Use of conditioned medium derived from cultures of umbilical cord mesenchymal stem cells to induce, stimulate, and promote hair growth and regeneration

Abstract

The present invention relates to a method for inducing, stimulating, and / or promoting hair growth and / or hair regeneration, comprising treating a subject's hair with conditioned medium derived from culturing umbilical cord mesenchymal stem cells. The present invention also relates to a method for alleviating and / or reducing hair loss and / or thinning, comprising treating a subject's hair with conditioned medium derived from culturing umbilical cord mesenchymal stem cells. The present invention also relates to a method for producing conditioned medium, comprising (a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), and (b) removing the umbilical cord mesenchymal stem cells from the culture medium, wherein the conditioned medium is obtained by recovering the cell culture medium. The present invention also relates to a conditioned medium obtained or obtainable by this production method, a composition thereof, and uses thereof. TIFF2025538460000011.tif174161

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from International Patent Application No. PCT / SG2022 / 050834, filed November 17, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to methods for inducing, stimulating, and / or promoting hair growth and / or hair regeneration, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. Similarly, the present invention relates to methods for alleviating and / or reducing hair loss and / or thinning, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. These methods are non-therapeutic and cosmetic, and can be used to treat hair loss and / or thinning in both men and women.

[0003] The present invention also relates to a conditioned medium, its use, compositions containing it, and a method 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 includes a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), and b) removing the umbilical cord mesenchymal stem cells from the culture medium, wherein the conditioned medium is obtained by recovering the cell culture medium. Following step b) of the method for producing a conditioned medium, the method can include one or more additional steps of culturing the umbilical cord mesenchymal stem cells in a second (further) cell culture medium, optionally containing a water-soluble antioxidant. The second cell culture medium may be growth factor- and / or serum-free. Furthermore, the cells can be cultured in this second cell culture medium at a concentration of about 1 million cells per ml. The culturing of umbilical cord mesenchymal stem cells in step c) can be carried out in the same culture medium as in step a), i.e., a culture 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 in step a). The present invention also relates to a conditioned medium derived from the culture of umbilical cord mesenchymal stem cells and obtained or obtainable by the production method of the present invention, uses thereof, and compositions comprising the conditioned medium.

[0004] The present invention also relates to the use of the conditioned medium and compositions thereof to induce, stimulate, and / or promote hair growth and / or hair regrowth.The present invention also relates to the use of the conditioned medium and compositions thereof to alleviate and / or reduce hair loss and / or thinning in a subject. [Background technology]

[0005] Background of the Invention Hair consists of a hair shaft and a hair root. The hair shaft is the part visible on the skin's surface, while the root is below the skin's surface. At the base of the hair follicle is the dermal papilla, a pear-shaped structure formed by a group of fibroblasts that supplies blood to the hair root, delivers nutrients for new hair production, and controls the regeneration of the hair follicle throughout its life cycle.

[0006] Normal hair follicles undergo a regeneration cycle defined by the growth phase (anagen), catagen, resting phase (telogen), and shedding phase (exogen). Anagen is the growth phase of the hair follicle and is responsible for the regrowth portion of the hair follicle cycle. During the anagen phase, fibroblasts in the dermal papilla secrete numerous growth factors to maintain the active proliferation and differentiation of keratinocytes in the proximal hair bulb, which form the hair fiber. At the end of the growth phase, the dermal papilla detaches from the follicle, cutting off its blood supply and potentially leading to hair fiber pluck and hair loss.

[0007] According to the American Hair Loss Association, two-thirds of people over 35 years of age experience signs of hair loss. While hair loss is more prevalent in men, it also commonly affects women in their 50s and 60s. Thus, in recent years, hair loss has become increasingly prevalent not only among men but also among women and young people. For example, hair loss can be caused by a variety of factors, including genetic traits and internal factors such as psychological stress in daily life. In relation to this, with industrial development, environmental pollution, stress, and the aging population, the signs of hair loss have become more severe. Furthermore, with the advent of the well-being era, interest in quality of life and appearance has also increased. Furthermore, hair loss can also be associated with other factors, such as illness, hormonal changes, or medication side effects. These can affect and inhibit hair production through abnormal hair follicle cycles and altered hair follicle morphology, leading to physical destruction of hair follicles.

[0008] For example, alopecia areata is thought to be an organ-specific autoimmune disease resulting from a loss of immune privilege in hair follicles (HFs), and therefore, treatments are mostly immunosuppressive. As another example, androgenetic alopecia (AGA), also known as male and female pattern baldness, is the most common cause of hair loss worldwide today. It can affect up to 50% of Caucasian men. AGA has many causes and is widely recognized as multifactorial, involving genetic, environmental, dietary, and hormonal influences. In fact, the hair follicle growth cycle shortens, and the hairs produced during that cycle become shorter and thinner. Over time, hair follicles become completely inactive within the scalp. Furthermore, hair loss can also be induced after viral infections such as COVID-19 or dengue virus infection, as recently reported in, for example, Hussain N, Agarwala P, Iqbal K, Omar HMS, et al. A systematic review of acute telogen effluvium, a harrowing post-COVID-19 manifestation. J Med Virol. 2022 Apr;94(4):1391-1401. doi: 10.1002 / jmv.27534 (Non-Patent Document 1) or Chu & Yang, Dengue-associated telogen effluvium: A report of 14 patients. Dermatologica Sinica, Volume 35, Issue 3, September 2017, Pages 124-126 (Non-Patent Document 2).

[0009] Many types of hair growth and restoration agents are commercially available to treat the above-mentioned hair loss phenomenon. The only drug therapies specifically approved by the U.S. Food and Drug Administration (FDA) are minoxidil and finasteride, both of which may have numerous side effects. Furthermore, while these two compounds are effective in preventing hair loss, they have minimal effect on hair growth. Therefore, there is a need to develop cost-effective and safe hair loss prevention technologies that can not only prevent hair loss but also effectively promote hair growth and regrowth.

[0010] Thus, there remains a need to find new compounds and methods for promoting hair growth and regrowth. It is therefore an object of the present invention to provide such compounds and methods. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Hussain N, Agarwala P, Iqbal K, Omar HMS, et al. A systematic review of acute telogen effluvium, a harrowing post-COVID-19 manifestation. J Med Virol. 2022 Apr;94(4):1391-1401. doi: 10.1002 / jmv.27534 [Non-patent document 2] Chu & Yang, Dengue-associated telogen effluvium: A report of 14 patients. Dermatologica Sinica, Volume 35, Issue 3, September 2017, Pages 124-126 Summary of the Invention

[0012] This object is achieved by a method, a conditioned medium and uses thereof having the features of the independent claims.

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

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

[0015] In a third aspect, the present invention provides a method for producing a conditioned medium, the method comprising the steps of: (a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum); (b) removing the umbilical cord mesenchymal stem cells from the culture medium; and (c) optionally culturing the umbilical cord mesenchymal stem cells in a further culture medium, preferably serum-free and optionally containing a water-soluble antioxidant, wherein the conditioned medium is obtained by recovering the cell culture medium. In step (a) of this method, the umbilical cord mesenchymal stem cells can be cultured until they reach about 70%, about 80%, about 90%, about 95%, or even 100% confluency (complete confluency). In optional step (c) of this method, the mesenchymal stem cells can be further cultured for a suitable period of time, which can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0016] In a fourth aspect, the present invention provides conditioned medium from umbilical cord mesenchymal stem cells obtained or obtainable by a method according to 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 use of conditioned medium derived from a culture of umbilical cord mesenchymal stem cells for inducing, stimulating and / or promoting hair growth and / or hair regrowth and / or for alleviating and / or reducing hair loss and / or thinning in a subject. [Brief explanation of the drawings]

