-1 (ICAM1), and insulin-like growth factor 1 (IGF1); interleukin-6 (IL-6); interleukin-8 (IL-8)); promoting angiogenesis (e.g., angiopoietin-1 (ANG-1); hepatocyte growth factor (HGF); vascular endothelial growth factor (VEGF); anti-inflammatory effects (e.g., transforming growth factor beta-1 (TGF-beta1); soluble tumor necrosis factor (TNF) receptor 1); promoting elastin and / or hyaluronic acid expression (e.g., transforming growth factor beta-1 (TGF-beta1); basic fibroblast growth factor (bFGF); insulin-like growth factor 1 (IGF1)); and other factors, including monocyte chemoattractant protein-1 (MCP-1); tissue inhibitor of matrix metalloproteinase 1 (TIMP1), and tumor necrosis factor receptor superfamily member 10C (TR10C).

[0049] The conditioned media of the present application can comprise at least one, and more typically, a combination of biological factors produced by umbilical cord mesenchymal stem cells (e.g., at least one, and more typically, a combination of the biological factors listed above). For example, these biological factors can comprise one or more (at least one) angiogenic cytokines and / or growth factors. These biological factors can include one or more selected from the group consisting of ANG-1, HGF, VEGF such as VEGFA and / or VEGFB, TGF such as TGF-beta1, PDGF such as PDGF-AA or PDGF-BB, bFGF, MCP-1, IL-6, IL-8, TNF, TIMP1, TR10C, UPAR, ICAM1, and IGF1.

[0050] In this case, the present invention has found that conditioned medium derived from umbilical cord mesenchymal stem cell culture improves hair growth, wherein the cells particularly secrete growth factors and other biological factors into the conditioned medium. In this way, it is believed that the conditioned medium of the present invention derives high concentrations of growth factors and / or other biological factors that can effectively help prolong the growth phase, induce cell growth and cell proliferation (particularly dermal papilla cell proliferation), and inhibit apoptosis signaling. In this context, it should be noted that culturing umbilical cord mesenchymal stem cells in a culture medium as described herein provides the isolation of extremely homogeneous and well-defined umbilical cord tissue mesenchymal stem cell populations (e.g., umbilical cord Wharton's jelly or umbilical cord amniotic membrane mesenchymal stem cell populations), as disclosed in WO 2019 / 199234A1 or WO 2018 / 067071A1. In particular, the isolation of the umbilical cord mesenchymal stem cell population resulted in greater than 90% or even 99% or more cells being positive for the three mesenchymal stem cell markers CD73, CD90, and at the same time these stem cells lacked expression of CD34, CD45 and HLA-DR (see, e.g., WO 2019 / 199234A1 or WO 2018 / 067071A1), meaning that 99% or even more of the cells in the population expressed the stem cell markers CD73, CD90 and CD105 but did not express the markers CD34, CD45 and HLA-DR. In the present context, such a population of umbilical cord (tissue) mesenchymal stem cells fully meets the generally accepted criteria for human mesenchymal stem cells for cell therapy, as defined, for example, by Dominici et al., “Minimal criteria for defining multipotent mesenchymalstromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol. 8, No. 4, 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. P. 75–79.This, in turn, means that conditioned medium derived from the culture of such an extremely homogeneous and well-defined cell population is ideal for producing highly homogeneous and well-defined components in the conditioned medium, such as highly concentrated growth factors. Furthermore, the use of the conditioned medium of the present invention in hair growth therapy has several other advantages over stem cell-based applications, as the donor-recipient matching issues associated with stem cell-based hair transplantation can be avoided, and the conditioned medium is easier to prepare and less expensive than stem cell-based hair therapy.

[0051] In addition, the optimal conditions for hair growth therapy and hair transplantation are equally dependent on healthy skin conditions. In this case, it is reported that the conditioned medium derived from umbilical cord mesenchymal stem cell culture has a beneficial effect on skin quality and regeneration. For example, the use of conditioned medium shows optimization of the healing process of moderate to severe light-damaged patients (Hoss et al., Red Deer Umbilical Cord-Derived Stem Cell Conditioned Media Combined With Ablative Resurfacing of the Face, Journal of Drugs in Dermatology, Volume 19, Issue 11, Nov 2020 and to reduce pain and discomfort after laser therapy (Dr.Cheryl Effron, Reduction of Pain and Discomfort post CO2 Fractional Laserresurfacing after the application of umbilical cord lining extract:A Single-Blinded Split-Face Trial, available at https: / / calecimprofessional.com / pages / clinical-abstract-dr-cheryl-effron). Without wishing to be bound by theory, the optimized healing and reduced pain of damaged skin areas achieved through the use of conditioned medium may be attributed to the presence of secreted products from umbilical cord mesenchymal stem cells contained in the conditioned medium. Such secreted compounds / products are disclosed, for example, in WO2019 / 199234A1, where increased expression and / or secretion of umbilical cord mesenchymal stem cell products into the culture medium can induce or improve wound healing properties. Thus, products secreted into the conditioned medium by umbilical cord mesenchymal stem cells contribute to the maintenance of healthy skin conditions, which, as discovered herein, also contribute to optimal hair growth and / or healing processes following hair transplantation. The beneficial effects of conditioned medium derived from umbilical cord mesenchymal stem cell cultures on skin conditions may be attributed to the immunomodulatory properties of the conditioned medium, which are also known to be exerted by umbilical cord mesenchymal stem cells. Based on these findings, it has been discovered herein that exposing conditioned medium derived from umbilical cord mesenchymal stem cell cultures to the scalp of a subject reduces inflammation.See Example 9 herein, in which the production of tumor necrosis factor-α (TNF-α), a key inflammatory cytokine produced by macrophages / monocytes during acute inflammation and responsible for various signaling pathways in cells (e.g., those leading to necrosis or apoptosis), was significantly downregulated. Thus, in addition to stimulating and / or promoting hair growth and / or hair regeneration, the present invention has the additional advantage that the conditioned medium can reduce inflammation and promote cell growth and cell proliferation.

[0052] Conditioned medium derived from umbilical cord mesenchymal stem cell cultures that is believed to be suitable for hair growth (e.g., by inducing and stimulating hair growth in patients with thinning hair / hair loss) can be derived from the culture of any suitable umbilical cord (tissue) mesenchymal stem cell population known in the art. For example, umbilical cord mesenchymal stem cells can be derived from any mammalian tissue or compartment / body part known to contain umbilical cord mesenchymal stem cells. In an illustrative example, the mesenchymal stem cells can be mesenchymal stem cells from the umbilical cord amniotic membrane. The effects of promoting hair growth and preventing hair loss are achieved, for example, because these stem cells secrete growth factors and other biological factors related to hair growth, such as by producing dermal papilla cells and / or increasing the number and size of hair follicles. For example, biological factors that can be in the conditioned medium include, but are not limited to, growth factors, nucleic acids such as mRNA or DNA, exosomes, hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and particulate matter. Such conditioned medium and any combination of biological factors contained therein (including but not limited to growth factors) can be used in the methods of the present invention.

[0053] The treatment / processing of the present invention can be used for example without dilution or dilution with conditioned medium.When using in diluted form, the conditioned medium can be used with a final concentration of, for example, about 10% to about 90% or about 10% to about 80%, including use at a final concentration of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or about 70% (v / v of the cumulative volume of the composition comprising conditioned medium).For this purpose, the conditioned medium of the present invention can be diluted (prepared) with any suitable diluent / carrier medium.Carrier medium can be a liquid, gel or cream-like preparation.For example, conditioned medium is diluted in carrier medium, and the carrier medium is such as an aqueous solution, basal medium or cream preparation of the physiological buffer solution, water, extracellular matrix components of PBS or saline, only enumerates several suitable culture media. Basal medium used herein refers to a mixture containing sugars, amino acids, water, etc. required for cell survival, including commercially prepared culture media, such as, but not limited to, Dulbecco's modified Eagle's medium (DMEM), endothelial differentiation medium (EDM), minimum essential medium (MEM), basal medium Eagle (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (G-MEM), and Iscove's modified Dulbecco's medium. Examples of aqueous solutions of extracellular matrix components are hyaluronic acid aqueous solutions, collagen aqueous solutions, or fibronectin aqueous solutions, all of which are commercially available for use in cosmetics. An illustrative example of such a composition of the present invention that can be used to induce, stimulate and / or promote hair growth and / or hair regeneration as described herein is a solution (liquid composition) containing about 90 to about 10% (v / v) conditioned medium, with the remainder (i.e., about 10 to about 90% (v / v)) being an aqueous solution of hyaluronic acid. In some other embodiments, the conditioned medium of the present invention can be contained in a solution containing DMEM as a basal medium; in such a solution, the conditioned medium can also be present in the range of about 90% to about 10% (v / v), with the remainder being DMEM. The conditioned medium of the present invention can further contain an antioxidant, such as a soluble antioxidant. Water-soluble antioxidants can be added to the culture medium to prevent oxidative damage. Examples of suitable water-soluble antioxidants include glutathione, uric acid, Trolox or Allicidin, to name a few.

[0054] The conditioned medium of the present invention can also be formulated to improve skin absorption of the conditioned medium after topical application to the skin / scalp of a subject. For this purpose, the conditioned medium can be encapsulated in a liposomal formulation. An illustrative example of such a liposomal formulation is a liposome made from a phospholipid mixture. Such a phospholipid mixture for cosmetic products is, for example, Lucas Meyer Cosmetics's Pro-Lipo TM Neo's liposome delivery system, which is a ready-to-use liposome delivery system that allows encapsulation of conditioned medium for topical administration (see Example 12). The composition for topical administration as described herein may, for example, contain about 30 to 70% (v / v) conditioned medium and about 20% (v / v) Pro-Lipo TM Neo (Liposomal Mixture), and the remainder / balance is any suitable aqueous solution.

[0055] The conditioned medium of the present application can be obtained after culturing mesenchymal stem cells in a culture medium comprising DMEM, F12, M171 and FBS, followed by culturing the umbilical cord mesenchymal stem cells in the above-mentioned further culture medium. The further culture medium can be any (further) basal medium suitable for the growth of animal cells, in particular a basal medium suitable for culturing mesenchymal stem cells. The further culture medium can be a serum-free culture medium. Non-limiting examples of such serum-free culture media include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Keratinocyte Medium (KM), KBM (Keratinocyte Basal Medium), EpiLife KM (Keratinocyte-EpiLife Medium), and the like. The further culture medium can also contain serum. As an example of such serum-containing culture medium, the conditioned medium used can be the conditioned medium described for and commonly used for isolating and culturing mesenchymal stem cell populations from umbilical cord amniotic membrane, such as medium PTT-4. This medium PTT-4 consists of 90% (v / v) CMRL 1066 and 10% (v / v) FBS, and has been used in US patent application US 2008 / 0248005 and corresponding international patent application WO 2007 / 046775 for isolating and culturing mesenchymal stem cell populations from umbilical cord amniotic membrane, in US patent application US 2008 / 0248005 and international patent application WO 2007 / 046775 the medium was shown to have superior wound healing properties. In some examples, the cell culture medium described herein, for example a medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum), can be used for culturing umbilical cord mesenchymal stem cells, wherein after culturing, for example once the cells have reached full confluency, the cell culture medium is removed and replaced with another culture medium / solution, such as a medium solution comprising, for example, DMEM, which can comprise an antioxidant component. Typically, during the optional culturing step in the further culture medium of the method of preparing a conditioned medium as described herein, the mesenchymal stem cells can be cultured for any suitable period of time. Such a suitable period of time can be, for example, a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or even longer, for example, up to a period of 10 days or 14 days, if desired. In the present context, it is noted that both the conditioned medium obtained in the first culturing step as well as the conditioned medium obtained in the second (further) culturing step can be used for inducing, stimulating and / or promoting hair growth and / or hair regeneration as described herein.