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

[0020] [Figure 1A] Figures 1A and 1B show representative images of immunohistochemical staining for elastin in human dermal fibroblasts (HDFs) after 48 hours of culture in DMEM / 10% FCS (control) and 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 various donor profiles (subjects aged 23-73 years, skin from various sites: forehead, eyelid, cheek, neck, and temple) 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). [Figure 1B] See legend to Figure 1A. [Figure 2A]Figure 2A and Figure 2B show representative images of immunohistochemical staining for hyaluronic acid (HA) in HDFs after 48 hours of culture in DMEM / 10% FCS (control) and RD-CLMSC-CM. To compare the effects of DMEM / 10% FCS and RD-CLMSC-CM on HA expression, HDFs (n = 14) with various donor profiles (subjects aged 23-73 years, skin from various sites: forehead, eyelid, cheek, neck, and temple) were cultured in DMEM / 10% FCS (control) and RD-CLMSC-CM for 48 hours, followed by immunocytochemical staining for HA. Optical density was measured using ImageJ. RD-CLMSC-CM upregulated hyaluronic acid expression in HDFs by 83% compared to HDFs cultured in DMEM / 10% FCS (control). [Figure 2B] See legend to Figure 2A. [Figure 3] Total cell counts were compared 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 HDFs from aged skin. Starting with the same seeding density, the mean total cell count was calculated for each group after 5 days of incubation in the test medium. Compared to the control, the greatest increase in mean 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%). [Figure 4] An in vitro "scratch" wound assay was performed to compare the migration-promoting 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 with a p200 pipette tip on a confluent human dermal fibroblast (HDF) monolayer, and images were taken at days 0 and 5 to track migration progress. The "scratch" wound was completely filled by HDFs cultured in RD-CLMSC-CM at day 5, compared with 80% in FSF-CM. [Figure 5] Shown are top views of the scalp before treatment (left) and after 6 weeks and 6 treatments (right) with a composition comprising conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. [Figure 6A-1] Figures 6A-G show top views of the scalp before and after treatment with a composition containing conditioned medium derived from cultures of mesenchymal stem cells from the amniotic membrane of red deer umbilical cords (red deer umbilical cord lining mesenchymal stem cell conditioned medium) (Figures 6B-G) or at weekly intervals during the treatment period (Figures 6A, B). [Figure 6A-2] See legend to Figure 6A-1. [Figure 6A-3] See legend to Figure 6A-1. [Figure 6A-4] See legend to Figure 6A-1. [Figure 6A-5] See legend to Figure 6A-1. [Figure 6B-1] See legend to Figure 6A-1. [Figure 6B-2] See legend to Figure 6A-1. [Figure 6C-1] See legend to Figure 6A-1. [Figure 6C-2] See legend to Figure 6A-1. [Figure 6C-3] See legend to Figure 6A-1. [Figure 6C-4] See legend to Figure 6A-1. [Figure 6C-5] See legend to Figure 6A-1. [Figure 6C-6] See legend to Figure 6A-1. [Figure 6C-7] See legend to Figure 6A-1. [Figure 6C-8] See legend to Figure 6A-1. [Figure 6C-9] See legend to Figure 6A-1. [Figure 6C-10] See legend to Figure 6A-1. [Figure 6C-11] See legend to Figure 6A-1. [Figure 6D] See legend to Figure 6A-1. [Figure 6E] See legend to Figure 6A-1. [Figure 6F] See legend to Figure 6A-1. [Figure 6G-1] See legend to Figure 6A-1. [Figure 6G-2] See legend to Figure 6A-1. [Figure 6G-3] See legend to Figure 6A-1. [Figure 6G-4] See legend to Figure 6A-1. [Figure 6G-5] See legend to Figure 6A-1. [Figure 6G-6] See legend to Figure 6A-1. [Figure 6G-7] See legend to Figure 6A-1. [Figure 6G-8] See legend to Figure 6A-1. [Figure 6G-9] See legend to Figure 6A-1. [Figure 6G-10] See legend to Figure 6A-1. [Figure 6G-11] See legend to Figure 6A-1. [Figure 6G-12] See legend to Figure 6A-1. [Figure 6G-13] See legend to Figure 6A-1. [Figure 6G-14] See legend to Figure 6A-1. [Figure 7] Top views of the scalp of a patient with COVID-19-induced hair loss, showing visible scalp, brittle hair, and poor hair density; the top photo shows the scalp before treatment. The middle photo shows the top view of the patient's scalp three months after treatment, showing less visible scalp and the beginning of regrowth at the frontal hairline. The bottom photo shows the results six months after five monthly applications of the stem cell serum, showing almost no visible scalp and restored hair density. [Figure 8-1]FIG. 8 shows human dermal papilla cells (HFDPCs) before treatment, after treatment with minoxidil at a concentration of 2 μg / mL, minoxidil at a concentration of 0.2 μg / mL, and after treatment with conditioned medium diluted 160-fold (legend "PC 160-fold dilution") and 320-fold (legend "PC 320-fold dilution"), compared to negative controls (NC) diluted 160-fold and 320-fold (legend "NC 320-fold dilution" and "PC 320-fold dilution"). [Figure 8-2] See description of Figure 8-1. [Figure 9] Figure 9 shows the release of TNF-α in normal human epidermal keratinocytes (NHEK) after exposure to UVB (UV+) and without exposure to UVB (UV-) as a negative control. Suppression of TNF-α production in the presence of 2-, 4-, 8-, 16-, 32-, and 64-fold diluted conditioned medium and in the presence of vitamin D. Figure 9 shows that in this experiment, the conditioned medium of the present invention reduced the expression / production of TNF-α, a key inflammatory cytokine, by 30-fold, comparable to vitamin D. [Figure 10-1] FIG. 10 shows the Lonza technical information sheet for Dulbecco's Modified Eagle's Medium (including the catalog number for DMEM) used in the preparation of the specific medium example (PTT-6) in the experimental section. [Figure 10-2] See description of Figure 10-1. [Figure 11] 1 shows Lonza's technical information sheet for Ham's F12 medium. [Figure 12] 1 shows the Lonza technical information sheet for DMEM:F12 (1:1) medium (including the catalog number for DMEM:F12 (1:1) medium) used in preparing the specific medium example (PTT-6) in the experimental section. [Figure 13-1] FIG. 13 shows the Life Technologies Corporation technical information sheet for M171 medium (including the catalog number for M171 medium) used in the preparation of the specific example of medium (PTT-6) in the experimental section. [Figure 13-2] See description of Figure 13-1. [Figure 14]A list of the components (including their commercial suppliers and catalog numbers) used in the experimental section for the preparation of medium PTT-6 is provided. [Figure 15] 1 shows the Norwood classification of patterned hair loss in men. [Figure 16] 1 shows the Ludwig classification of patterned hair loss in women. [Figure 17A] FIG. 17 shows the objective evaluation (Hairmetrix®) of 10 patients with hair loss who had a composition comprising the conditioned medium of the present invention applied to their scalp (capillitium); FIG. 17A shows the follicular units per square centimeter (FU / sqcm) over a 12-week treatment period, FIG. 17B shows the intra-follicular distance (mm) over a 12-week treatment period, FIG. 17C shows the total hair count per square centimeter over a 12-week treatment period, and FIG. 17D shows the total hair diameter per square centimeter (millions / sqcm) over a 12-week treatment period. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 18A] FIG. 18 shows the subjective assessment of patients by standardized questionnaire; FIG. 18A shows patient satisfaction with hair growth, FIG. 18B shows the possibility of patients hiding their hair, and FIG. 18C shows the quality of life of patients affected by hair loss. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19]Table 6 shows the primary outcome parameters (mean and standard deviation (mean / SD)) over the 12-week treatment period and the results of the objective scalp assessment (Hairmetrix®) by linear regression model P-value. The primary outcome parameters shown in Table 6 are (from top to bottom) intrafollicular distance (mm), T:V ratio, follicular units (FU) per sq cm, mean hair count per FU, mean hair diameter (micm), terminal hair count per sq cm, total hair count per sq cm (Total), sum of terminal hair diameters (micm) per sq cm, and sum of total hair diameters (micm) per sq cm. [Figure 20] Table 7 contains the results of patients' subjective assessments using a standardized questionnaire. The primary outcome parameters (means and standard deviations (mean, SD)) over the 12-week treatment period and P-values ​​for linear regression models are shown. The primary 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 texture, satisfaction with results, comfort with hair, self-confidence, friends / family's reaction to hair texture, thoughts about hair appearance, hiding hair, rearranging hair, and impact on quality of life. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description The present invention is directed to a method suitable for inducing, stimulating, and / or promoting hair growth and / or hair regeneration by using a conditioned medium derived from the culture of mesenchymal stem cells from the amniotic membrane of the umbilical cord. Furthermore, the method of the present invention is suitable for alleviating and / or reducing hair loss and / or thinning by using a conditioned medium derived from the culture of mesenchymal stem cells from the umbilical cord. Thus, the present invention is based on the discovery that conditioned medium derived from the culture of mesenchymal stem cells from the umbilical cord secretes biological factors (e.g., proteins), and these factors contribute to the excellent hair loss prevention and hair growth effects of conditioned medium used to treat a subject's hair, particularly the skin at the hairline. In this regard, the inventors have surprisingly determined that umbilical cord mesenchymal stem cells secrete biological factors effective in promoting hair growth, and further confirmed that conditioned medium obtained by culturing umbilical cord mesenchymal stem cells exhibits highly effective hair growth-promoting effects.

[0022] As shown in the Experimental Section, the present invention utilizes conditioned medium derived from cultures of umbilical cord mesenchymal stem cells, which has been demonstrated to effectively and consistently improve hair quality, density, and coverage from inactive or dormant hair follicles in patients suffering from hair thinning / hair loss. For example, as demonstrated in the Experimental Section, 6 weeks of treatment with conditioned medium derived from cultures of umbilical cord mesenchymal stem cells (CALECIM®) objectively improved hair density and coverage in patients experiencing hair thinning / hair loss (Example 6; Figures 6A-K). As another example, conditioned medium derived from cultures of umbilical cord mesenchymal stem cells (CALECIM®) stimulates hair growth in patients suffering from androgenetic alopecia (AGA) (Example 7, Figure 5)—AGA, also known as male-pattern and female-pattern baldness, is the most common cause of hair loss worldwide today. Furthermore, when patients with telogen effluvium (TE), a diffuse alopecia following COVID-19 infection, were treated five times monthly with conditioned medium (CALECIM®) derived from cultures of umbilical cord mesenchymal stem cells after failing to respond to conventional hair loss treatments, they experienced progressive improvement in hair density with regrowth of villi at the frontal hairline and prefrontal cortex (Example 8, Figure 7). In this context, the present invention has found that conditioned medium derived from cultures of umbilical cord mesenchymal stem cells has a significant effect on hair growth.

[0023] Thus, the present invention relates to a medium conditioned by culturing umbilical cord mesenchymal stem cells. Thus, this conditioned medium is obtained by culturing umbilical cord mesenchymal stem cells in a cell culture medium as described herein and separating the resulting medium from the cells; thus, the conditioned medium of the present invention contains secreted stem cell products (called biological factors). Therefore, the conditioned medium contains biological factors but is substantially free of, or completely free of, stem cells. Biological factors that may be present in the conditioned medium include, but are not limited to, growth factors, exosomes, hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. Any combination of such conditioned medium and the biological factors contained therein (including, but not limited to, growth factors) can be used in the methods of the present invention.

[0024] As demonstrated in the experimental examples herein, conditioned medium derived from cultures of umbilical cord mesenchymal stem cells is particularly rich in growth factors and is proposed to be useful in restoring disrupted hair follicle activity in conditions of hair thinning / hair loss, such as traditional alopecia and COVID-19-induced hair loss. For example, epidermal growth factor (EGF) is known to be actively involved in hair growth because it promotes the anagen phase (growth phase) of hair, regulates hair follicle elongation, and co-stimulates 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 8 (FGF) is known to be actively involved in hair follicle development. As shown in the experimental section of this specification (Example 2), conditioned medium—derived from cultures of red deer umbilical cord mesenchymal stem cells (RD-CLMSCs) in a medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum)—when applied to human dermal fibroblasts (HDFs) to evaluate the effect of conditioned medium on human skin, significantly up-regulates the expression of elastin and hyaluronic acid compared to application of DMEM / 10% FCS to HDFs (Example 3, Figures 1 and 2). Without wishing to be bound by theory, the upregulation of elastin and hyaluronic acid expression present in RD-CLMSC-CM is due to the presence of growth factors such as transforming growth factor (TGF) β1 and TGF β2, insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and fibroblast growth factor 7 (FGF-7) present in RD-CLMSC-CM.