[0056] The umbilical cord (tissue) mesenchymal stem cells used herein to prepare the conditioned media can be from (derived from) any compartment of umbilical cord tissue that contains mesenchymal stem cells. The mesenchymal stem cell population can be an amniotic membrane (AM), perivascular (PV), Wharton's Jelly (WJ), umbilical cord amniotic membrane (also referred to as umbilical cord lining mesenchymal cells), and mixed (MC) mesenchymal stem cell population of the umbilical cord, which means a mesenchymal stem cell population that includes stem cells from two or more of these compartments. In some examples, the mesenchymal stem cells are umbilical cord amniotic membrane mesenchymal stem cells. Isolation of mesenchymal stem cells from these compartments and thereof are known to those skilled in the art and described by, e.g., 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. The mixed mesenchymal stem cell population of the umbilical cord can be obtained, e.g., by stripping the arteries and veins from the umbilical cord tissue, cutting the remaining tissue and Wharton's Jelly into pieces and culturing the umbilical cord tissue in a culture medium, such as PTT-6 (by tissue dissociation piece method).Mixed umbilical cord mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue with intact umbilical cord vasculature by the tissue dissociation block method under conditions as described in Schugar et al. "High harvest yield, high expansion, and phenotypestability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009: 789526 (cultured in DMEM supplemented with 10% fetal bovine serum, 10% horse serum and 1% penicillin / streptomycin serum). In this context, it should be noted that mesenchymal stem cell populations from the umbilical cord-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.

[0057] According to the above, it is noted here that the conditioned medium is derived from culturing mesenchymal stem cells of the umbilical cord tissue, wherein the stem cells can be cultured in a medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum), and the stem cells can be isolated from their natural environment prior to culturing in the medium as described herein. This method is particularly useful for mesenchymal stem cell populations of the umbilical cord. Such stem cell populations (e.g. Wharton's jelly mesenchymal stem cell populations) can first be isolated as described in Subramanian et al., 2015, PLoS ONE, previously incorporated by reference or International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord" and then the isolated mesenchymal stem cell population is cultured in a medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). Placental mesenchymal stem cell populations can also be isolated from the placenta, for example, as described in European Patent Application EP1288293, Talwadekar et al, "Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood 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 subsequently cultured in the medium described herein.

[0058] In this context, it should be noted that the culture medium described herein also allows for the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from their natural environment. Thus, the culture medium described herein also allows for the isolation of a population of mesenchymal stem cells under conditions that allow the proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. In some instances, the cell culture medium used to isolate and / or culture umbilical cord mesenchymal stem cells can be the culture medium used to culture cells to produce the conditioned medium of the present invention.

[0059] In light of the above disclosure, it is noted that the mesenchymal stem cell populations described herein can be isolated and cultured from any umbilical cord tissue (i.e., can be derived from any umbilical cord tissue) as long as the umbilical cord tissue contains the amniotic membrane (also known as the "cord lining"). Thus, as described in the experimental section of this application, the mesenchymal stem cell populations can be isolated from (a portion of) the entire umbilical cord. Thus, in addition to the amniotic membrane, the umbilical cord tissue can comprise any other tissue / component of the umbilical cord. For example, as described in U.S. Patent Application No. 2006 / 0078993 or International Patent Application No. WO2006 / 019357 Figure 16 As shown, the amniotic membrane of the umbilical cord is the outermost portion of the umbilical cord, covering it. Furthermore, the umbilical cord contains a vein (which delivers oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's jelly, a jelly-like substance primarily composed of mucopolysaccharides. Therefore, the umbilical cord tissue used in the present invention may also include this vein, two arteries, and Wharton's jelly. Using this entire (intact) portion of the umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the number of separation steps, making the method of the present invention simpler, faster, less error-prone, and more economical. Therefore, the isolation of mesenchymal stem cells can begin with tissue explantation, and if a larger number of mesenchymal stem cells is desired, the isolated mesenchymal stem cells can then be subcultured (cultivated). Alternatively, the amniotic membrane can be first separated from the other components of the umbilical cord, and then the mesenchymal umbilical cord lining stem cells can be isolated from the amniotic membrane by culturing the amniotic membrane in the culture medium described herein. The culture may also be performed by a tissue dissociation block method, optionally followed by subculturing the isolated mesenchymal stem cells.

[0060] In this context, the term "tissue separation block method" or "tissue separation block method" is used with its conventional meaning in the art and refers to a method in which, once tissue (e.g., umbilical cord tissue) is harvested, the tissue or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, and over time, stem cells migrate from the tissue to the surface of the culture dish. These primary stem cells can then be further amplified and transferred to a fresh culture dish by micropropagation (passage culture) as described herein. In this context, it is noted that in the first step of isolating / obtaining a population of umbilical cord mesenchymal stem cells (such as amniotic membrane or Wharton's jelly mesenchymal stem cells), a master cell bank of isolated mesenchymal stem cells is obtained, and subsequent passage culture can obtain a working cell bank. If the conditioned medium derived from umbilical cord mesenchymal stem cell culture of the present invention is used in an application that promotes hair growth, the cell population of the working cell bank is typically used for this purpose. The stem cell population of the separation step (which can constitute the master cell bank) and the stem cell population of the passage culture step (which can constitute the working cell bank) can both be stored, for example, in the form of cryopreservation.

[0061] In the present context "inducing, stimulating and / or promoting hair growth and / or hair re-growth" means that the conditioned medium enhances and / or initiates (induces) the ability of hair follicles to grow hair, particularly in patients with low active or dormant hair follicles and / or patients with hair thinning / hair loss (such as androgenetic alopecia). This enhanced and / or initiated ability to grow hair can be attributed to the presence of growth factors, as mentioned above, which are known in the art to be involved in inducing, stimulating and / or promoting hair growth. In the experimental examples herein, the secretion of growth factors in the conditioned medium derived from umbilical cord mesenchymal stem cells, which are positively associated with "inducing, stimulating and / or promoting hair growth and / or hair re-growth" and present in the conditioned medium, was evaluated in relation to human foreskin fibroblasts (FSF) and DMEM / 10% FCS (control). In this sense, it was demonstrated that culturing human dermal fibroblasts (HDF) in conditioned medium derived from umbilical cord mesenchymal stem cell cultures showed superior proliferation and pro-migration properties of human dermal fibroblasts compared to culturing in DMEM / 10% FCS (control) or human foreskin fibroblasts (FSF), which can be attributed to the secretion of a high amount (corresponding to high secretion levels or high concentrations) of growth factors into the supernatant / culture medium by the umbilical cord mesenchymal stem cells compared to FSF cultures. The secretion of growth factors into the culture medium (and thus the growth factors contained in the conditioned medium of the present application) can be measured / determined by any suitable method, for example by measuring the amount of growth factors by means of commercially available antibodies / immunoassays (see experimental section). Such measurements can be performed in an automated fashion using, for example, systems such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA). The ability of the conditioned medium described herein to induce, stimulate and / or promote hair growth and / or hair re-growth can also be determined by cellular assays, for example, by examining the growth promoting effect of the conditioned medium on cultured human hair follicle dermal papilla cells as described herein (see Example 9), or by applying the conditioned medium to patients diagnosed with hair loss and examining, for example, its effect on the number of hair follicle units per square centimeter, interfollicular distance or total hair count per square centimeter as described herein (see Example 11).

[0062] "Norwood classification or grade" or "Norwood-Hamilton classification or grade" refers to the classification criteria proposed by O'Tar Norwood to divide the stages of male pattern baldness. The Norwood classification / grade is the most widely used classification criteria for male hair loss, which defines two main patterns and several less common types (see, e.g., Gupta & Mysore, Classifications of Patterned Hair Loss: A Review, J Cutan Aesthet Surg. 2016 Jan-Mar;9(1):3–12). Norwood observed that thinning began in the temporal and vertex / top of the head and slowly progressed to encompass the entire top of the scalp. Therefore, this classification criteria is based on this pattern and is also shown in Figure 15 Medium. The degree of hair loss according to the Norwood scale is as follows (Roman and Arabic numerals are used interchangeably in this article when referring to the Norwood scale):

[0063] Type I: There is slight or no receding hairline.

[0064] Type II: There is a triangular, usually symmetrical, area of ​​receding forehead at the temporal hairline.

[0065] Type III: This represents the minimum amount of hair loss required to be considered Norwood baldness. There is deep, symmetrical receding of the temporal areas, which are either bare or only sparsely covered with hair. In type III, hair loss is primarily concentrated in the vault area, and the frontotemporal hairline does not recede as much as seen in type III.

[0066] Type IV: The frontotemporal recession is more severe than in type III, and there is sparse or no hair on the top of the head. The two areas of hair loss are separated by a band of moderately dense hair that extends across the top of the head. This band of hair connects to the complete hairline on both sides of the scalp.

[0067] Type V: The baldness area on the top of the head is still separated from the frontotemporal area, but the boundary is blurred. The hair band across the top of the head becomes narrower and thinner, and the baldness areas on the top of the head and the frontotemporal area become larger.

[0068] Type VI: The bridge of hair across the top of the head disappears, leaving only sparse hair. The frontotemporal and parietal regions are connected, and the degree of hair loss is greater.

[0069] Type VII: The most severe form of hair loss and only a narrow, horseshoe-shaped band of hair remains on the sides and back of the scalp. This hair is usually not dense and is quite fine.

[0070] The Norwood grade also defines a Type A variant outside of its standard classification system, which is distinguished by two major and two minor characteristics.

[0071] The main features are: 1) the anterior edge of the hairline advances posteriorly, leaving no islands of hair in the mid-front region, and 2) the absence of concurrent parietal balding. Instead, the anterior hairline continues to advance posteriorly toward the scalp.

[0072] Secondary features are: 1) the presence of scattered thinning hair in the area of ​​hair loss, and 2) the horseshoe-shaped fringe of hair remaining on the sides and back of the scalp tends to be wider and reach higher on the head compared to Norwood's criteria. The various variations of Type A described by Norwood are as follows:

[0073] Type IIA: The hairline is in front of the coronal plane and 2 cm in front of the external auditory canal.

[0074] Type IIIA: The hairline has receded to a point between the limits of type IIA and the level of the external auditory canal.

[0075] Type IVA: The hairline has receded beyond the external auditory canal but has not yet reached the top of the skull.

[0076] Type VA: The exposed area includes the top of the head. More severe hair loss than type VA cannot be distinguished from types VI or VII.

[0077] In this context, it is noted that it has been found herein that the conditioned medium described herein can be preferably used to treat hair loss in men who have been diagnosed with type I, II, III, IV, V, or VI hair loss according to the Norwood classification. Or, in other words, hair loss of any of type I, II, III, IV, V, or VI according to the Norwood classification can be treated with the conditioned medium of the present invention.

[0078] "Ludwig classification or grade" refers to the classification criteria proposed by E. Ludwig in 1979 for classifying female pattern hair loss or baldness (androgenetic alopecia). It ranges from Type I / Grade I to Type III / Grade III (see also Figure 16 ), wherein the terms "type" and "grade" are used interchangeably herein. When referring to the Ludwig grade, Roman numerals and Arabic numerals are used interchangeably herein.

[0079] Grade I: Perceptible thinning of hair occurs on the top of the head, with the front being defined by a line 1–3 cm behind the front hairline. Figure 16 As shown, grade I is further classified into grades Ia, Ib, Ic, and Id, and grade II is further classified into grades IIa and IIb.

[0080] Grade II: Marked thinning of the hair on the top of the head within the Grade I area.