[0025] In this regard, those skilled in the art are aware that the presence of growth factors is involved in inducing increased expression of elastin and hyaluronic acid. In humans, transforming growth factor-β1 (TGF-β1) plays a central role in the expression of tropoelastin (TE), a soluble form of elastin; see Kuang, PP, et al., Activation of elastin transcription by transforming growth factor-β 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 Kaehaeri, V.M., 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): pp. 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): pp. 10-6. Interestingly, the combination of TGF-β1 and hyaluronic acid oligomers synergistically increased the levels 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): pp. 5698-707.TGF-β1 further inhibited elastin degradation through a reduction in the levels and activity of elastinolytic proteases, including matrix metalloproteinases (MMP)-2 and -9; see Dai, J., et al., Overexpression of transforming growth factor-beta1 stabilizes already-formed aortic aneurysms: 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 at 1 ng / ml; see Kaehaeri, 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.In mammals, three hyaluronan synthase (HAS) enzymes, HAS-1, 2, and 3, synthesize hyaluronan chains of various lengths; see Weigel, P.H., V.C. Hascall, and M. Tammi, Hyaluronan synthases. J. Biol. Chem., 1997. 272(22): pp. 13997-4000; Itano, N., et al., Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J. Biol. Chem., 1999. 274(35): pp. 25085-92; and Itano, N. and K. Kimata, Mammalian hyaluronan synthases. IUBMB Life, 2002. 54(4): pp. 195-9. Gene expression of HAS-1 and HAS-2 in the dermis and epidermis is differentially upregulated by TGF-β1; see Stuhlmeier, KM and C. Pollaschek, 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): pp. 8753-60; and Stern, R. and HI Maibach, Hyaluronan in skin: aspects of aging and its pharmacologic modulation. Clin Dermatol, 2008. 26(2): pp. 106-22. HAS-2 and HAS-3 mRNA expression is stimulated by fibroblast growth factor (FGF)-7, which activates keratinocyte migration and promotes wound healing, leading to the accumulation of intermediate-sized hyaluronan in the culture medium and within keratinocytes.TGF-β2 and platelet-derived growth factor (PDGF)-BB have been shown to induce the expression of HAS-2 and hyaluronan synthase by osteoblasts; see Nikitovic, D., et al., Transforming Growth Factor-β as a key molecule triggering the expression of versican isoforms v0 and v1, Hyaluronan Synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells. IUBMB Life, 2006. 58(1): p. 47-53.

[0026] Therefore, the significant upregulation of elastin and hyaluronan expression observed in all exposed HDFs may be due to the presence of TGF-βs, PDGF-BB, and FGF-7 present in RD-CLMSC-CM. In addition to the known involvement of growth factors in enhancing elastin and hyaluronan expression, it has been found herein that growth factors, including but not limited to transforming growth factor (TGF) β1 and TGF β2, insulin-like growth factor 1 (IGF-1), and platelet-derived growth factor (PDGF) (including PDGFaa, PDGFbb, and / or PDGFab, etc.), are actively involved in hair growth.

[0027] Furthermore, the Examples show that conditioned medium derived from mesenchymal stem cells of red deer and human umbilical cord amniotic membranes applied to human dermal fibroblasts (HDFs) significantly improved the proliferation capacity of HDFs, which could not be achieved with, for example, DMEM / 10% FCS (control) or human foreskin fibroblasts (FSFs) (Example 4, Figure 3). Furthermore, cell migration assessed in an in vitro "scratch" assay showed that the migration-promoting properties of conditioned medium derived from cultures of mesenchymal stem cells of red deer umbilical cord amniotic membranes were stronger than those of cultures of human foreskin fibroblasts (FSFs) (Example 5, Figure 4). The superior proliferation and migration-promoting properties of conditioned medium derived from cultures of mesenchymal stem cells of umbilical cord (red deer and human) amniotic membranes compared to DMEM / 10% FCS (control) or human foreskin fibroblasts (FSFs) suggest the presence of other proteins and / or growth factors in such conditioned medium that may positively affect 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): pp. 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 has been 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 Biophysica Acta (BBA) - Molecular Cell Research, 2005. 1743(1): p. 49-56).

[0028] Therefore, the significant proliferation and increased mobility of HDFs cultured in RD-CLMSC-CM may be due to the proliferative and migration-promoting effects of VEGF, TGF-β1, and HGF. In addition to the expression of growth factors, conditioned medium derived from the culture of umbilical cord mesenchymal stem cells contains anti-apoptotic and maintenance factors that delay cellular senescence, slow growth arrest, and inhibit apoptosis. Furthermore, in this context, stimulation of hair growth may also be due to VEGF and HGF, as these growth factors are known to those skilled in the art to be actively involved in hair growth.In this context, VEGF is known to be involved in hair follicle growth and cycle, where hair growth, hair follicle increase, and hair size are promoted by improved angiogenesis, especially during the anagen phase of the hair cycle; 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 hair dermal 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 transplantation improves the revascularization of transplanted hair growth mediated by angiogenesis. Forum 2007:2.

[0029] Growth factors are agents, such as naturally occurring substances, that can stimulate cell growth and / or proliferation and / or cell differentiation. Typically, growth factors are proteins or steroid hormones. While "growth factor" and "factor" are used interchangeably, the term "biological factor" as used herein is not limited to growth factors. For example, umbilical cord mesenchymal stem cells can produce one or more factors involved in the following: 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-type plasminogen activator receptor (UPAR), intercellular adhesion molecule-1 (ICAM1), insulin-like growth factor 1 (IGF1), interleukin-6 (IL-6), interleukin-8 (IL-8)); promotion of angiogenesis (e.g., angiopoietin-1 (ANG-1), hepatocyte proliferation); factors (HGF, vascular endothelial growth factor (VEGF)); anti-inflammatory (e.g., transforming growth factor beta 1 (TGF-β1), soluble tumor necrosis factor (TNF) receptor 1); promoting the expression of elastin and / or hyaluronic acid (e.g., transforming growth factor beta 1 (TGF-β1), 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).

[0030] The conditioned medium of the present invention may contain at least one biological factor produced by umbilical cord mesenchymal stem cells, more typically a combination thereof (e.g., at least one of the above biological factors, more typically a combination thereof). For example, it may contain one or more (at least one) angiogenic cytokines and / or growth factors. It may contain one or more selected from the group consisting of ANG-1, HGF, VEGF such as VEGFA and / or VEGFB, TGF such as TGF-β1, PDGF such as PDGF-AA or PDGF-BB, bFGF, MCP-1, IL-6, IL-8, TNF, TIMP1, TR10C, UPAR, ICAM1, and IGF1.

[0031] In this regard, the present invention has discovered that conditioned medium derived from the culture of umbilical cord mesenchymal stem cells (the cells secrete, inter alia, growth factors and other biological factors into the conditioned medium) improves hair growth. Thus, it is believed that the conditioned medium of the present invention induces high concentrations of growth factors and / or other biological factors, which may effectively help to prolong the anagen phase, induce cell growth and proliferation, particularly proliferation of dermal papilla cells, and simultaneously suppress apoptotic signals. In this regard, it should be noted that culturing umbilical cord mesenchymal stem cells in the culture medium described herein results in the isolation of highly homogeneous, well-defined mesenchymal stem cell populations from umbilical cord tissue (e.g., Wharton's Jelly from umbilical cord or mesenchymal stem cell populations from amniotic membrane), as disclosed in WO 2019 / 199234 A1 or WO 2018 / 067071 A1. In particular, isolation of umbilical cord mesenchymal stem cell populations yields more than 90%, even more than 99%, of cells that are positive for the three mesenchymal stem cell markers CD73, CD90, while at the same time these stem cells lack expression of CD34, CD45 and HLA-DR (see, for example, WO 2019 / 199234 A1 or WO 2018 / 067071 A1), i.e., 99% or more of the cells in this population express the stem cell markers CD73, CD90 and CD105, but not the markers CD34, CD45 and HLA-DR.In this regard, such mesenchymal stem cell populations from umbilical cord tissue fully meet the generally accepted criteria for human mesenchymal stem cells to be used in cell therapy, as defined, for example, by Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Cytotherapy (2006) Vol. 8, No. 4, pp. 315-317; Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," Stem Cell Research & Therapy 2013, 4:66; Vonk et al., Stem Cell Research & Therapy (2015) 6:94; or Kundrotas Acta Medica Lituanica. 2012, Vol. 19, No. 2, pp. 75-79. This, in turn, means that conditioned medium derived from the culture of such highly homogeneous and well-defined cell populations is ideal for inducing highly homogeneous and well-defined components, such as high concentrations of growth factors, into the conditioned medium. Furthermore, the use of the conditioned medium of the present invention in hair growth treatments has several additional advantages over stem cell-based therapies: the conditioned medium can avoid the donor-recipient compatibility issues associated with stem cell-based hair transplantation, and it is easier to prepare and less expensive than stem cell-based hair treatments.

[0032] Furthermore, optimal conditions for hair growth treatments and hair transplants may be provided by healthy skin conditions. In this regard, conditioned medium derived from cultures of umbilical cord mesenchymal stem cells has been reported to have beneficial effects on skin quality and regeneration. For example, the use of conditioned medium has been shown to optimize the healing process in patients with moderate to severe photodamage (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 reduce pain and discomfort after laser treatment (Dr. Cheryl Effron, Reduction of Pain and Discomfort Post CO2 Fractional Laser Resurfacing 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 optimization of healing and reduction of pain in damaged skin areas by the use of conditioned medium may be due to the presence of secretory products from umbilical cord mesenchymal stem cells 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 have the effect of inducing or improving wound healing properties. Thus, products secreted by umbilical cord mesenchymal stem cells into the conditioned medium can contribute to a healthy skin condition, which, as found herein, is also advantageous for optimal hair growth and / or the healing process after hair transplantation.The beneficial effects of conditioned medium derived from culturing umbilical cord mesenchymal stem cells on skin conditions may be due, inter alia, to the immunomodulatory properties of the conditioned medium, which are also known to be achieved by umbilical cord mesenchymal stem cells. In accordance with these findings, it has been found herein that exposing a subject's scalp to conditioned medium derived from culturing umbilical cord mesenchymal stem cells suppresses inflammation. See Example 9 herein; there, the production of tumor necrosis factor alpha (TNFα), an important inflammatory cytokine produced by macrophages / monocytes during acute inflammation and involved in various intracellular signaling pathways, such as those leading to necrosis and 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 inflammation can be suppressed by the conditioned medium, promoting cell growth and proliferation.

[0033] Conditioned medium derived from the culture of umbilical cord mesenchymal stem cells, which is suitable for hair growth (e.g., by inducing and promoting hair growth in patients suffering from thinning / hair loss), can be derived from the culture of any suitable mesenchymal stem cell population from umbilical cord tissues known in the art. For example, umbilical cord mesenchymal stem cells can be derived from any mammalian tissue or compartment / body part known to contain umbilical cord mesenchymal stem cells. In a specific example, mesenchymal stem cells can be mesenchymal stem cells from the amniotic membrane of the umbilical cord. These stem cells secrete growth factors and other biological factors associated with hair growth, for example, by generating dermal papilla cells and / or increasing the number and size of hair follicles, thereby promoting hair growth and preventing hair loss. For example, biological factors that may be contained in the conditioned medium include, but are not limited to, growth factors, nucleic acids such as mRNA or DNA, exosomes, hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. Any combination of such conditioned media and the biological factors contained therein (including, but not limited to, growth factors) can be used in the methods of the present invention.

[0034] The conditioned medium of the present invention used for treatment and therapy can be applied, for example, undiluted or diluted. When used in diluted form, the conditioned medium can be used, for example, at a final concentration of about 10% to about 90% or about 10% to about 80%, e.g., 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 relative to the total volume of the composition containing the conditioned medium). For this purpose, the conditioned medium of the present invention can be diluted (formulated) with any suitable diluent / carrier medium. The carrier medium may be a liquid, gel, or cream formulation. For example, the conditioned medium can be diluted with a carrier medium such as a physiological buffer solution such as PBS or saline, water, an aqueous solution of extracellular matrix components, a basal medium, or a cream formulation, to name just a few suitable media. As used herein, the term "basal medium" refers to a mixture containing sugars, amino acids, water, and other components required for cell survival. Examples include commercially available prepared media such as Dulbecco's Modified Eagle's Medium (DMEM), endothelial cell differentiation medium (EDM), minimal essential medium (MEM), Basal Medium Eagle's Medium (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. Examples of aqueous solutions of extracellular matrix components include aqueous solutions of hyaluronic acid, collagen, or fibronectin, all of which are commercially available for cosmetic use. A specific 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) of a conditioned medium and the remainder being an aqueous solution of hyaluronic acid (i.e., about 10% to about 90% (v / v)). In some other examples, the conditioned medium of the present invention may be contained in a solution containing DMEM as a basal medium. In such a solution, the conditioned medium is present, for example, in a range of about 90% to about 10% (v / v), with the remainder being DMEM. The conditioned medium of the present invention may further contain an antioxidant, for example, a soluble antioxidant. Adding a water-soluble antioxidant to the medium can prevent oxidative damage.Examples of suitable water-soluble antioxidants include glutathione, uric acid, Trolox, or Allicidin, to name just a few.

[0035] The conditioned medium of the present invention can also be formulated to improve dermal absorption of the conditioned medium after topical application to the skin / scalp of a subject. To this end, the conditioned medium can be encapsulated in a liposomal preparation. A specific example of such a liposomal preparation is liposomes made from a phospholipid mixture. An example of such a phospholipid mixture for cosmetic use is Lucas Meyer Cosmetics' Pro-Lipo™ Neo liposomal delivery system, a ready-to-use liposomal delivery system capable of encapsulating conditioned medium for topical administration (see Example 12). The topical compositions described herein may contain, for example, about 30-70% (v / v) conditioned medium and about 20% (v / v) Pro-Lipo™ Neo (liposomal mixture), with the remainder being any suitable aqueous solution.

[0036] The conditioned medium of the present invention can be derived from culturing umbilical cord mesenchymal stem cells in the aforementioned additional culture medium after culturing mesenchymal stem cells in a medium containing DMEM, F12, M171, and FBS. This additional culture medium can be any (additional) basal culture medium suitable for the growth of animal cells, particularly a basal medium suitable for culturing mesenchymal stem cells. This additional medium can be serum-free. Non-limiting examples of such serum-free media include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), and keratinocyte medium (KM), KBM (keratinocyte basal medium), EpiLife KM (keratinocyte-EpiLife medium), etc. Alternatively, this additional culture medium can contain serum. As an example of such a serum-containing medium, the medium used can be one commonly described and used for the isolation and culture of mesenchymal stem cell populations from the amniotic membrane of the umbilical cord, such as medium PTT-4. This medium, PTT-4, consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS and has been used in U.S. Patent Application US2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 for the isolation and culture of mesenchymal stem cell populations from the amniotic membrane of the umbilical cord, and has been shown to have excellent wound healing properties in U.S. Patent Application US2008 / 0248005 and International Patent Application WO2007 / 046775. In some examples, cell culture media described herein, such as media containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), can be used to culture umbilical cord mesenchymal stem cells, where the cell culture medium is removed after culturing, for example, after the cells reach full confluency, and replaced with another culture medium / solution, such as a culture medium / solution containing DMEM (which may contain antioxidant components). Generally, during any culture step in the additional medium of the conditioned medium preparation method described herein, the mesenchymal stem cells can be cultured for an appropriate period of time. Such an appropriate period can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or even longer, for example, up to 10 or 14 days, as desired.In this regard, it should be noted for clarity that both the conditioned medium obtained in the first culture step and the conditioned medium obtained in the second (further) culture step can be used to induce, stimulate and / or promote hair growth and / or hair regeneration as described herein.

[0037] The umbilical cord (tissue) mesenchymal stem cells used herein to prepare conditioned medium may be from any compartment of umbilical cord tissue that contains mesenchymal stem cells (or may be derived from any compartment). The mesenchymal stem cell population may be an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, a mesenchymal stem cell population from the amniotic membrane of the umbilical cord (also referred to as umbilical cord lining mesenchymal cells), or even a mixed umbilical cord mesenchymal stem cell population (MC), i.e., a mesenchymal stem cell population containing stem cells from two or more of these compartments. In some examples, the mesenchymal stem cells are from the amniotic membrane of the umbilical cord. Mesenchymal stem cells from these compartments and their isolation are known to those skilled in the art and are described, for example, in Subramanian et al., "Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells," PLoS ONE 10(6): e0127992, 2015 and references cited therein; Van Pham et al., "Isolation and Proliferation of Umbilical Cord Tissue-Derived Mesenchymal Stem Cells for Clinical Applications," Cell Tissue Bank (2016) 17:289-302, 2016. Mixed mesenchymal stem cell populations from the umbilical cord can be obtained (by tissue explantation), for example, by removing arteries and veins from umbilical cord tissue, dissecting the remaining tissue and Wharton's jelly, and culturing the umbilical cord tissue in a culture medium such as PTT-6.Mixed umbilical cord mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue, including intact umbilical vessels, as tissue explants under conditions such as those described in Schugar et al. "High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue" Journal of biomedicine & biotechnology. 2009; 2009:789526 (culture in serum-supplemented DMEM containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin / streptomycin). In this regard, it should be noted that mesenchymal stem cell populations from the umbilical-placental junction can be isolated as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224.

[0038] In accordance with the above, it should be noted that the conditioned medium is derived from the culture of mesenchymal stem cells from umbilical cord tissue, and these stem cells can be cultured in culture media including DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (171 Medium), and FBS (fetal bovine serum), or can be isolated from their natural environment prior to culture in the culture media described herein. Such an approach is particularly useful for umbilical cord mesenchymal stem cell populations. Such stem cell populations, for example, mesenchymal stem cell populations from Wharton's jelly, can be first isolated as described in Subramanian et al., 2015, PLoS ONE, supra, or International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord", and the isolated mesenchymal stem cell population can then be cultured in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).Placental mesenchymal stem cell populations may also be isolated from the placenta as described, for example, in European Patent Application EP1 288 293; Talwadekar et al, "Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma" Biomed Res J 2014;1(2):126-136; Talwadekar et al, "Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts - a paired sample study" Scientific Reports 5:15784 (2015); or Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017;(122):55224, and then cultured in the culture medium described herein.

[0039] In this regard, it should be noted that the culture medium described herein therefore also allows for the isolation of a mesenchymal stem cell population (also referred to herein as "mesenchymal stem cells") from its natural environment. Thus, the culture medium described herein also allows for the isolation of a mesenchymal stem cell population under conditions that allow cell proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. In some examples, the cell culture medium used to isolate and / or culture umbilical cord mesenchymal stem cells can be the medium used to culture the cells to produce the conditioned medium of the present invention.

[0040] In accordance with the above disclosure, it should be noted that the mesenchymal stem cell populations described herein can be isolated and cultured (i.e., derived) from any umbilical cord tissue, so long as the umbilical cord tissue contains the amniotic membrane (also referred to as the "cord lining"). Thus, as described in the Experimental Section of this application, mesenchymal stem cell populations can be isolated from the entire umbilical cord (or a portion thereof). Therefore, the umbilical cord tissue may contain other tissues / components of the umbilical cord in addition to the amniotic membrane. For example, as shown in Figure 16 of U.S. Patent Application Publication No. 2006 / 0078993 or International Patent Application No. WO2006 / 019357, the amniotic membrane of the umbilical cord is the outermost portion of the umbilical cord and covers it. Additionally, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-poor blood away from the fetus). These three blood vessels are embedded in Wharton's jelly, a gelatinous substance composed primarily of mucopolysaccharides, for protection and mechanical support. Therefore, umbilical cord tissue, as used herein, can also include this one vein, two arteries, and Wharton's jelly. Using such an entire (intact) portion of the umbilical cord has the advantage that it is not necessary to separate the amniotic membrane from other components of the umbilical cord. This reduces the isolation steps, thus making the method of the present invention simpler, faster, less error-prone, and more economical. Thus, mesenchymal stem cell isolation can begin with tissue explantation, and if larger quantities of mesenchymal stem cells are required, the isolated mesenchymal stem cells can be subsequently subcultured (cultured). Alternatively, mesenchymal umbilical cord lining stem cells can be isolated from the amniotic membrane by first separating the amniotic membrane from other components of the umbilical cord and culturing the amniotic membrane in the culture medium described herein. This culture can also be performed by tissue explantation, followed by optional subculture of the isolated mesenchymal stem cells.