[0081] Grade III: Complete baldness (completely bald) in the areas seen in grades I and II.

[0082] Grade I begins with thinning on the crown of the head. In Grade II, the scalp begins to show. By the time hair loss progresses to Grade III, all the hair on the crown of the head may have fallen out.

[0083] In this context, it is noted that it has been found herein that the conditioned medium as described herein can be preferably used to treat hair loss in women, wherein the women have been diagnosed with type I (including type Ia, type Ib, type Ic, and type Id), type II (type IIa and type IIb) hair loss, or type III hair loss according to the Ludwig classification. Or, in other words, according to the Ludwig classification, any of type I (including type Ia, type Ib, type Ic, and type Id), type II (type IIa and type IIb), or type III hair loss can be treated with the conditioned medium of the present invention.

[0084] "DMEM" refers to Dulbecco's Modified Eagle's Medium, a modification of Basal Eagle's Medium (BME) developed in 1969 (see Figure 10 , which shows a data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for culturing embryonic mouse cells. Since then, DMEM has become the standard culture medium for cell culture and is commercially available from a variety of sources, such as ThermoFisher Scientific (Cat. No. 11965-084), Sigma Aldrich (Cat. No. D5546), or Lonza, to name just a few. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is the DMEM culture medium available from Lonza as Catalog No. 12-604F. This culture medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is the DMEM culture medium from Sigma Aldrich as Catalog No. D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0085] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed for culturing a variety of mammalian and hybridoma cells when used with serum and in combination with hormones and transferrin (see Figure 11which shows a data sheet for Ham’s F12 medium from Lonza). Any commercially available (e.g., from ThermoFisher Scientific (cat. no. 11765-054), Sigma Aldrich (cat. no. N4888), or Lonza, to name a few suppliers) Ham’s F12 medium can be used in the present application. In preferred embodiments, Ham’s F12 medium from Lonza is employed.

[0086] “DMEM / F12” or “DMEM:F12” means a 1 : 1 mixture of DMEM and Ham’s F12 medium (see Figure 12 which shows a data sheet for DMEM:F12 (1 : 1) from Lonza). DMEM / F12 (1 : 1) medium is also a widely adopted basal medium for supporting the growth of many different mammalian cells and is commercially available from multiple suppliers such as ThermoFisher Scientific (cat. no. 11330057), Sigma Aldrich (cat. no. D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present application. In preferred embodiments, the DMEM:F12 medium used herein is DMEM / F12 (1 : 1) medium available from Lonza under cat. no. 12-719F (which is DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0087] “M171” means medium 171, which has been developed as a basal medium for culturing normal human mammary epithelial cell growth (see Figure 13 which shows a data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely adopted and commercially available from suppliers such as ThermoFisher Scientific or, for example, Life Technologies Corporation (cat. no. M171500). Any commercially available M171 medium can be used in the present application. In preferred embodiments, the M171 medium used herein is M171 medium available from Life Technologies Corporation under cat. no. M171500.

[0088] " FBS " means fetal bovine serum (also referred to as " fetal bovine (calf) serum "), i.e., the portion of blood retained after the blood naturally coagulates and is then centrifuged to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement in eukaryotic cell in vitro cell culture because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. Preferably, FBS is obtained from members of the International Serum Industry Association (ISIA), which focuses on ensuring the safety and safe use of serum and animal-derived products by proper traceability of origin, authenticity of labels, and proper standardization and supervision. Suppliers of FBS as members of ISIA include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare with catalog number A15-151.

[0089] The culture medium for deriving a conditioned medium from umbilical cord mesenchymal stem cell culture as described herein may include DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v). The numerical values ​​of "% (v / v)" used herein refer to the volume of a single component relative to the final volume of the culture medium. This means, for example, if DMEM is present in the culture medium at a final concentration of about 55 to 65% (v / v), then 1 liter of culture medium contains about 550 to 650 ml of DMEM. In other embodiments, the culture medium may comprise DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In further embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0090] In addition to the above components, the culture medium may include supplements to the umbilical cord mesenchymal stem cells that are beneficial for culturing and for deriving a conditioned medium for inducing, stimulating and / or promoting hair growth and / or hair regeneration. For example, the culture medium as described herein may include epidermal growth factor (EGF). If present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some such embodiments, the culture medium may include EGF at a final concentration of about 10 ng / ml.

[0091] The culture medium described herein may also contain insulin. If present, the insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some such embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0092] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may comprise adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0093] As described herein, umbilical cord tissue can be cultured until a suitable number of (primary) mesenchymal stem cells (such as umbilical cord lining stem cells, Wharton's jelly or placental stem cells) grow out of the tissue. Typically, umbilical cord tissue is cultured until the cell outgrowth of the mesenchymal stem cells of the corresponding tissue reaches about 70% to about 80% confluence. It should be noted here that the term "confluency" is used in its conventional sense in the field of cell culture and is intended to be an estimate / indicator of the number of adherent cells in a culture dish or culture flask, referring to the proportion of the surface covered by cells. For example, 50% confluence means that approximately half of the surface is covered and there is still room for cells to grow. 100% confluence means that the surface is completely covered with cells and there is no longer room for cell monolayer growth.

[0094] In case the primary cells (mesenchymal stem cells) of appropriate quantity are obtained from corresponding tissue by the tissue separation block method, the mesenchymal stem cells are removed from the culture vessel for cultivating. By doing this, it is possible to obtain the master cell bank containing (primary) separation of for example umbilical cord or placenta mesenchymal stem cells. Usually, because mesenchymal stem cells are adherent cells, standard enzyme treatment is used to harvest cells. For example, the enzyme treatment can include trypsin digestion as described in International U.S. Patent Application 2006 / 0078993, International Patent Application WO2006 / 019357 or International Patent Application WO2007 / 046775, meaning that the cells grown outward can be harvested for further amplification by trypsin digestion (0.125% trypsin / 0.05% EDTA). If the mesenchymal stem cells gathered in the crops are for example used to produce master cell bank, then cell freezing preservation and storage can also be carried out for further use, as described below.

[0095] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculture. If a population of umbilical cord mesenchymal stem cells previously isolated from its natural environment is used, subculture or culture (the two terms are used interchangeably hereinafter) will also be performed. The subculture can also be started from frozen primary cells, i.e., from the master cell bank. For subculture, any suitable number of cells can be seeded in a culture vessel such as a cell culture plate. For this purpose, the mesenchymal stem cells can be grown at, for example, about 0.5 x 10 6 cells / ml to approximately 5.0 x 10 6 Cells are suspended in a suitable culture medium for subculture (most conveniently, the culture medium of the present invention) at a concentration of about 1.0 x 10 cells / ml. 6The cells are suspended at a concentration of 10 cells / ml for subculture. The subculture can be carried out by culturing in a simple culture bottle or in a multi-layer system that can be stacked in an incubator, such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (part of Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA). Alternatively, the subculture can also be carried out in a closed self-contained system (such as a bioreactor). Bioreactors of different designs are known to those skilled in the art, for example, parallel plates, hollow fibers or microfluidic bioreactors. See, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", cited above. Illustrative examples of commercially available hollow fiber bioreactors are Cell Expansion System (Terumo BCT, Inc), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048–1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell populations described herein is the Xuri Cell Expansion System available from GE Healthcare. If a working cell bank is to be produced under GMP conditions and a large number of cells are required, then, for example, It is particularly beneficial to culture mesenchymal stem cell populations in an automated system such as the Cell Expansion System.

[0096] The subculture of the mesenchymal stem cells of the present invention can be carried out in a culture medium as described herein. Therefore, the culture medium can be used to isolate a mesenchymal stem cell population from, for example, the amniotic membrane of the umbilical cord or Wharton's jelly and for subsequent cultivation of isolated primary cells by culture. Similarly, for cultivation, mesenchymal stem cells can be cultured until a suitable amount of cells are grown. In some embodiments, mesenchymal stem cells are cultured until the mesenchymal stem cells reach about 70% confluence, about 80% confluence, about 90% confluence, about 95% confluence, to about 96% or even 100% confluence.

[0097] The isolation / culturing of the mesenchymal stem cell population can be carried out under standard conditions for culturing mammalian cells. Typically, the method of isolating a mesenchymal stem cell population is typically carried out under the following conditions (temperature, atmosphere): the conditions are generally used to culture cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37° C. in a normal atmosphere containing 5% CO 2 , respectively. In this context, it is noted that the mesenchymal cell populations described herein can be derived from any mammalian species, such as humans, red deer, mice, rats, guinea pigs, pigs, rabbits, goats, horses, dogs, cats, sheep or monkeys. In an exemplary embodiment, the umbilical cord mesenchymal stem cells are red deer or human.

[0098] Once the desired / appropriate amount of mesenchymal stem cells are obtained from self-culture or passage culture, the mesenchymal stem cells are harvested by removing them from the culture container for cultivation. Usually, the mesenchymal stem cells are harvested again by enzyme treatment, including trypsin digestion of cells. The separated mesenchymal stem cells are subsequently collected and used directly or preserved for further use. Usually, they are preserved by cryopreservation. The term "cryopreservation" is used in this article with its conventional meaning to describe the process of preserving mesenchymal stem cells by cooling to a low subzero temperature (such as (usually) -80°C or -196°C (boiling point of liquid nitrogen)). Cryopreservation can be performed as known to those skilled in the art, and may include the use of a cryoprotectant such as dimethyl sulfoxide (DMSO) or glycerol, which slows down the formation of ice crystals in umbilical cord cells.

[0099] The isolated mesenchymal stem cell populations obtained by the culture and / or isolation methods described herein are highly defined and homogeneous. Such highly homogeneous mesenchymal stem cell populations derived from tissues such as umbilical cord amniotic membrane or Wharton's jelly are reported in WO 2018 / 067071A1 and WO 2019 / 199234A1. For example, as disclosed and shown in the experimental section of WO 2019 / 199234A1A1, at least about 90%, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in a population of umbilical cord mesenchymal stem cells (e.g., isolated from umbilical cord Wharton's jelly or amniotic membrane) express each of the following markers: CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR, meaning that this percentage of the isolated cell population expresses each of CD73, CD90, and CD105, and lacks expression of the following markers: CD34, CD45, and HLA-DR. This highly homogenous population of mesenchymal stem cells derived from the umbilical cord amniotic membrane meets the criteria for mesenchymal stem cells for cell therapy (see also the experimental section in WO 2019 / 199234A1, and, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra, cited above). In this context, it is noted that this population of mesenchymal stem cells can be obtained by the isolation method described in WO 2019 / 199234A1, or, if desired, by a different method (such as cell sorting).

[0100] In another aspect, the present invention relates to a method for inducing, stimulating and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating the hair of a subject with a conditioned medium derived from umbilical cord mesenchymal stem cells cultured in a medium as described herein, wherein the culture medium may further comprise mixing to obtain a final volume of 500 mL of culture medium:

[0101] i. 250 ml of DMEM

[0102] ii.118ml M171

[0103] iii. 118ml DMEM / F12

[0104] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0105] As mentioned above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, when using this method of preparing the medium, the final concentrations (v / v) in a total volume of 500 ml are as follows:

[0106] DMEM: 250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v)

[0107] M171: 118 ml, corresponding to 118 / 500 = 23.6% (v / v)

[0108] F12: 59 ml, corresponding to 59 / 500=11.8% (v / v).

[0109] The culture medium as described herein further comprises the addition of:

[0110] v. 1 ml of EGF stock solution (5 μg / ml) to give a final EGF concentration of 10 ng / ml, and

[0111] vi. Insulin 0.175 ml stock solution (14.28 mg / ml) to give a final insulin concentration of 5 μg / ml.

[0112] It should be noted here that, when mixing, the volume of above-mentioned these components i to vi obtains the substratum that final volume is 499.675ml.If no longer in substratum, add other components, then remaining 0.325ml (volume being added to 500ml) can be for example arbitrarily among component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of substratum is 500ml.In addition, also note that component i to iv need not add according to the order that they are listed, but certainly can use any order to make these components mix to obtain substratum of the present invention. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to achieve final concentrations as described herein, i.e., a final concentration of about 55 to 65% (v / v) of DMEM, a final concentration of about 5 to 15% (v / v) of F12, a final concentration of about 15 to 30% (v / v) of M171, and a final concentration of about 1 to 8% (v / v) of FBS.

[0113] In other embodiments, the method further comprises adding one or more of the following supplements to the DMEM: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) in a volume of 0.325 ml, thereby obtaining a total volume of 500 ml of culture medium. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows:

[0114] about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine,

[0115] About 1 to 10 μg / ml hydrocortisone,

[0116] About 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0117] In some embodiments, umbilical cord mesenchymal stem cells are cultured in a cell culture medium described herein, for example, in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 medium), M171 (culture medium 171), and FBS (fetal bovine serum), and the conditioned medium of the present invention is produced by removing the umbilical cord mesenchymal stem cells from the culture medium, wherein the conditioned medium is obtained by collecting the cell culture medium. In addition, in some instances, the culture medium in which the stem cells have been cultured can be replaced by a second cell culture medium, and the cells can be cultured in the second cell culture medium, which can be a cell culture medium that is the same as or different from the first cell culture medium. In some instances, the second cell culture medium can be a culture medium without growth factors and / or a serum-free culture medium. Alternatively, previously cultured umbilical cord mesenchymal stem cells (e.g., stored cultured stem cells) can be further cultured in a culture medium without growth factors and / or serum. In either case, the resulting culture medium is referred to as a conditioned medium. The resulting conditioned medium is harvested and can be further processed to produce a composition comprising a conditioned medium of the present invention.

[0118] In some embodiments, the conditioned medium of the present invention or a composition comprising the conditioned medium of the present invention is diluted in a carrier medium and has a final concentration of, for example, about 10% to about 80% (v / v), or has a final concentration of, for example, about 25% to about 50% (v / v). The conditioned medium of the present invention or a composition comprising the conditioned medium of the present invention can be diluted in any suitable diluent / carrier medium, as long as the carrier medium does not affect the function and safety of the conditioned medium when topically applied to the skin / scalp of the subject. For example, the conditioned medium can be diluted in PBS, water, basal medium or cream formulation. The basal medium used herein refers to a mixture containing sugars, amino acids, water, etc. required for cell survival, including commercially prepared culture media, such as, but not limited to, Dulbecco's modified Eagle's medium (DMEM), endothelial differentiation medium (EDM), minimum essential medium (MEM), basal medium Eagle (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (G-MEM) and Iscove's modified Dulbecco's medium. In addition, the conditioned medium to be used can be any basic medium suitable for growing animal cells, non-limiting examples of which include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Resveratrol Memorial Institute Medium (RPMI), Keratinocyte Medium (KM), KBM (Keratinocyte Basal Medium), EpiLife KM (Keratinocyte-EpiLife Medium), etc. In some examples, the conditioned medium comprises the cell culture medium described herein, such as a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).

[0119] As described above, the conditioned medium derived from umbilical cord mesenchymal stem cell culture of the present invention can also be included in the composition. The conditioned medium as described herein and according to the present invention or a composition comprising the conditioned medium can be applied to the scalp, face and other areas of the skin where hair can grow, particularly at the hairline. As used herein, the hairline should be understood as the hair roots on the surface of the subject's skin, and is not limited to the edge of the subject's hair (e.g., along the top of the forehead), but also includes the hair roots at any surface of the subject's skin, particularly the scalp. The area for applying the conditioned medium as described herein and according to the present invention or a composition comprising the conditioned medium includes not only the scalp, but also any part of the body skin where hair growth is desired. The skin to be treated with the conditioned medium of the present invention can be any skin area where hair can grow, for example, it can be an area where hair or other body hair is damaged due to scars caused by injury, or an area where a beauty care effect is desired (e.g., a wide forehead or M-shaped forehead, eyelashes or eyebrows). Therefore, the skilled person knows and can identify skin areas that can grow hair and are therefore suitable for inducing, stimulating and / or promoting hair growth and / or hair regeneration using the conditioned medium of the present invention.

[0120] In some embodiments, the composition described herein is a pharmaceutical or cosmetic composition for inhibiting hair loss and promoting hair growth. The composition can be formulated in any formulation conventionally prepared in the art and generally comprises a conditioned medium of the present invention and a cosmetic or pharmaceutically suitable acceptable carrier / receptor. The composition of the present invention may also contain conventional additives and adjuvants in the cosmetic, pharmaceutical or dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, spices, fillers, bactericides, odor absorbers and dyes or colorants. Typically, the composition can be used by directly applying it to the skin (particularly the scalp) or to the hair (such as hair grafts before hair transplantation) via dermal administration. Preferably, the composition comprising the conditioned medium of the present invention is a cosmetic composition. In some embodiments, the composition is suitable for topical application. Compositions suitable for topical application can be in liquid or viscous form. Examples include ointments, creams and lotions. The formulation 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 containing the conditioned medium of the present invention for topical administration should be understood as a formulation that can be absorbed into the hair follicle structure in a targeted manner, in particular, wherein the active ingredients in the conditioned medium of the present invention or the composition containing the conditioned medium of the present invention are able to fully penetrate the epidermis and dermis to reach the hair follicle and the hair follicle bulb matrix, but are not absorbed into the systemic circulation (see the Examples section for this). In some examples, the conditioned medium of the present invention or the composition containing the conditioned medium of the present invention passes through the skin surface and enters the hair follicle by diffusion through the epidermis or diffusion through the dermis. In some examples, the conditioned medium of the present invention is superficially injected under the scalp without being absorbed into the systemic circulation. The method of administering the conditioned medium of the present invention or the composition containing the conditioned medium can include any method disclosed in the art, and the frequency of its application can vary depending on the skin condition. Additionally, prior to administering the conditioned medium of the present invention or a composition comprising the conditioned medium, the subject's skin can be treated with microneedles. Prior to administering the conditioned medium of the present invention or a composition comprising the conditioned medium, any skin penetration enhancing component can be used. As shown in the experimental section, the subject's skin can be treated with Dermapen, a microneedle device that uses microneedles 1-3 mm in length to penetrate the skin.

[0121] As another example, the conditioned media derived from the culture of umbilical cord mesenchymal stem cells of the present application can be used in the preparation of hair grafts. As is generally known to those skilled in the art, a conventional hair transplant procedure involves taking hair from an area of the patient's scalp that contains permanent growth hair and implanting the hair into an area of the patient's scalp that is bald. In some examples of the present application, the conditioned media or composition comprising the conditioned media for the treatment of hair loss / hair thinning of the present application can be used in the preparation of hair grafts prior to implanting the hair into the bald area of the patient to treat hair loss / hair thinning, wherein the hair has been taken from the subject and incubated in the conditioned media of the present application to induce hair follicle growth, and then the hair is implanted into the bald area of the patient's skin, such as the scalp.

[0122] Thus, methods of treating a subject with conditioned media derived from the culture of umbilical cord mesenchymal stem cells are described. The method comprises administering to the hair of the subject or to isolated hair used in the preparation of hair grafts an effective amount of the conditioned media of the present application. Likewise, the present application relates to the use of the conditioned media and compositions comprising said conditioned media in inducing, stimulating and / or promoting hair growth and / or hair regeneration. Alternatively or additionally, the present application relates to the use of the conditioned media and compositions comprising said conditioned media in alleviating and / or reducing hair loss and / or hair thinning. According to the present application, said use comprises treating the hair of a subject with conditioned media derived from the culture of umbilical cord mesenchymal stem cells. As illustrated in the Examples section (see, for example, Examples 6-8), an "effective amount of conditioned media" can be the amount of conditioned media administered to the skin / scalp of the subject per treatment, such as 1 ml, 2.5 ml or 5 ml of conditioned media (either undiluted or diluted in carrier medium to a concentration of, for example, 50% (v / v)). The treatment process can comprise repeated administration of such effective amount over a period of time as described herein.

[0123] In principle, any subject is suitable for treatment by means of the conditioned media of the present application. For example, the subject to be treated can suffer from hair loss and / or hair thinning. Finally, the present application also provides a method of treating a non-human mammal or a human subject, in particular a subject or patient suffering from hair loss and / or hair thinning, said method comprising administering to the hair of the subject an effective amount of the conditioned media of the present application or a composition comprising said conditioned media.

[0124] Hair loss and / or thinning hair can be male or female hair loss, and can be caused by any disease or condition, particularly any disease or condition associated with hair loss and / or thinning hair and wishing / needing hair regeneration. Hair loss can be hormone-related hair loss, such as perimenopausal-related hair loss or postpartum-related hair loss. The subject may suffer from hair loss caused by a disease, including inflammation-induced diseases, such as alopecia, such as androgenic alopecia or alopecia areata; or telogen effluvium, such as viral-induced telogen effluvium, including the telogen effluvium induced by COVID-19 or dengue virus-induced telogen effluvium. The example of alopecia areata that can be treated with conditioned medium as described herein includes unifocal alopecia areata, multifocal alopecia areata, serpiginous alopecia, alopecia totalis or alopecia universalis. Hair loss can also be caused by illness, such as hair loss caused by the effect of systemic medical problems (such as thyroid disease), drug side effects or drug treatments (such as cancer chemotherapy) and nutritional deficiency states. In this regard, hair loss can be chronic or acute. In some instances, hair loss can be chronic. In some embodiments, the subject suffers from COVID-19 induced hair loss, particularly hair loss that begins after COVID-19 infection, for example, as described in the experimental section herein, the patient suffers from COVID-19 induced telogen effluvium (TE) (which refers to diffuse scalp hair loss). In some other embodiments, the subject suffers from androgenetic alopecia (AGA).

[0125] The present invention is further illustrated by the following non-limiting experimental examples.

[0126] The sequences used herein are depicted in Table 1 below.

[0127] Table 1. Sequences used in this article.

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Experimental Examples

[0135] Example 1: Isolation and culture of umbilical cord mesenchymal stem cells (CLMSCs)

[0136] 1. Preparation of Culture Medium for Treatment of CLMSCs

[0137] a. Prepare 500 ml of PTT-6 (culture / growth medium) by adding the following substances in the order listed:

[0138] i.DMEM, 250ml

[0139] ii.M171 118ml

[0140] iii. DMEM F12 118ml

[0141] iv. FBS 12.5ml (final concentration 2.5%)

[0142] v. EGF 1ml (final concentration 10ng / ml)

[0143] vi. Insulin 0.175 ml (final concentration 5 μg / ml)

[0144] The volume of above-mentioned components i to vi obtains the culture medium that final volume is 499.675ml.If no longer add other components in culture medium, then remaining 0.325ml (volume added to 500ml) can be any one among for example component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of culture medium is 500ml.Alternatively, can add the storing solution of antibiotic (such as penicillin-streptomycin-amphotericin (Amphotericin)) to obtain final volume is 500ml.Can also add one or more in the following fill-in of 0.325ml volume in described culture medium: adenine, hydrocortisone and 3,3 ', 5-triiodo-L-thyronine sodium salt (T3), obtain thus the culture medium that cumulative volume is 500ml.

[0145] vii. Label the bottle with "PTT-6," the date the medium was prepared, the operator's initials, and the expiration date followed by the phrase "Expiration." The expiration date is the earliest expiration date of any component or one month from the preparation date, whichever comes first.