[0041] In this context, the terms "tissue explant" or "tissue explant method" are used in their usual sense in the art to refer to a method in which, once harvested, a tissue (e.g., umbilical cord tissue) or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, allowing stem cells to migrate from the tissue to the surface of the culture dish over time. These primary stem cells can then be further expanded by micropropagation (subculture), as also described herein, and transferred to a new dish. In this context, it should be noted that the initial step of isolating / obtaining a mesenchymal stem cell population from the umbilical cord, such as amniotic membrane or Wharton's jelly mesenchymal stem cells, results in a master cell bank of the isolated mesenchymal stem cells, and subsequent subcultures result in a working cell bank. When the conditioned medium derived from the culture of umbilical cord mesenchymal stem cells of the present invention is used to promote hair growth, a cell population from the working cell bank is typically used for this purpose. Both the stem cell population from the isolation step (which may constitute a master cell bank) and the stem cell population from the subculture step (which may constitute a working cell bank) may be stored, for example, in a cryopreserved form.

[0042] "Inducing, stimulating, and / or promoting hair growth and / or hair regeneration" as used herein refers to the ability of a conditioned medium to increase and / or initiate (induce) hair growth from hair follicles, particularly in patients with inactive or dormant hair follicles and / or patients with thinning hair / hair loss, such as androgenetic alopecia. The ability to increase and / or initiate hair growth may be due to the presence of growth factors known to those skilled in the art to be involved in inducing, stimulating, and / or promoting hair growth, as explained above. In the experimental example herein, the secretion of growth factors actively involved in "inducing, stimulating, and / or promoting hair growth and / or hair regeneration" and present in conditioned medium derived from umbilical cord mesenchymal stem cells is evaluated compared to conditioned medium from human foreskin fibroblasts (FSFs) and DMEM / 10% FCS (control). In this context, culturing human dermal fibroblasts (HDFs) in conditioned medium derived from umbilical cord mesenchymal stem cell cultures demonstrated superior proliferation and migration-promoting properties for HDFs compared to cultures in DMEM / 10% FCS (control) or human foreskin fibroblasts (FSFs); this may be due to the fact that umbilical cord mesenchymal stem cells secrete larger amounts (corresponding to higher secretion levels or concentrations) of growth factors into the supernatant / culture medium 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 invention) can be measured / determined in any suitable manner, for example, by measuring the amount of growth factors using commercially available antibodies / immunoassays (see Experimental Section). Such measurements can be performed in an automated manner, for example, using a system 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 regeneration can also be determined by cellular assays, such as examining the growth-promoting effect of the conditioned medium on cultured human dermal papilla cells, as described herein (see Example 9), or by administering the conditioned medium to a patient diagnosed with alopecia and examining its effect, for example, on the number of follicular units per square centimeter, intrafollicular distance, or total number of hairs per square centimeter, as described herein (see Example 11).

[0043] "Norwood classification or scale" or "Norwood-Hamilton classification or scale" refers to the classification introduced by O'Tar Norwood to classify the stages of male pattern baldness. The Norwood classification / scale is the most widely used classification of male hair loss, defining two major patterns and several less common types (see, for example, Gupta & Mysore, Classifications of Patterned Hair Loss: A Review, J Cutan Aesthet Surg. 2016 Jan-Mar; 9(1): 3-12). Norwood observed that hair thinning begins at the temples and crown / vertex and slowly progresses to spread across the entire upper scalp. Therefore, this classification is based on this pattern and is also shown in Figure 15. The stages of hair loss according to the Norwood scale are as follows (Roman numerals and Arabic numerals are used interchangeably herein when referring to the Norwood scale): Type I: Little or no hairline recession. Type II: There is a triangular, usually symmetrical, area of ​​recession at the frontotemporal hairline. Type III: This represents minimal hair loss sufficient to be considered baldness according to Norwood. There is deep symmetrical recession at the temples, which are bare or sparsely covered with hair. In type III vertex, hair loss begins primarily at the vertex, with limited frontotemporal hairline recession, not exceeding the extent seen in type III. Type IV: Frontotemporal recession is more severe than in Type III, and hair on the top of the head is sparse or absent. The two bald areas are separated by a band of moderately dense hair across the top, which connects to a well-haired fringe on the sides of the scalp. Type V: The bald area on the top of the head is still separated from the frontotemporal area but is less distinct. The band of hair across the top is narrower and sparser, while the bald areas on the top and frontotemporal areas become wider. Type VI: The bridge of hair across the top of the head is gone, leaving only sparse hair. The frontotemporal and parietal areas are connected, and the degree of hair loss is greater. Type VII: This is the most severe form of hair loss, leaving only a narrow, horseshoe-shaped band of hair on the sides and back of the scalp. This hair is usually not dense and can be quite thin.

[0044] The Norwood scale also defines a type A variant from his standard classification system, which is distinguished by two major and two minor features.

[0045] The key features are 1) the progression of the anterior hairline backward without leaving an island of hair in the central frontal area, and 2) the lack of a simultaneous development of a bald area on the parietal area. Instead, the frontal hairline continues to progress backward on the scalp.

[0046] Minor features are 1) persistent sparse hairs scattered across the bald areas, and 2) the remaining horseshoe-shaped fringe of hair on the sides and back of the scalp tends to be wider and reach higher up the head compared to Norwood's norm. The various Type A variants described by Norwood are as follows: Type IIA: The hairline is located 2 cm anterior to the ear canal in the coronal plane. Type IIIA: The hairline recedes to a point between the limit of type IIA and the level of the ear canal. Type IVA: The hairline recedes beyond the ear canal but does not reach the top of the head. Type VA: The exposed area includes the vertex. Hair loss more severe than type VA is indistinguishable from types VI or VII.

[0047] In this regard, it should be noted that it has been found herein that the conditioned medium described herein can be preferably used to treat male hair loss diagnosed as type I, type II, type III, type IV, type V, or type VI according to the Norwood classification. In other words, any type I, type II, type III, type IV, type V, or type VI hair loss according to the Norwood classification can be treated with the conditioned medium of the present invention.

[0048] "Ludwig classification or scale" refers to the classification introduced by E. Ludwig in 1979 to classify female pattern hair loss or baldness (androgenetic alopecia). It ranges from Type / Stage I to Type / Stage III and is defined as follows (see also Figure 16); the terms "type" and "stage" are used interchangeably herein. When referring to the Ludwig scale, Roman numerals and Arabic numerals are used interchangeably herein. Stage I: Visible thinning of hair on the crown, limited in front by a line located 1-3 cm behind the frontal hairline. As shown in Figure 16, stage I is further subdivided into stages Ia, Ib, Ic, and Id, while stage II is further subdivided into stages IIa and IIb. Stage II: Significant thinning of hair on the top of the head in the area seen in Stage I. Stage III: Complete baldness (total exposure) within the areas seen in stages I and II.

[0049] Stage I begins with thinning hair on the top of the head. In stage II, the scalp becomes more visible. As hair loss progresses to stage III, the hair on the top of the head may be completely lost.

[0050] In this regard, it should be noted that the conditioned medium described herein can be preferably used to treat female hair loss diagnosed as type I (including type Ia, type Ib, type Ic and type Id), type II (type IIa and type IIb) or type III according to the Ludwig classification.In other words, any type I (including type Ia, type Ib, type Ic and type Id), type II (type IIa and type IIb) or type III according to the Ludwig classification can be treated with the conditioned medium of the present invention.

[0051] "DMEM" refers to Dulbecco's Modified Eagle's Medium, developed in 1969 and modified from Basal Medium Eagle (BME) (see Figure 10, which shows the data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from various sources, including ThermoFisher Scientific (catalog number 11965-084), Sigma-Aldrich (catalog number D5546), and Lonza, to name just a few. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich DMEM medium, catalog number D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0052] "FI2" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed to cultivate a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 11, which shows the data sheet for Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., the medium from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number N4888), or Lonza, to name just a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0053] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 culture medium (see FIG. 12, which shows the data sheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is a widely used basal medium for supporting the growth of various mammalian cells and is commercially available from various sources, such as ThermoFisher Scientific (catalog number 11330057), Sigma-Aldrich (catalog number D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium available from Lonza under catalog number 12-719F (which is DMEM:F12 containing L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0054] "M171" refers to Culture Medium 171, which was developed as a basal medium for the culture or growth of normal human mammary epithelial cells (see Figure 13, which shows the data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely used and is commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is M171 medium available from Life Technologies Corporation under catalog number M171500.

[0055] "FBS" refers to fetal bovine serum (also known as "fetal calf serum"), i.e., the blood fraction remaining after natural clotting of blood and subsequent centrifugation (to remove any remaining red blood cells). Fetal bovine serum is the most widely used serum supplement for in vitro eukaryotic cell culture because of its extremely low antibody levels, higher growth factor content, and versatility for a variety of cell culture applications. FBS is preferably obtained from members of the International Serum Industry Association (ISIA), whose primary focus is the safety and safe use of serum and animal-derived products through proper origin traceability, label authenticity, and appropriate standardization and oversight. Suppliers of FBS that are ISIA members include, to name just a few, Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, Inc. In a currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0056] The culture medium described herein may contain a final concentration of approximately 55-65% (v / v) DMEM, approximately 5-15% (v / v) F12, approximately 15-30% (v / v) M171, and approximately 1-8% (v / v) FBS to produce conditioned medium from the culture of umbilical cord mesenchymal stem cells. As used herein, the "% (v / v)" values ​​refer to the volume of each component relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium at a final concentration of approximately 55-65% (v / v), for example, one liter of culture medium will contain approximately 550-650 ml of DMEM. In another embodiment, the culture medium may contain DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v). In a further embodiment, the culture medium may contain DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0057] In addition to the above components, the culture medium may contain supplements advantageous for culturing umbilical cord mesenchymal stem cells and for producing a conditioned medium for inducing, stimulating, and / or promoting hair growth and / or hair regeneration. The culture medium described herein may contain, for example, epidermal growth factor (EGF). When present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0058] The culture medium described herein may also contain insulin. When present, insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0059] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0060] As described herein, umbilical cord tissue can be cultured until an appropriate number of (primary) mesenchymal stem cells, such as umbilical cord lining stem cells, Wharton's jelly, or placental stem cells, outgrow the tissue. Typically, umbilical cord tissue is cultured until cell outgrowth of the mesenchymal stem cells from each tissue reaches approximately 70% to 80% confluency. Note that, as used herein, the terms "confluency" and "confluence" are used in their usual sense in the cell culture art, as an estimate / indicator of the number of adherent cells in a culture dish or flask, and refer to the percentage of the surface covered by cells. For example, 50% confluence means that approximately half of the surface is covered, with additional room for cell growth. 100% confluence means that the surface is completely covered with cells, with no room left for cells to grow as a monolayer.