[0146] b. In a 50 ml centrifuge tube, add 2.5 ml FBS to 47.5 ml HBSS to prepare rinse medium (Hank's buffered saline solution (HBSS) with 5% FBS without calcium or magnesium). Label the tube "Rinse Medium," the operator's initials, and the date the medium was prepared.

[0147] c. All media were sterile tested using Bactec Lytic / 10 (Dickinson & Company) and Bactec Pluc+Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared culture medium was injected into each bottle.

[0148] 2. Collection and Isolation of Human and Red Deer (RD) Umbilical Cord Tissue

[0149] Red deer (RD) umbilical cords were obtained from a New Zealand farm raising these animals for horn velvet, according to specific protocols. RD-CLMSCs were isolated from the umbilical cords according to the protocol described in International Patent Application WO 2006 / 019357A1. Human umbilical cord tissue (donated with the mother's informed consent) was processed for subsequent isolation of mesenchymal stem cells from the umbilical cords, as described in International Patent Applications WO 2018 / 067071A1 or WO 2019 / 199234A1.

[0150] Briefly, the umbilical cords of red deer and humans were rinsed clean and immediately transferred to a 500 ml sterile glass bottle containing culture transport medium (L-15 medium supplemented with 50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml amphotericin (fungizone), 50 μg / ml gentamicin; all reagents were purchased from Invitrogen) and then transferred to the laboratory. In the laboratory, stem cell extraction was performed in a laminar flow hood under sterile conditions. The umbilical cord was first transferred to a sterile stainless steel tray. All residual blood in the umbilical cord vessels was removed by multiple syringe washes using warm phosphate-buffered saline (PBS) supplemented with 5 IU / ml heparin (from Sigma). Pure PBS without heparin was used in the final wash. The umbilical cord tissue samples were then cut into 2 cm long segments and transferred to 10 cm diameter cell culture dishes, where they were further washed and sterilized with 70% ethanol and then washed several times with PBS containing an antibiotic cocktail (50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml amphotericin, 50 μg / ml gentamicin; all purchased from Invitrogen) until the solution became clear.

[0151] 3. Isolation and Culture of RD-CLMSCs and H-CLMSCs

[0152] First, the umbilical cord tissue is dissected to separate the amniotic membrane from Wharton's jelly (i.e., the umbilical cord stroma) and other internal components. The separated amniotic membrane is then cut into small segments (0.5 cm x 0.5 cm) for cell isolation. These small segments are then placed on tissue culture dishes under different cell culture conditions for tissue explant isolation to isolate mesenchymal stem cells.

[0153] To isolate / culture mesenchymal cells, tissue explants were immersed in PTT-6 medium (CellResearch Corp, Singapore) and maintained in a CO2 cell culture incubator at 37°C. The medium was changed every 2 or 3 days. Cell outgrowth was monitored under a light microscope. At approximately 70% confluence, cells were treated with trypsin (0.05% trypsin / 0.02% EDTA) for further expansion or cryopreservation. H-CLMSCs (#CLMC43) were also obtained from a CRC tissue bank.

[0154] Example 2: Derived from red deer umbilical cord lining mesenchymal stem cells (RD-CLMSC), human umbilical cord lining mesenchymal stem cells (H- Conditioned culture medium of CLMSCs and human foreskin fibroblasts (FSF)

[0155] Frozen vials containing RD-CLMSCs, H-CLMSCs, and human foreskin fibroblasts (FSF) as a control were retrieved from storage and quickly thawed in a water bath at 37°C. FSF were donated to CRC (CellResearch Corporation, Singapore) by the Stem Cell and Wound Healing Research Group, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore.

[0156] RD-CLMSC and H-CLMSC were cultured using PTT-6 medium (CellResearch Corporation, Singapore) at 37°C and 5% CO2. FSF were cultured using DMEM with 10% FCS at 37°C and 5% CO2. The cell culture medium was replaced every 2 to 3 days. When 80% to 100% confluent, the cell culture medium was removed, the cells were optionally rinsed in PBS, and then cultured in DMEM basal medium, wherein the culture medium optionally contained a water-soluble antioxidant but did not contain growth factors or serum. The culture dish was incubated for another 48 hours, and the spent culture medium was collected into a centrifuge tube and centrifuged at 1800 rpm for 10 minutes. The supernatant was collected as conditioned medium (CM) into labeled test tubes: red deer umbilical cord lining mesenchymal stem cell conditioned medium (RD-CLMSC-CM), human umbilical cord lining mesenchymal stem cell conditioned medium (H-CLMSC-CM), and human foreskin fibroblast conditioned medium (FSF-CM). The conditioned medium was stored at -80°C until use.

[0157] Example 3: Human dermal fibroblasts cultured in conditioned medium of RD-CLMSCs and FSF vs. DMEM / 10% FCS Expression of elastin and hyaluronic acid (HA) in human high density fibroblasts (HDFs)

[0158] The effects of conditioned media derived from cultures of red deer umbilical cord mesenchymal stem cells (RD-CLMSC) and human foreskin fibroblasts (FSF) on human skin were evaluated to elucidate the potential mechanisms of action of conditioned media from different sources (RD-CLMSC and FSF) on human skin and whether these mechanisms are related to the promotion of hair growth. To evaluate the effects of conditioned media on human skin, human dermal fibroblasts (HDFs) from the tissue bank of CellResearch Corporation (CRC) with the sources listed in the table below were selected and used. These HDFs were divided into normal fibroblasts (NF) obtained from individuals under 60 years old and aged skin fibroblasts (asF) obtained from individuals 60 years old and older.

[0159] Table 2: HDF and donor information

[0160]

[0161] method

[0162] HDFs were seeded in 96-well plates at a density of 10,000 cells / well in DMEM / 10% FCS (LifeTech Holdings, cat. no. 10270106). At approximately 80% confluence, the medium was removed and the remaining cells in the wells were washed once with PBS. The cells were then cultured in DMEM / 10% FCS (negative control) or RD-CLMSC-CM for 48 hours, followed by immunocytochemical staining for hyaluronic acid (HA) (My BioSource, cat. no. MBS2025717) and elastin (LifeTech Holdings, cat. no. MA 127129). Figure 1 A and Figure 2A. Briefly, cells were washed with 1 x PBS and then fixed in cold methanol for 10 minutes, followed by blocking with 2.5% normal horse serum (Vector Laboratories cat. no. PK-7200) for 20 minutes in a humidified box. Cells were then incubated with primary HA antibody (1 :50) and elastin antibody (1 :50) for 2 hours at room temperature. Cells were washed with TBS (VWR Life Science cat. no. 788) and 1% Tween solution (Sigma Cat. no. P9416) and then incubated with secondary antibody (Vector Laboratories cat. no. PK-7200) for 15 minutes. Cells were washed with 1 x TBS / 1% tween solution three more times and then incubated with ABC reagent (Vector Laboratories cat. no. PK-7200) for 15 minutes at room temperature. Finally, DAB (Dako cat. no. K3468) was added for colorimetric development. Hematoxylin (Sigma Cat. No. HHS16) was used for counterstaining. Images were taken with a brightfield phase-contrast microscope (Olympus) at 10x magnification. Expression of elastin and hyaluronic acid was quantified with ImageJ (National Institutes of Health, United States).

[0163] result

[0164] To compare the effects of RD-CLMSC-CM and DMEM / 10% FCS (control) on the modulation of elastin and HA expression in HDF, and to assess whether these effects are dependent on donor age and site of skin tissue procurement, this set of experiments selected HDF (n=14) from donors aged 23 to 73 years and multiple donor sites (forehead, eyelid, cheek, neck, temple) isolated from the skin bank. Cells were cultured in DMEM / 10% FCS or RD-CLMSC-CM for 48 hours, followed by immunocytochemical staining for elastin and HA. Expression levels were quantified by optical density using ImageJ (National Institutes of Health, United States). As shown in Figures Figure 1 A and 1B and Figure 2 As shown in Figures A and 2B, RD-CLMSC-CM upregulated elastin expression in HDF by 56% and HA expression by 83% compared to DMEM / 10% FCS. In summary, this set of experiments demonstrated that the conditioned medium of the present invention significantly upregulated HDF elastin and hyaluronic acid expression. This effect appeared to be independent of donor age or site of skin origin.

[0165] in conclusion

[0166] Without wishing to be bound by theory, it can be hypothesized that the upregulation of elastin and HA expression present in RD-CLMSC-CM is due to the presence of growth factors present in RD-CLMSC-CM including TGF-β, IGF, PDGF-BB, and FGF-7.

[0167] Example 4: Conditioned medium derived from H-CLMSC, RD-CLMSC and FSF cultures vs. DMEM / 10% FCS Proliferation of human dermal fibroblasts (HDF)

[0168] background

[0169] The proliferation properties of red deer umbilical cord lining mesenchymal stem cell-conditioned medium (RD-CLMSC-CM), human umbilical cord lining mesenchymal stem cell-conditioned medium (H-CLMSC-CM), and human foreskin fibroblast-conditioned medium (FSF-CM) were studied by exposing aged human dermal fibroblasts (HDFs) to the following media for 5 days and compared with DMEM / 10% FCS (control) ( Figure 3 In this experiment, we used slowly proliferating aging HDFs rather than normally proliferating HDFs to amplify the differences in the proliferation effects of different CMs. The superior proliferation properties suggest the presence of other protein growth factors in the conditioned medium that may have a positive impact on HDF proliferation.

[0170] method

[0171] Aged skin dermal fibroblasts (asF) (asF74 / asF75 / asF76, Table 2) were isolated from the skin of donors aged 60 years and above. Equal amounts of asF were seeded (in triplicate) into 24-well plates containing DMEM / 10% FCS and incubated at 37°C and 5% CO2 for 24 hours to allow cell adhesion. After 24 hours, the culture medium was removed from the culture plate and the wells were rinsed once with PBS. On day 0, HDFs were cultured in DMEM / 10% FCS (control), FSF-CM, H-CLMSC-CM or RD-CLMSC-CM. The culture medium was changed on day 2. The culture medium was removed on day 5 and the cells were rinsed once with PBS. The cells were trypsinized and observed under a microscope to ensure that the cells were completely detached, and then neutralized by adding 1 ml of DMEM / 10% FCS. The cell suspension was centrifuged at 1800 rpm for 10 minutes, the supernatant was discarded, and the pellet at the bottom of each tube was resuspended with 1 ml of DMEM. 50 μl of cell suspension was mixed with 50 μl of trypan blue (Sigma cat. no. T8154) and incubated for 10 minutes. Total cell counts were performed using a Countess-II automated cell counter system (Life technologies cat no. AMQAX10001).

[0172] result

[0173] like Figure 3 As shown, the highest increase in mean total cell counts was observed in the RD-CLMSC-CM group (113% increase over control) compared to DMEM / 10% FCS (control), followed by H-CLMSC-CM (112%) and FSF-CM (16%). These results indicate that the proliferation factors in RD-CLMSCs are equally potent as those in H-CLMSC-CM and demonstrate cross-species efficacy without toxic effects on HDFs. Without being bound by theory, the superior proliferation properties of CLMSC-CM (RD and H) compared to FSF-CM and FBS controls suggest the presence of additional protein growth factors in CLMSC-CM that may have a positive impact on HDF proliferation.

[0174] Example 5: Pro-migratory properties of RD-CLMSC-CM compared to FSF-CM evaluated in an in vitro "scratch" wound assay sex

[0175] background

[0176] The in vitro "scratch" assay mimics cell migration during wound healing and is a well-established method for measuring cell migration.

[0177] method

[0178] To establish a monolayer of cells, 100,000 HDFs were seeded per 100-mm dish and cultured to 100% confluence in DMEM / 10% FCS at 37°C and 5% CO2. A "scratch" was created by scraping the monolayer in a straight line using a p200 pipette tip. The dish was then rinsed once with PBS to remove any loose cells. The cells were immersed in FSF-CM or RD-CLMSC-CM for 5 days, and images were collected on days 0 and 5 to record cell migration from the gap edge.

[0179] result

[0180] like Figure 4 As shown, the pro-migratory properties of RD-CLMSC-CM and FSF-CM were compared by measuring the area remaining unfilled by HDFs after 5 days of culture in the test CM. On day 5, all "scratches" in the RD-CLMSC-CM-treated group were completely filled, while only approximately 80% were filled in the FSF-CM-treated group. These results demonstrate that the pro-migratory properties of RD-CLMSC-CM are more effective than those of FSF-CM. The significant increase in HDF migration in RD-CLMSC-CM can be attributed to the pro-migratory effects of VEGF, TGF-β1, and HGF.

[0181] Example 6: Promoting Hair Induction Ability Using Conditioned Medium Derived from RD-MLSC Culture

[0182] background

[0183] The purpose of this study was to evaluate the ability of the conditioned medium derived from umbilical cord mesenchymal stem cell culture as described in Example 2 to stimulate inactive or dormant hair follicles to improve hair growth in men with androgenetic alopecia (AGA). As an exemplary conditioned medium of the present invention, a novel cosmetic product formulated with the conditioned medium of the present invention was used. It contains proteins (especially growth factors) and exosomes naturally secreted by umbilical cord mesenchymal stem cells.

[0184] method

[0185] Select patients with thinning hair / hair loss. Patients who will not be able to return for follow-up examinations as planned; Patients who exhibited any adverse reactions to the product; patients with thyroid dysfunction; patients with vitamin and iron deficiencies; patients with hormonal imbalances; smokers; pregnant and lactating women; male patients with a Norwood grade of 5 or higher; and female patients with a Ludwig grade of 3 or higher were excluded from the study.

[0186] Once the patient has been selected, photographs are taken before the first treatment; see Figures 6A to 6GTreatments are given weekly, usually for a total of 6 treatments. A second set of photographs is taken during and / or upon completion of treatment; see Figures 6A to 6G .

[0187] The plan for each treatment is as follows:

[0188] ●Disinfect the scalp with 0.5% chlorhexidine.

[0189] ● Apply 2.5ml The Advanced Hair Growth System (formulated with conditioned medium derived from cultured mesenchymal stem cells derived from red deer umbilical cord) was needled into the treatment area using a derma stamp with 1mm, 1.2mm, and 1.5mm long microneedles.

[0190] ●After the microneedle stamp treatment, apply an additional 2.5ml and gently massage into the scalp.

[0191] ●Instruct patients not to wash their hair for at least 24 hours, and ideally 48 hours, after treatment.

[0192] result

[0193] like Figures 6A-6G As shown, the treating physician evaluated All patients experienced objective improvements in hair density and coverage within the area treated with the Advanced Hair Growth System. All patients also reported improvements in hair quality and coverage. Patients were followed up at three and six months to assess their progress.

[0194] Example 7: Conditioned medium derived from RD-MLSC culture stimulates improvement in patients with androgenetic alopecia (AGA) Hair growth

[0195] background

[0196] Androgenetic alopecia (AGA), also known as male and female pattern baldness, is the most common cause of hair loss in the world today. It can affect up to 50% of Caucasian men. The causes of AGA are complex and are widely considered to be a multifactorial disease influenced by genetics, environment, diet, and hormones. The essential manifestation is that the hair follicle growth cycle gradually shortens, and the hair produced during the cycle becomes shorter and thinner. Over time, the hair follicles in the scalp become completely inactive. The purpose of this study was to evaluate the preparation of a conditioned medium derived from umbilical cord red deer mesenchymal stem cells cultured as described in Example 2. The product's ability to stimulate underactive or dormant hair follicles to improve hair growth in men with AGA.

[0197] method

[0198] Five male patients with AGA, grade 3-4 on the Norwood Alopecia Scale, were selected. All participants were screened for vitamin deficiency, iron deficiency, and thyroid function. Patients with abnormal results were excluded from the study. Smokers were also excluded from the study. Once the patients were selected, photographs were taken before the first treatment. Treatments were administered weekly for a total of six treatments. A second set of photographs was taken at the completion of the six weekly treatments; see Figure 5 .

[0199] The plan for each treatment is as follows:

[0200] ●Disinfect the scalp with 0.5% chlorhexidine.

[0201] ● Apply 2.5 ml of a composition containing 80% conditioned medium and 20% aqueous hyaluronic acid ( The Advanced Hair Growth System, formulated with conditioned medium derived from cultured mesenchymal stem cells derived from red deer umbilical cord, is injected into the treatment area using a derma stamp with 2mm-long microneedles.

[0202] ●After the microneedle stamp treatment, apply an additional 2.5ml and gently massage into the scalp.

[0203] ●Instruct patients not to wash their hair for at least 24 hours, and ideally 48 hours, after treatment.

[0204] result

[0205] like Figure 5 As shown, at week 6, all patients had objective improvements in hair density and coverage within the area treated with the composition containing conditioned medium, as assessed by the treating physician. All patients also reported improvements in hair quality and coverage. Patients were followed up at 3 and 6 months to assess their progress.

[0206] Example 8: Case Study - Conditioned Medium from RD-MLSC Culture in Patients with COVID-19 Induced Scalp Stimulates improved hair growth in patients with hair loss

[0207] background

[0208] COVID-19-induced scalp hair loss can induce telogen effluvium (TE), which refers to diffuse scalp hair loss. A patient with severe, chronic TE who had not recovered spontaneously after 6 months of hair loss and had not responded to conventional treatments such as minoxidil and LED light therapy (Hairmax) was treated with a composition containing conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0209] Methods and Results

[0210] The patient's scalp was gradually treated by wiping with an alcohol swab, then 1 ml of a composition comprising conditioned medium derived from culture of umbilical cord mesenchymal stem cells was applied, massaged in until dry. The patient was instructed to wait until evening to wash the hair. Once a month, 5 treatments of conditioned medium derived from culture of umbilical cord mesenchymal stem cells were applied. The patient's hair density gradually increased with the regrowth of the frontal hairline and the vellus hair of the prefrontal region Figure 7 ) The hair fragility was reversed, with no breakage upon combing.

[0211] Example 9: Growth-promoting effect of conditioned medium on cultured human hair follicle dermal papilla cells (HFDPCs)

[0212] Testing plan and process

[0213] Hair growth is controlled by a unique repetitive cycle consisting of a growth phase, a catagen phase and a telogen phase. Dermal papilla cells (DPCs) are a group of specialized fibroblasts within the hair follicle bulb that have an important function in controlling hair growth, not only in the normal hair cycle but also in the pathogenesis of specific disorders such as androgenetic alopecia. Therefore, factors that affect the function of DPCs in hair loss are of great importance from a therapeutic point of view. The proliferative capacity of HFDPCs was evaluated by determining their metabolic activity using a high water-soluble tetrazolium salt - 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazolium salt (WST-8). HFDPCs were treated with different concentrations of minoxidil (0.0125-200 μg / mL; see Table 3, left column, used as a reference) or different dilutions of conditioned medium of the application (PC, dilution range 10-1280 fold; see Table 3, third column). The negative control (NC) refers to HFDPCs incubated with basal medium only.

[0214] Analysis results

[0215] The results are shown in Table 3 and, as shown in Figure 9 , for HFDPCs treated with the crude extract, there was an increase of about 10% in the number of cells at a concentration of 0.048 mg / mL compared to the control. For HFDPCs treated with minoxidil (reference), there was an increase of about 20% in the number of cells compared to the negative control at a dilution of 320 fold.

[0216] Table 3. Cell count of HFDPCs treated with conditioned medium (right), minoxidil (left, as a reference compound) and negative control (NC)

[0217]

[0218] Statistical analysis

[0219] All data are presented as mean ± standard deviation (SD). To compare the different treatments between the two groups, Student's t-test was used. p < 0.05 was considered significant, p < 0.01 was generally considered highly significant, and p < 0.001 was extremely significant. After calculation, p was 0.03.

[0220] in conclusion

[0221] When the samples treated with the conditioned medium according to the present invention were diluted 160 and 320 times, approximately 10% and 20% more cells were observed compared to the cells treated with the negative control (see also Figure 8 ); this growth-promoting effect in this model experiment was similar to that of the reference compound minoxidil, which has an optimal concentration of 0.2 to 2 μg / mL. Alternatively, in this experiment, the conditioned medium of the present invention was shown to increase dermal papilla cell proliferation by up to approximately 24%, which is comparable to the most effective concentration of minoxidil. It is noted that minoxidil is known to have undesirable and serious side effects, including but not limited to itching or rash, burning of the scalp, inflammation and pain at the hair roots, skin redness, facial swelling, and increased hair loss; see, for example, https: / / www.drugs.com / sfx / rogaine-side-effects.html. In contrast, no side effects have been observed to date with the conditioned medium of the present invention. Furthermore, while minoxidil is reportedly effective in treating hair loss in only 40% of patients, the conditioned medium of the present application has been found to have no side effects and to effectively induce, stimulate, and / or promote hair growth and hair regeneration in up to 95% of patients included in this study (data not shown). Therefore, this example demonstrates that the conditioned medium of the present invention offers a significant improvement over minoxidil.

[0222] Example 10: Effect of conditioned medium (CALECIM) on UV-induced primary normal human epidermal keratinocytes Anti-inflammatory activity of (NHEK)

[0223] Testing Process

[0224] The anti-inflammatory activity of the samples was tested against UVB-induced NHEK cells in 6-well culture plates. First, NHEK cells were seeded into 6-well plates at a density of 100,000 cells / well. After 24 hours, the cells were washed once with warm PBS and induced by UVB (40mJ / cm3). Then, PBS was removed and replaced with fresh culture medium containing the sample or vitamin D (4μM). The cells were incubated for another 24 hours at 37°C, 5% CO2. The cell culture supernatant was collected and stored at -80°C before use. Sample preparation: 1ml B was added to 49ml A to prepare the sample to be tested. The sample was diluted 2, 4, 8, 16, 32 and 64 times using NHEK culture medium.

[0225] TNF-α detection

[0226] a. Label a removable 8-well strip for the experiment. Wash the strip twice with approximately 400 μl of wash buffer per well, and aspirate the contents of the wells thoroughly after each wash.

[0227] b. Add 100 μL of each standard to the standard wells. Add 50 μL of sample diluent and 50 μL of sample to the appropriate wells. Then add 50 μL of biotin-conjugate to all wells.

[0228] c. Cover the wells and incubate with gentle shaking at room temperature for 2 hours.

[0229] d. Discard the solution and wash four times with 1X wash solution. Wash by injecting 400 μl of wash buffer into each well using a multichannel pipette. Complete removal of liquid at each step is crucial for the effectiveness of the experiment. After the final wash, remove any remaining wash buffer by aspiration or pouring. Invert the plate and blot dry with a clean paper towel.

[0230] e. Add 100 μl of the prepared streptavidin solution to each well and incubate at room temperature with gentle shaking for 1 hour.

[0231] f. Discard the solution and repeat the washing steps in step d.

[0232] g. Add 100 μl of TMB substrate solution to each well and incubate in the dark at room temperature for 30 minutes.

[0233] h. Add 100 μL of stop solution to each well and immediately read the sample at 450 nm.