[0061] Once an appropriate number of primary cells (mesenchymal stem cells) are obtained from each tissue by tissue explantation, the mesenchymal stem cells are removed from the culture vessel used for culturing. This allows for the creation of a master cell bank containing isolated (primary) mesenchymal stem cells, for example, from the umbilical cord or placenta. Generally, because such mesenchymal stem cells are adherent cells, the cells are harvested using standard enzyme treatments. For example, enzyme treatments can include trypsin treatment as described in International Patent Application No. 2006 / 0078993, International Patent Application No. WO2006 / 019357, or International Patent Application No. WO2007 / 046775. That is, growing cells can be harvested by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further expansion. When the harvested mesenchymal stem cells are used, for example, to create a master cell bank, they can also be cryopreserved as described below and stored for further use.

[0062] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculturing. Subculturing or culturing (hereinafter, both terms are used interchangeably) may also be performed when using a population of mesenchymal stem cells from the umbilical cord that have previously been isolated from their natural environment. Subculturing can also be initiated from frozen primary cells, i.e., from a master cell bank. For subculturing, an appropriate amount of cells is seeded into a culture vessel, such as a cell culture plate. For this purpose, the mesenchymal cells are cultured in an appropriate medium for subculturing (most conveniently, the culture medium described herein), at a concentration of, for example, about 0.5 × 10 6 pieces / ml ~ approx. 5.0×10 6 For example, the cells can be suspended at a cell concentration of about 1.0 x 10 cells / ml for subculture. 6The cells are suspended at a concentration of 10 ... An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell populations described herein is the Xuri Cell Expansion System available from GE Heathcare. Culturing mesenchymal stem cell populations in an automated system such as the Quantum® Cell Expansion System is particularly beneficial when working cell banks are to be generated under GMP conditions and large numbers of cells are desired.

[0063] The subculture of the mesenchymal stem cells of the present invention can be carried out in the culture medium described herein.Therefore, this culture medium can be used for both the isolation of mesenchymal stem cell populations, for example, from the amniotic membrane of the umbilical cord or Wharton's jelly, and the subsequent culture of the isolated primary cells.In addition, in culture, mesenchymal stem cells can be cultured until they proliferate to an appropriate amount.In some examples, mesenchymal stem cells are cultured until they reach about 70% confluency, about 80% confluency, about 90% confluency, about 95% confluency, about 96% confluency, or 100% confluency.

[0064] Isolation / culturing of mesenchymal stem cell populations can be performed under standard mammalian cell culture conditions. Generally, methods for isolating mesenchymal stem cell populations are performed under conditions (temperature, atmosphere) typically used for culturing cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37°C in a standard atmosphere of 5% CO2, respectively. In this regard, it should be noted that the mesenchymal cell populations described herein can be derived from any mammalian species, such as humans, red deer, mice, rats, guinea pigs, pigs, rabbits, goats, horses, dogs, cats, sheep, or monkeys. In exemplary embodiments, umbilical cord mesenchymal stem cells are red deer or human.

[0065] Once a desired / appropriate number of mesenchymal stem cells have been obtained from culture or subculture, they are harvested by removing them from the culture vessel used for culturing. Harvesting of mesenchymal stem cells is typically performed by enzymatic treatment, such as trypsinization of the cells. The isolated mesenchymal stem cells are then recovered and used directly or preserved for further use. Preservation is typically achieved by cryopreservation. The term "cryopreservation" is used herein in its ordinary sense to refer to the process of preserving mesenchymal stem cells by cooling them to subzero temperatures, such as -80°C (typically) or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be performed as known to those skilled in the art and can include the use of cryoprotectants, such as dimethyl sulfoxide (DMSO) or glycerol, which retard the formation of ice crystals within the umbilical cord cells.

[0066] The isolated populations of mesenchymal stem cells obtained by the culture and / or isolation methods described herein are highly defined and homogeneous. Such highly homogeneous populations of mesenchymal stem cells derived from tissues such as the amniotic membrane of the umbilical cord or Wharton's jelly are reported in WO 2018 / 067071 A1 and WO 2019 / 199234 A1. For example, as disclosed and shown in the experimental section of WO 2019 / 199234 A1, at least about 90%, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in a population of umbilical cord mesenchymal stem cells (e.g., isolated from umbilical cord Wharton's jelly or amniotic membrane) express each of the following markers: CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR; that is, this percentage of the isolated cell population expresses each of CD73, CD90, and CD105, and lacks expression of the following markers: CD34, CD45, and HLA-DR. Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord meets the criteria for mesenchymal stem cells to be used in cell therapy (see the experimental section of WO 2019 / 199234 A1 and, e.g., Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," supra). In this regard, it should be noted that this mesenchymal stem cell population can be obtained not only by the isolation method described in WO 2019 / 199234 A1, but also by different methods, such as cell sorting, if desired.

[0067] In a further aspect, the present invention is directed to a method of inducing, stimulating, and / or promoting hair growth and / or hair regeneration, comprising treating hair of a subject with conditioned medium derived from culturing umbilical cord mesenchymal stem cells in a culture medium described herein; wherein the culture medium may further comprise mixing the following ingredients to obtain a final volume of 500 ml of culture medium: i. 250 ml of DMEM ii. 118ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) (to a final concentration of 2.5% (v / v)).

[0068] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, if you use this method to make culture medium, the final concentrations (v / v) for a total volume of 500 ml are as follows: DMEM: 250ml+59ml=309ml, equivalent to 309 / 500=61.8%(v / v); M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v); F12: 59ml, equivalent to 59 / 500=11.8% (v / v).

[0069] The culture media described herein may further comprise the addition of the following components: v. 1 ml of EGF stock solution (5 μg / ml) (resulting in a final EGF concentration of 10 ng / ml), and vi. 0.175 ml of insulin stock solution (14.28 mg / ml) (to give a final insulin concentration of 5 μg / ml).

[0070] It should be noted that the above amounts of components i-vi, when mixed, result in a final culture medium volume of 499.675 ml. If no further components are added to the culture medium, the remaining 0.325 ml (added to a volume of 500 ml) can be, for example, any of components i-iv, i.e., DMEM, M171, DMEM / F12, or FBS. Alternatively, it is possible to adjust the concentration of the EGF or insulin stock solution so that the total volume of the culture medium is 500 ml. Furthermore, it should be understood that components i-iv do not necessarily have to be added in the order listed, and that these components can be mixed in any order to form the culture medium described herein. That is, for example, M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to reach the final concentrations described herein, i.e., a final DMEM concentration of about 55-65% (v / v), a final F12 concentration of about 5-15% (v / v), a final M171 concentration of about 15-30% (v / v), and a final FBS concentration of about 1-8% (v / v).

[0071] In other embodiments, the method further comprises adding 0.325 ml of one or more of the following supplements to the DMEM: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine sodium salt (T3), thereby bringing the total volume of the culture medium to 500 ml. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows: about 0.05-0.1 μg / ml adenine, for example about 0.025 μg / ml adenine; hydrocortisone at approximately 1–10 μg / ml; About 0.5-5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0072] In some embodiments, the conditioned medium of the present invention is produced by culturing umbilical cord mesenchymal stem cells in a cell culture medium described herein, such as a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), and then removing the umbilical cord mesenchymal stem cells from the culture medium. In this case, the conditioned medium is obtained by recovering the cell culture medium. Furthermore, in some instances, the culture medium in which the stem cells were cultured can be replaced with a second cell culture medium, and the cells can be cultured in the second medium; the second medium can be the same as or different from the first medium. In some instances, the second cell culture medium can be growth factor-free and / or serum-free. Alternatively, previously cultured umbilical cord mesenchymal stem cells (e.g., stored cultured stem cells) can be further cultured in growth factor-free and / or serum-free medium. In either case, the resulting medium is referred to as conditioned medium. The resulting conditioned medium can be collected and further processed to prepare a composition comprising the conditioned medium of the present invention.

[0073] In some embodiments, the conditioned medium of the present invention or a composition comprising the conditioned medium of the present invention is diluted with a carrier medium, for example, to a final concentration of about 10% to about 80% (v / v), or, for example, to a final concentration of 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 with any suitable diluent / carrier medium, as long as the carrier medium does not affect the function and safety of the conditioned medium when topically applied to the skin / scalp of a subject. For example, the conditioned medium can be diluted with PBS, water, a basal medium, or a cream formulation. As used herein, the term "basal medium" refers to a mixture containing sugars, amino acids, water, etc., necessary for cell survival, and includes, but is not limited to, commercially prepared media such as Dulbecco's Modified Eagle Medium (DMEM), Endothelial Cell Differentiation Medium (EDM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. Furthermore, the conditioned medium used may be any basal culture medium suitable for the growth of animal cells, including, but not limited to, Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), and Keratinocyte Medium (KM), KBM (Keratinocyte Basal Medium), EpiLife KM (Keratinocyte-EpiLife Medium), etc. In some examples, the conditioned medium comprises a cell culture medium described herein, such as DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and culture medium containing FBS (fetal bovine serum).

[0074] In accordance with the above, conditioned medium derived from the culture of umbilical cord mesenchymal stem cells of the present invention can also be included in the composition. The conditioned medium or compositions comprising the conditioned medium described herein and of the present invention can be applied to the scalp, face, and other areas of the skin where hair growth is likely to occur, particularly the hairline. As used herein, "hairline" should be understood as the base of hair on a subject's skin surface, and is not limited to the hair follicle along the top of the forehead, but includes the base of hair on any skin surface of a subject, particularly the scalp. Areas for application of the conditioned medium or compositions comprising the conditioned medium described herein and of the present invention include not only the scalp, but also any skin area of ​​the body requiring hair growth. The skin treated with the conditioned medium of the present invention can be any skin area capable of hair growth, including areas where hair or other body hair is damaged due to scarring or injury, or areas requiring cosmetic treatment, such as a wide or M-shaped forehead, eyelashes, or eyebrows. Thus, one skilled in the art will be able to recognize and identify skin sites that are capable of hair growth and are therefore suitable for methods of inducing, stimulating, and / or promoting hair growth and / or regrowth using the conditioned medium of the present invention.

[0075] In some embodiments, the compositions described herein are pharmaceutical or cosmetic compositions for inhibiting hair loss and enhancing hair growth. The compositions can be prepared in any formulation conventionally produced in the art and typically comprise the conditioned medium of the present invention and a suitable cosmetically or pharmaceutically acceptable carrier / recipient. The compositions of the present invention can also comprise additives and adjuvants commonly used in cosmetics, pharmaceuticals, or dermatology, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, disinfectants, odor absorbers, dyes or colorants, etc. Typically, the compositions are used as dermatological applications by directly applying them to the skin, particularly the scalp, or to transplanted hair prior to hair transplantation. Preferably, the compositions comprising the conditioned medium of the present invention are cosmetic compositions. In some embodiments, the compositions are adapted for topical application. Compositions adapted for topical application can be in liquid or viscous form. Examples include ointments, creams, lotions, etc. The preparation of such compositions is within the knowledge of those skilled in the art and is described, for example, in Gennaro, AL and Gennaro, AR (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA. In this context, a composition containing the conditioned medium of the present invention for topical application should be understood as a formulation capable of being absorbed into the hair follicle structure in a targeted manner, particularly in this case, formulated so that the active ingredients of the conditioned medium of the present invention or a composition containing the conditioned medium of the present invention can sufficiently penetrate the epidermal and dermal layers to reach the hair follicle and hair follicle bulb matrix, but are not absorbed into the systemic circulation (see the Examples section for this point). In some examples, the conditioned medium of the present invention or a composition containing the conditioned medium of the present invention passes through the surface of the skin and reaches the hair follicle by transepidermal or transdermal diffusion. In some examples, the conditioned medium of the present invention is injected superficially under the scalp without being absorbed into the systemic circulation.Methods for applying the conditioned medium or a composition containing the conditioned medium of the present invention include any method disclosed in the art, and the frequency of application may vary depending on the condition of the skin. Furthermore, before applying the conditioned medium or a composition containing the conditioned medium of the present invention, the subject's skin can be treated with microneedles. Any skin-piercing device can be used before applying the conditioned medium or a composition containing the conditioned medium of the present invention. As shown in the experimental section, the subject's skin can be treated with a Dermapen, a microneedling device for piercing the skin using microneedles 1 to 3 mm in length.

[0076] As another example, the conditioned medium derived from the culture of umbilical cord mesenchymal stem cells of the present invention can be used to prepare hair for transplantation.As is generally known to those skilled in the art, conventional hair transplantation methods involve plucking hair from areas of a patient's scalp that contain growing hair and planting the hair in bald areas of the patient's scalp.In some examples of the present invention, the conditioned medium of the present invention for treating hair loss / thinning hair or a composition comprising the conditioned medium can be used in the preparation stage of hair for transplantation before transplanting the hair into bald areas of a patient to treat hair loss / thinning hair; in this case, the hair is harvested from a subject and incubated in the conditioned medium of the present invention until hair follicle growth is induced before being planted in bald areas of the patient's skin, such as the scalp.

[0077] Thus, a method of treating a subject with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells is described. The method comprises administering an effective amount of the conditioned medium of the present invention to the subject's hair or to hair isolated for preparation for hair transplantation. Similarly, the present invention relates to conditioned medium and compositions comprising the conditioned medium for use in inducing, stimulating, and / or promoting hair growth and / or hair regeneration. Alternatively, or in addition, the present invention relates to conditioned medium and compositions comprising the conditioned medium for use in alleviating and / or reducing hair loss and / or thinning. According to the present invention, the use comprises treating the subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. As explained in the Examples section (see, e.g., Examples 6-8), an "effective amount of conditioned medium" can be a volume, such as 1 ml, 2.5 ml, or 5 ml, of conditioned medium (undiluted or diluted with a carrier vehicle to, e.g., a 50% (v / v) concentration) applied to the subject's skin / scalp per treatment. A course of treatment involves repeated administration of an effective amount as described herein over a period of time.

[0078] In principle, any subject is suitable for treatment with the conditioned medium of the present invention.For example, the subject to be treated suffers from hair loss and / or thinning hair.Finally, the present invention also provides a method for treating non-human mammal or human subject, particularly the subject or patient suffering from hair loss and / or thinning hair, which method comprises administering an effective amount of the conditioned medium of the present invention or a composition comprising the conditioned medium to the hair of the subject.

[0079] Hair loss and / or thinning may be male-pattern or female-pattern hair loss, and may be caused by any disease or condition, particularly one that involves hair loss / thinning and for which hair regrowth is desired / required. Hair loss may be hormone-related hair loss, such as menopause-related hair loss or postpartum-related hair loss. The subject may suffer from hair loss caused by a disease, including inflammatory-induced alopecia, such as androgenetic alopecia or alopecia areata; or telogen effluvium, such as virus-induced telogen effluvium, such as COVID-19-induced telogen effluvium or dengue virus-induced telogen effluvium. Examples of alopecia areata that can be treated with the conditioned medium described herein include alopecia areata, alopecia areata multiplex, alopecia serpiginosa, alopecia totalis, or alopecia universalis. Hair loss may also be caused by systemic medical problems, such as thyroid disease, adverse drug effects or the effects of drug treatments such as cancer chemotherapy, and hair loss due to nutritional deficiency. In this regard, hair loss may be acute or chronic. In some cases, hair loss is 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 of this specification, the patient suffers from COVID-19-induced telogen effluvium (TE) (referring to diffuse scalp alopecia).In other some embodiments, the subject suffers from androgenetic alopecia (AGA).

[0080] The present invention is further illustrated by the following non-limiting experimental examples.

[0081] The sequences used herein are shown in Table 1 below.

[0082] Table 1: Sequences used herein TIFF2025538460000002.tif206150TIFF2025538460000003.tif219150TIFF20255384600 00004.tif224150TIFF2025538460000005.tif226150TIFF2025538460000006.tif111150 [Example]

[0083] Experimental example Example 1: Isolation and culture of umbilical cord mesenchymal stem cells (CLMSCs) 1. Preparation of medium for treating CLMSCs a. To prepare 500 ml of PTT-6 (culture / growth medium), add the following ingredients in the order listed: i. DMEM, 250ml ii. M171 118ml iii. 118ml of DMEM F12 iv. FBS 12.5ml (final concentration 2.5%) v. EGF 1ml (final concentration 10ng / ml) vi. Insulin 0.175ml (final concentration 5μg / ml)

[0084] The combined amounts of components i through vi above result in a final volume of 499.675 ml of culture medium. If no additional components are added to this culture medium, the remaining 0.325 ml (to reach a volume of 500 ml) can be, for example, any of components i through iv, i.e., DMEM, M171, DMEM / F12, or FBS. Alternatively, it is possible to adjust the concentration of an EGF or insulin stock solution to bring the total volume of culture medium to 500 ml. Alternatively, a stock solution of an antibiotic, such as penicillin-streptomycin-amphotericin, can be added to bring the final volume to 500 ml. It is also possible to add 0.325 ml of one or more of the following supplements to the culture medium: adenine, hydrocortisone, or 3,3',5-triiodo-F thyronine sodium salt (T3), thereby bringing the total volume of culture medium to 500 ml.

[0085] vii. Label the bottle "PTT-6" and write the date the medium was prepared, the operator's initials, and "expires on" followed by the expiration date, which should be the earliest expiration date of any of the ingredients or one month from the date of preparation, whichever comes first.

[0086] b. To prepare rinse medium (Hank's Buffered Salt Solution (HBSS) without calcium or magnesium and containing 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube "Rinse Medium" and include your initials and the date the medium was prepared.

[0087] c. All media / culture media are tested for sterility using Bactec Fytic / 10 (Dickinson & Company) and Bactec Pluc + Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared media / culture media is poured into each bottle.

[0088] 2. Collection and isolation of human and red deer (RD) umbilical cord tissue Umbilical cords from red deer (RD) were collected from a farm in New Zealand where these animals are raised for horn velvet harvesting, according to specific instructions. RD-CLMSCs were isolated from the umbilical cords according to the protocol described in International Patent Application WO 2006 / 019357 A1. Human umbilical cord tissue (provided with maternal informed consent) was processed as described in International Patent Application WO 2018 / 067071 A1 or WO 2019 / 199234 A1 for subsequent isolation of mesenchymal stem cells from the umbilical cord.

[0089] Briefly, red deer and human umbilical cords were washed thoroughly and immediately transferred to 500 ml sterile glass bottles containing culture transport medium (L-15 medium supplemented with 50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml fungizone, and 50 μg / ml gentamicin; all reagents purchased from Invitrogen) before being transported to the laboratory. In the laboratory, stem cell extraction was performed under sterile conditions in a laminar flow hood. First, the umbilical cords were transferred to a sterile stainless steel tray. Any blood remaining in the umbilical vessels was removed by multiple syringe washes with warm phosphate-buffered saline (PBS) supplemented with 5 IU / ml heparin (Sigma). The final wash was performed with pure PBS without heparin. The umbilical cord tissue specimens were then cut into 2 cm lengths and transferred to 10 cm diameter cell culture dishes, further washed and disinfected with 70% ethanol, followed by multiple washes with PBS containing an antibiotic mixture (50 IU / ml penicillin, 50 μg / ml streptomycin, 250 μg / ml fungizone, 50 μg / ml gentamicin; all purchased from Invitrogen) until the solution became clear.