[0234] Analysis results

[0235] Keratinocytes are the main target of UVB and play a key role in inflammation and immune regulation by inducing the release of proinflammatory cytokines (IL-1, IL-6, IL-8, IL-10, GM-CSF and TNF-α), cyclooxygenase products (PGE2) and matrix degrading enzymes such as metalloproteinases (MMPs). 2 Production of exemplary inflammatory cytokines TNF-α after UVB exposure. As expected, exposure to UVB induced an increase in TNF-α release (1.6 pg / ml vs. 485 pg / ml). Samples at dilutions of 2 and 4 showed the strongest inhibitory effect on TNF-α production, which was comparable to the inhibitory effect of vitamin D; see Figure 9 .

[0236] in conclusion

[0237] In this experiment, the conditioned medium of the present invention reduced the expression / production of a key inflammatory cytokine (TNF-α) by 30-fold, an effect comparable to that of vitamin D.

[0238] Example 11: Conditioned medium derived from RD-MLSC culture can effectively improve androgenic alopecia in men and women Hair growth in patients with AGA

[0239] Participants

[0240] Study participants (n=5 females and n=5 males) with a mean age of 34.1±14.2 years were recruited at a single site in Vienna, Austria. Patients with symptoms of hair thinning / hair loss and subjective perception of hair thinning / hair loss were recruited primarily through social media and study recruitment platforms.

[0241] On-site screening was performed to identify eligible subjects according to the exclusion and inclusion criteria shown in Table 4, the latter including the Hamilton-Norwood and Ludwig grades for male and female hair loss patients, respectively. All subjects consented to the use of their demographic and study outcome data for scientific and marketing purposes.

[0242] Table 4: Inclusion and Exclusion Criteria

[0243]

[0244]

[0245] Treatment options

[0246] Each of the 10 patients had their scalp disinfected with 0.5% chlorhexidine before application. The treatment area was selected subjectively based on the patient's area of ​​concern and there were slight differences between different patients. 2.5 ml of the conditioned medium composition of the present invention (containing 80% conditioned medium, 20% hyaluronic acid aqueous solution) was applied to the treatment area. Then, the corresponding area was microneedled using a microneedle stamp with a needle length of 0.5 mm (including 3 operations in alternating directions), and an additional 2.5 ml of the conditioned medium composition was applied and gently rubbed into the scalp. Patients were informed not to wash their hair for at least 24 hours after treatment, and preferably within 48 hours.

[0247] The treatment was repeated weekly for 12 weeks. Patients continued weekly treatment at home, administered the microneedle stamp and product weekly, and returned to the clinic for on-site evaluations at weeks 4, 6, 7, 10, and 12. This evaluation included subject-reported outcomes (e.g., psychological questionnaires) and a Canfield Scientific Software-based objective measurement of hair growth.

[0248] Measurement

[0249] Two trained assessors The D200-evo system was used with a Canfield Scientific (4 Wood Hollow Road, Parsippany, NJ 07054, USA) The software records all patient photos taken during treatment to assess hair changes. Canfield's non-invasive hair diagnostic tool provides immediate results during the diagnostic process without the need for hair cutting. To ensure objective data, photographs can be taken by researchers without the need for additional laboratory evaluation.

[0250] Standardized questionnaire

[0251] Standardized questionnaires were used to assess patients' quality of life and subjective treatment experience (not shown).

[0252] result

[0253] As shown in Table 5, 10 patients (mean age 34.1 ± 14.2 years, 50% female) were administered conditioned medium according to the treatment protocol described in the Methods section. Notably, male patients with Norwood alopecia types 4, 5, and 6 (meaning severe hair loss) participated in the study.

[0254] Table 5. Basic demographics of patients with alopecia (n=10) who had conditioned medium applied to their capillaries for 12 weeks according to the treatment regimen presented in the Methods section.

[0255]

[0256] After 12 weeks of treatment, if Figure 17 A and Figure 17 As shown in B, a significant increase in follicular units per square centimeter (sqcm) was detected (P < .05), with an associated decrease in interfollicular distance (P < .05). Figure 17 C and Figure 17 As shown in Table 7 and Table 7, the total hair count per square centimeter increased (P < .05), and thus the sum of the hair widths per square centimeter increased (P < .05). Of note, the number of hairs per follicular unit and the mean hair width did not change. No adverse reactions were reported. Figure 18 Based on the patients' subjective assessments, standardized questionnaires demonstrated significant improvement in hair growth, less need for hair covering, and improved quality of life related to hair loss, as shown in Figures A to 18C. Nine out of ten patients said they would recommend this conditioned medium product, indicating that this conditioned medium is also effective in treating severe hair loss, such as Norwood types 4, 5, and 6.

[0257] Example 12: Encapsulation of Conditioned Medium from RD-MLSC Cultures in Liposome Formulations and Modification of the Formulations Application of the drug in hair growth in patients with androgenic alopecia (AGA)

[0258] The phospholipid mixture Pro-Lipo was commercially available from Lucas Meyer Cosmetics. TM Neo encapsulated conditioned medium from RD-MLSC cultures to form bilayer liposomes suitable for topical administration. This liposome formulation was then used to treat patients with AGA. In this study, the conditioned medium was used in conjunction with other compounds reported to prevent and stop hair loss and / or stimulate hair growth. The use of Capixyl®, obtained from Lucas Meyer Cosmetics, has been reported. TM (a biomimetic peptide (acetyl tetrapeptide-3)), ribose, menthol and caffeine as compounds that stimulate hair growth. The encapsulated hair serum produced in this experiment has the following components:

[0259] Encapsulated hair growth serum

[0260] a. Conditioned medium: 50% (v / v)

[0261] b.Capixyl TM (Acetyl tetrapeptide-3): 2% (labeled amount, v / v)

[0262] c. Ribose: 0.5% (labeled amount, v / v)

[0263] d. Pro-Lipo Neo (liposome mixture)-delivery system: 20% (v / v)

[0264] e. Menthol: 0.1% (labeled amount, v (v))

[0265] f. Caffeine: 2% (labeled amount, v / v)

[0266] The balance is excipients.

[0267] The encapsulated hair growth serum was formulated as follows:

[0268] Step 1: Liposomal encapsulation of active ingredients

[0269] 1. Thaw conditioned medium (CM).

[0270] 2. Mix CM and Capixyl under low shear TM , ribose, caffeine and PLN.

[0271] 3. Stir for 30 minutes until smooth.

[0272] Step 2: Incorporate menthol into the serum matrix

[0273] 1. Dissolve menthol in the excipient matrix

[0274] 2. Turn off the heat to cool the excipient matrix.

[0275] Step 3: Incorporation of liposome-encapsulated active substances into the serum matrix

[0276] 1. Mix the liposome-encapsulated active ingredient, excipient matrix, and preservatives under low shear stirring until homogeneous.

[0277] 2. Make the pH of the final product 7.

[0278] Because this liposomal formulation improves the skin absorption of conditioned medium after topical application to the scalp, the hair growth serum thus produced was topically applied to the scalp of patients with AGA without the need for microneedling and the use of a microneedle stamp. During treatment with this liposomal formulation of conditioned medium, significant improvements in hair growth were observed (data not shown), demonstrating the effectiveness of this formulation for inducing and promoting hair growth.

[0279] The present invention is also characterized by the following items.

[0280] 1. A method for inducing, stimulating and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0281] 2. A method for alleviating and / or reducing hair loss and / or hair thinning, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

[0282] 3. The method according to item 1 or 2, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

[0283] 4. The method of claim 3, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

[0284] 5. The method of claim 3 or 4, 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).

[0285] 6. The method of any one of items 3-5, 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).

[0286] 7. The method according to any one of items 3 to 6, wherein the culture medium further comprises

[0287] (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or

[0288] (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or

[0289] (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or

[0290] (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or

[0291] (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or

[0292] (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), wherein optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

[0293] 8. The method according to any one of items 1 to 7, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

[0294] 9. The method according to any one of items 1 to 8, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population of the umbilical cord amniotic membrane, a culture of a mesenchymal stem cell population of the umbilical cord Wharton's jelly (WJ), a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed (MC) mesenchymal stem cell population of the umbilical cord.

[0295] 10. The method according to any one of items 1 to 9, wherein the conditioned medium is topically administered to the skin, in particular the scalp, of the subject.

[0296] 11. The method of any one of items 1-10, wherein the conditioned medium is administered to the hair line of the subject.

[0297] 12. The method of any one of items 1-11, wherein the conditioned medium is administered about once, twice, or three times per week for a period of three weeks, four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or ten weeks or more.

[0298] 13. The method according to any one of items 1 to 12, wherein the subject suffers from hair loss / thinning, in particular inflammation-induced hair loss / thinning, such as chronic inflammation-induced hair loss.

[0299] 14. A method according to any one of items 1 to 13, wherein the subject suffers from a disease or condition associated with hair loss / thinning hair, in particular alopecia such as androgenic alopecia, alopecia areata, alopecia totalis, alopecia universalis or COVID-19 induced alopecia.

[0300] 15. A method for producing a conditioned medium, the method comprising

[0301] (a) Umbilical cord mesenchymal stem cells were cultured in a medium containing DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum);

[0302] (b) removing the umbilical cord mesenchymal stem cells from the culture medium;

[0303] (c) optionally, culturing the umbilical cord mesenchymal stem cells in another culture medium, optionally, wherein the cell culture medium comprises a water-soluble antioxidant,

[0304] The conditioned medium is obtained by collecting the cell culture medium.

[0305] 16. The method according to item 15, wherein the cell culture medium does not contain growth factors and / or serum, and / or wherein the cells are cultured at a concentration of about 1 million cells / 1 mL.

[0306] 17. The method according to item 16, wherein the umbilical cord mesenchymal stem cells are red deer umbilical cord mesenchymal stem cells.

[0307] 18. The method according to item 16 or 17, wherein the conditioned medium derived from umbilical cord mesenchymal stem cells is used to induce, stimulate and / or promote hair growth and / or hair regeneration; and / or to alleviate and / or reduce hair loss and / or hair thinning in a subject.

[0308] 19. The method according to any one of items 16-18, wherein the conditioned medium is diluted in control medium.

[0309] 20. The method according to any one of items 1 to 14, wherein the conditioned medium is obtained or obtainable by the method according to any one of items 15 to 19.

[0310] 21. A conditioned medium derived from umbilical cord mesenchymal stem cells, the conditioned medium being obtained or obtainable by the method according to any one of items 15 to 19.

[0311] 22. A composition comprising the conditioned medium of item 21.

[0312] 23. Use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture for inducing, stimulating and / or promoting hair growth and / or hair regeneration; and / or for alleviating and / or reducing hair loss and / or hair thinning in a subject.

[0313] 24. The use according to item 23, wherein the conditioned medium derived from umbilical cord mesenchymal stem cell culture is obtained or obtainable by the method according to any one of items 15 to 19.

[0314] 25. Use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture in the preparation of a medicament for inducing, stimulating and / or promoting hair growth and / or hair regeneration; and / or for alleviating and / or reducing hair loss and / or hair thinning in a subject.

[0315] 26. The use of the conditioned medium according to item 25, wherein the use comprises treating the hair of a subject with the conditioned medium derived from umbilical cord mesenchymal stem cell culture.

[0316] 27. The use according to item 25 or 26, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

[0317] 28. The use according to item 27, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

[0318] 29. The use according to item 27 or 28, 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).

[0319] 30. The use according to any one of items 26-28, 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).

[0320] 31. The use according to any one of items 26-30, wherein the culture medium further comprises

[0321] (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or

[0322] (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or

[0323] (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or

[0324] (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or

[0325] (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or

[0326] (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3),

[0327] Optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

[0328] 32. The use according to any one of items 25 to 31, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

[0329] 33. The use according to any one of items 25 to 32, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population of the umbilical cord amniotic membrane, a culture of a mesenchymal stem cell population of the umbilical cord Wharton's jelly (WJ), a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed (MC) mesenchymal stem cell population of the umbilical cord.

[0330] 34. The use according to any one of items 25 to 33, wherein the use comprises topically administering the conditioned medium to the skin, in particular the scalp, of a subject.

[0331] 35. The use according to any one of items 25-34, wherein the use comprises administering the conditioned medium to the hair line of a subject.

[0332] 36. The use according to any one of items 25-35, wherein the conditioned medium is administered about once, twice or three times per week for a period of three weeks, four weeks, or five weeks or six weeks, or seven weeks, or eight weeks or ten weeks or more.

[0333] 37. The use according to any one of items 25-36, wherein the subject suffers from hair loss / thinning, in particular inflammation-induced hair loss / thinning, such as chronic inflammation-induced hair loss.

[0334] 38. The use according to any one of items 25-37, wherein the subject suffers from a disease or condition associated with hair loss / thinning hair, in particular alopecia such as androgenic alopecia, alopecia areata, alopecia totalis, alopecia universalis or COVID-19 induced alopecia.

[0335] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0336] All patents and publications mentioned in this specification represent the levels of ordinary skill in the art to which the invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually incorporated by reference.

[0337] The invention described herein in an illustrative manner can be practiced in the absence of any element or elements, or any limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprise," "include," and "contain" are to be construed broadly and without limitation. Furthermore, the terms and expressions employed herein have been used as words of description, not limitation, and their use is not intended to exclude any equivalents of the features shown and described, or portions thereof, but rather to recognize that various modifications may be made within the scope of the claimed invention. Thus, it should be understood that while the invention has been specifically disclosed with reference to preferred embodiments and optional features, modifications and variations of the invention presented herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the claimed invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This generic description of the invention is encompassed, without precondition or negative limitation, by removing any subject matter from the genus, whether or not the deleted matter is specifically described herein. Furthermore, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will be apparent from the following claims.

Claims

1. A method for inducing, stimulating and / or promoting hair growth and / or hair regeneration, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

2. A method for alleviating and / or reducing hair loss and / or hair thinning, wherein the method comprises treating a subject's hair with a conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

3. The method according to claim 1 or 2, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

4. The method of claim 3, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

5. The method of claim 3 or 4, 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).

6. The method of any one of claims 3 to 5, 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).

7. The method according to any one of claims 3 to 6, wherein the culture medium further comprises (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), Optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

8. The method according to any one of claims 3 to 7, further comprising removing the umbilical cord mesenchymal stem cells from the culture medium; and culturing the umbilical cord mesenchymal stem cells in an additional culture medium, optionally wherein the additional cell culture medium comprises a water-soluble antioxidant, wherein said conditioned medium is obtained by collecting said additional cell culture medium.

9. The method according to claim 8, wherein the additional cell culture medium does not comprise growth factors and / or is a serum-free medium and / or wherein the cells are cultured at a concentration of about 1 million cells / 1 ml.

10. The method according to claim 9, wherein the additional culture medium is a basal medium suitable for culturing mesenchymal stem cells, preferably a serum-free medium.

11. The method according to claim 10, wherein the basal 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.

12. The method of any one of claims 8 to 11, wherein the conditioned medium is diluted in a carrier medium, wherein the final concentration of the conditioned medium is preferably about 10% to about 90% (v / v) of the total volume of the composition containing the conditioned medium, including a final concentration of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%. 13 . The method according to claim 1 , wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

14. The method according to any one of claims 1 to 12, wherein the conditioned medium is derived from a culture of an umbilical cord amniotic membrane mesenchymal stem cell population, a culture of an umbilical cord Wharton's jelly (WJ) mesenchymal stem cell population, a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of an umbilical cord mixed (MC) mesenchymal stem cell population.

15. The method according to any one of claims 1 to 14, wherein treating comprises topically applying the conditioned medium to the skin, in particular the scalp, of the subject, or injecting the conditioned medium into the skin, in particular the scalp, of the subject.

16. The method of any one of claims 1-15, wherein treating comprises applying the conditioned medium to the hair line of the subject.

17. The method of any one of claims 1-16, wherein the conditioned medium is administered about once, twice, or three times per week for a period of three, four, five, six, seven, eight, ten, or more weeks.

18. The method according to any one of claims 1 to 17, wherein the subject suffers from hair loss / thinning, in particular inflammation-induced hair loss / thinning, hair loss / thinning due to viral or bacterial infection, hair loss due to medication or hormone-related hair loss.

19. The method of claim 18, wherein the inflammation-induced hair loss / hair thinning is chronic inflammation-induced hair loss.

20. The method of claim 18, wherein the hormone-related hair loss / hair thinning is perimenopause-related hair loss or postpartum-related hair loss.

21. The method of claim 18, wherein the hair loss due to drug therapy is hair loss due to cancer chemotherapy.

22. The method according to any one of claims 1 to 21, wherein the subject suffers from a disease or condition associated with hair loss / thinning hair, in particular telogen effluvium, alopecia such as androgenic alopecia, alopecia areata, alopecia universalis or COVID-19 induced alopecia.

23. The method of claim 22, wherein the alopecia areata is unifocal alopecia areata, multifocal alopecia areata, serpiginous alopecia, alopecia totalis, or alopecia universalis.

24. The method of any one of claims 1 to 23, wherein the hair loss is male hair loss or female hair loss.

25. The method of claim 24, wherein the subject is a male, and wherein the male has been diagnosed with type I, type II, type III, type IV, type V hair loss, or type VI hair loss according to the Norwood classification.

26. The method of claim 24, wherein the subject is female, and wherein the female has been diagnosed with type I (including type Ia, type Ib, type Ic, and type Id), type II (type IIa and type IIb) hair loss, or type III hair loss according to the Ludwig classification.

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

28. The method of claim 27, wherein the additional cell culture medium does not comprise growth factors and / or is a serum-free medium and / or wherein the cells are cultured at a concentration of about 1 million cells / 1 ml.

29. The method according to claim 28, wherein the umbilical cord mesenchymal stem cells are derived from an umbilical cord amniotic membrane mesenchymal stem cell population, an umbilical cord Wharton's jelly (WJ) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, or an umbilical cord mixed (MC) mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cells are preferably human or red deer umbilical cord mesenchymal stem cells.

30. The method according to any one of claims 28 or 29, wherein the additional culture medium is a basal medium suitable for culturing mesenchymal stem cells, preferably a serum-free medium.

31. The method of claim 30, wherein the basal 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.

32. according to the method described in any one of claims 28 to 31, wherein the culture medium of step (a) comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

33. The method of claim 32, 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).

34. The method of claim 33, 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).

35. The method of any one of claims 27 to 34, wherein the conditioned medium is diluted in a carrier medium, wherein the final concentration of the conditioned medium is preferably about 10% to about 90% (v / v) of the total volume of the composition containing the conditioned medium, including a final concentration of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%.

36. The method of claim 35, wherein the carrier medium is a physiological buffer, water, an aqueous solution of extracellular matrix components, or a basal medium.

37. The method of claim 36, wherein the aqueous solution of the extracellular matrix component is an aqueous solution of hyaluronic acid, an aqueous solution of collagen, or an aqueous solution of fibronectin.

38. The method of any one of claims 27 to 37, wherein the conditioned medium is encapsulated in liposomes.

39. The method of claim 38, wherein the liposomes are made from a mixture of phospholipids.

40. The method according to any one of claims 28 to 39, wherein the conditioned medium derived from umbilical cord mesenchymal stem cells is used to induce, stimulate and / or promote hair growth and / or hair regeneration; and / or to alleviate and / or reduce hair loss and / or hair thinning in a subject.

41. The method of any one of claims 1 to 26, wherein the conditioned medium is obtained or obtainable by the method of any one of claims 27 to 40.

42. A conditioned medium derived from umbilical cord mesenchymal stem cells, obtained or obtainable by the method according to any one of claims 27 to 40.

43. A composition comprising the conditioned medium of claim 42.

44. Use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture for inducing, stimulating and / or promoting hair growth and / or hair regeneration; and / or for alleviating and / or reducing hair loss and / or hair thinning in a subject, wherein the use is preferably for cosmetic purposes.

45. The use according to claim 43, wherein the conditioned medium derived from umbilical cord mesenchymal stem cell culture is obtained or obtainable by the method according to any one of claims 27 to 40.

46. ​​Use of a conditioned medium derived from umbilical cord mesenchymal stem cell culture in the preparation of a medicament for inducing, stimulating and / or promoting hair growth and / or hair regeneration; and / or for alleviating and / or reducing hair loss and / or hair thinning in a subject.

47. The use according to claim 46, wherein the use comprises treating the hair of a subject with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

48. The use according to claim 46 or 47, wherein the conditioned medium is obtained or obtainable by culturing umbilical cord mesenchymal stem cells in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

49. The use according to claim 48, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

50. The use according to claim 48 or 49, 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).

51. The method of claim 46, 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).

52. The use according to any one of claims 46 to 51, wherein the culture medium further comprises (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml, or (ii) wherein the culture medium contains EGF at a final concentration of 10 ng / ml, or (iii) wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml, or (iv) wherein the culture medium contains insulin at a final concentration of 5 μg / ml, or (v) wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), or (vi) wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), Optionally, the culture medium comprises adenine at a final concentration of 0.01 to 0.1 μg / ml adenine, hydrocortisone at a final concentration of 0.1 to 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

53. The use according to any one of claims 44 to 52, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

54. The method of any one of claims 44 to 53, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population of the umbilical cord amniotic membrane, a culture of a mesenchymal stem cell population of the umbilical cord Wharton's jelly (WJ), a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed (MC) mesenchymal stem cell population of the umbilical cord.

55. The use according to any one of claims 44 to 54, wherein the use comprises topically administering the conditioned medium to the skin, particularly the scalp, of a subject.

56. The use of any one of claims 44 to 55, wherein the use comprises administering the conditioned medium to the hair line of a subject.

57. The use of any one of claims 44 to 56, wherein the conditioned medium is administered about once, twice, or three times per week for a period of three weeks, four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or ten weeks or more.

58. The use according to any one of claims 44 to 57, wherein the subject suffers from hair loss / thinning, in particular inflammation-induced hair loss / thinning, hair loss / thinning due to viral or bacterial infection, hair loss due to medication or hormone-related hair loss.

59. The use according to claim 58, wherein the inflammation-induced hair loss / hair thinning is chronic inflammation-induced hair loss.

60. The use according to claim 59, wherein the hormone-related hair loss / hair thinning is perimenopause-related hair loss or postpartum-related hair loss.

61. The method of claim 58, wherein the hair loss due to drug therapy is hair loss due to cancer chemotherapy.

62. The use according to any one of claims 42 to 59, wherein the subject suffers from a disease or condition associated with hair loss / thinning hair, in particular telogen effluvium, alopecia such as androgenic alopecia, alopecia areata, alopecia universalis or COVID-19 induced alopecia.

63. The method of claim 60, wherein the alopecia areata is unifocal alopecia areata, multifocal alopecia areata, serpiginous alopecia, alopecia totalis or alopecia universalis.

64. The use according to any one of claims 42 to 61, wherein the hair loss is male pattern balding or female pattern balding.

65. The use of claim 62, wherein the subject is a male, and wherein the male has been diagnosed with type I, type II, type III, type IV hair loss, or type V hair loss according to the Norwood classification.

66. The use of claim 63, wherein the subject is a female, and wherein the female has been diagnosed with type I (including type Ia, type Ib, type Ic, and type Id), type II (type IIa and type IIb) hair loss, or type III hair loss according to the Ludwig classification.

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