[0090] 3. Isolation and culture of RD-CLMSCs and H-CLMSCs First, umbilical cord tissue is dissected to separate the amnion from Wharton's jelly (i.e., the umbilical cord matrix) and other internal components. The isolated amnion is then cut into small pieces (0.5 cm x 0.5 cm) for cell isolation. For mesenchymal stem cell isolation, explantation is performed by placing the small pieces of amnion on tissue culture dishes under different cell culture conditions.

[0091] For mesenchymal cell isolation and culture, explants were submerged in PTT-6 medium (CellResearch Corp, Singapore) and maintained in a CO2 cell culture incubator at 37°C. The medium was changed every 2–3 days. Cell outgrowth was monitored under a light microscope. At approximately 70% confluence, cells were trypsinized (0.05% trypsin / 0.02% EDTA) for further expansion or cryopreservation. H-CLMSCs (#CLMC43) were also obtained from the CRC tissue bank.

[0092] Example 2: Conditioned media from red deer umbilical cord lining mesenchymal stem cells (RD-CLMSC), human umbilical cord lining mesenchymal stem cells (H-CLMSC) and human foreskin fibroblasts (FSF) Cryovials containing RD-CLMSCs, H-CLMSCs, and human foreskin fibroblasts (FSFs) as a control were removed from storage and rapidly thawed in a 37°C water bath. FSFs were a gift from the Stem Cell and Wound Healing Research Group, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, to CellResearch Corporation (CRC, Singapore).

[0093] PTT-6 medium (Cell Research Corporation, Singapore) was used to culture RD-CLMSCs and H-CLMSCs at 37°C and 5% CO2. DMEM containing 10% FCS was used to culture FSFs at 37°C and 5% CO2. The cell culture medium was changed every 2–3 days. At 80–100% confluency, the cell culture medium was removed, and the cells were optionally rinsed in PBS and then cultured in DMEM basal medium; this medium optionally contained water-soluble antioxidants but did not contain growth factors or serum. The culture dishes were incubated for an additional 48 hours, and the spent medium was collected in a centrifuge tube and centrifuged at 1800 rpm for 10 minutes. The supernatants were collected into labeled tubes as conditioned medium (CM): 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.

[0094] Example 3: Elastin and hyaluronic acid (HA) expression in human dermal fibroblasts (HDF) cultured in conditioned medium of RD-CLMSC and FSF compared to DMEM / 10% FCS The effects of conditioned medium derived from cultures of red deer umbilical cord mesenchymal stem cells (RD-CLMSCs) and human foreskin fibroblasts (FSFs) on human skin were evaluated to clarify the potential mechanisms of action of conditioned medium from different sources (RD-CLMSCs and FSFs) on human skin and whether such mechanisms are related to promoting hair growth. To evaluate the effects of conditioned medium on human skin, human dermal fibroblasts (HDFs) were used, selected from the CellResearch Corporation (CRC) tissue bank from the sources listed in the table below; the dermal fibroblasts were divided into normal fibroblasts (NFs) from individuals under 60 years of age and senescent dermal fibroblasts (asFs) from individuals over 60 years of age.

[0095] Table 2. HDF and donor profile TIFF2025538460000007.tif187150

[0096] method HDFs were seeded in 96-well plates at a cell density of 10,000 cells / well in DMEM / 10% FCS (LifeTech Holdings, catalog 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, catalog no. MBS2025717) and elastin (LifeTech Holdings, catalog no. MA 127129); Figures 1A and 2A. Briefly, cells were washed with 1X PBS, fixed in cold methanol for 10 minutes, and then blocked with 2.5% normal horse serum (Vector Laboratories, catalog no. PK-7200) for 20 minutes in a humidified chamber. The cells were then incubated with primary HA antibody (1:50) and elastin antibody (1:50) for 2 hours at room temperature. After washing with TBS (VWR Life Sciences, Catalog No. 788) and 1% Tween solution (Sigma, Catalog No. P9416), they were incubated with secondary antibody (Vector Laboratories, Catalog No. PK-7200) for 15 minutes. After three additional washes with 1X TBS / 1% Tween solution, the cells were incubated with ABC Reagent (Vector Laboratories, Catalog No. PK-7200) for 15 minutes at room temperature. Finally, DAB (Dako, Catalog No. K3468) was added for colorimetric development. Harris hematoxylin (Sigma, Catalog No. HHS16) was used for counterstaining. Images were taken at 10x magnification using a bright-field, phase-contrast microscope (Olympus). Elastin and hyaluronan expression were quantified using ImageJ (National Institutes of Health, USA).

[0097] result To compare the effects of RD-CLMSC-CM and DMEM / 10% FCS (control) on the regulation of elastin and HA expression in HDFs and assess whether these effects depended on the donor's age and the site from which the skin tissue was harvested, HDFs (n = 14) isolated from donors aged 23 to 73 years and from multiple donor sites (forehead, eyelid, cheek, neck, and temple) were selected from a skin library for this series of experiments. After culturing cells in DMEM / 10% FCS or RD-CLMSC-CM for 48 hours, immunocytochemical staining for elastin and HA was performed. Expression levels were quantified as optical density using ImageJ (National Institutes of Health, USA). As shown in Figures 1A and 1B and 2A and 2B, RD-CLMSC-CM upregulated elastin expression by 56% and HA expression by 83% in HDFs compared to DMEM / 10% FCS. Overall, this series of experiments demonstrates a significant upregulation of elastin and hyaluronan expression in HDFs by the conditioned medium of the present invention, an effect that did not appear to be related to donor age or the donor site from which the skin was derived.

[0098] conclusion Without wishing to be bound by theory, it can be hypothesized that the upregulation of elastin and HA expression seen in RD-CLMSC-CM is due to the presence of growth factors, including TGF-β, IGF, PDGF-BB, and FGF-7, present in RD-CLMSC-CM.

[0099] Example 4: Proliferative effect of human dermal fibroblasts (HDFs) in conditioned medium derived from cultures of H-CLMSCs, RD-CLMSCs, and FSFs compared to DMEM / 10% FCS background The proliferation characteristics 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 examined by exposing senescent human dermal fibroblasts (HDFs) to these media for 5 days and compared with DMEM / 10% FCS (control) (Figure 3). In this assay, slowly growing senescent HDFs were used instead of normally growing HDFs to amplify the differences in the proliferation effects of the different CMs. The superior proliferation characteristics suggest the presence of other protein growth factors in the conditioned medium that may be positively affecting HDF proliferation.

[0100] method Senescent dermal fibroblasts (asF) were isolated from the skin of donors over 60 years old (asF74 / asF75 / asF76, Table 2). The asFs were evenly 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 attachment. After 24 hours, the medium was removed from the plates, 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 medium was changed on day 2. On day 5, the medium was removed, and the cells were washed once with PBS. The cells were trypsinized and observed under a microscope to confirm that all cells had been removed, followed by neutralization with the addition of 1 ml DMEM / 10% FCS. The cell suspension was centrifuged at 1800 rpm for 10 minutes, the supernatant discarded, and the pellet at the bottom of each tube resuspended in 1 ml of DMEM. 50 μl of the cell suspension was mixed with 50 μl of trypan blue (Sigma, Cat. No. T8154) and incubated for 10 minutes. Total cell numbers were counted using a Countess-II automated cell counter system (Life Technologies, Cat. No. AMQAX10001).

[0101] result As shown in Figure 3, compared with DMEM / 10% FCS (control), the highest increase in mean total cell number was observed in the RD-CLMSC-CM group (113% increase compared to the control), followed by H-CLMSC-CM (112%) and FSF-CM (16%). These results demonstrate that the growth factors in RD-CLMSCs are as effective as those in H-CLMSC-CM and do not exert toxic effects on HDFs, demonstrating cross-species efficacy. Without wishing to be bound by theory, the superior growth characteristics of CLMSC-CM (RD and H) compared with FSF-CM and FBS controls suggest the presence of other protein growth factors in CLMSC-CM that may be positively influencing HDF proliferation.

[0102] Example 5: Pro-migratory properties of RD-CLMSC-CM compared to FSF-CM assessed in an in vitro "scratch" wound assay background The in vitro "scratch" assay mimics cell mobility during wound healing and is a well-developed method for measuring cell migration.

[0103] method To generate a cell monolayer, 100,000 HDFs were seeded in each 100 mm dish and cultured in DMEM / 10% FCS at 37°C and 5% CO2 until 100% confluent. A p200 pipette tip was used to create a linear scratch in the monolayer, creating a "scratch." The dish was then rinsed once with PBS to remove loose cells. Cells were immersed in FSF-CM or RD-CLMSC-CM for 5 days, and images were taken on days 0 and 5 to document cell migration from the gap edge.

[0104] result As shown in Figure 4, the migration-promoting properties of RD-CLMSC-CM were compared with those of FSF-CM by measuring the area remaining unfilled with HDFs after 5 days of culture in the test CM. By day 5, all "scratch" areas in the RD-CLMSC-CM-treated group were completely filled, whereas only approximately 80% of the scratches were filled in the FSF-CM-treated group. These results demonstrate that the migration-promoting properties of RD-CLMSC-CM are stronger than those of FSF-CM. The significant increase in migration of HDFs cultured in RD-CLMSC-CM may be due to the migration-promoting effects of VEGF, TGF-β1, and HGF.

[0105] Example 6: Promotion of hair inductive potential by conditioned medium derived from culture of RD-MLSCs background The purpose of this study was to evaluate the ability of conditioned medium derived from cultures of umbilical cord mesenchymal stem cells, as shown in Example 2, to stimulate improved hair growth from inactive or dormant hair follicles in men suffering from androgenetic alopecia (AGA). CALECIM®, a novel cosmetic formulated from the conditioned medium of the present invention, was used as an exemplary conditioned medium of the present invention; it contains proteins, particularly growth factors, and exosomes naturally secreted by umbilical cord mesenchymal stem cells.

[0106] method Patients were selected if they showed signs of hair thinning / hair loss. Patients who missed scheduled follow-up visits; patients who had any adverse reactions to CALECIM® products; patients with thyroid dysfunction, vitamin and iron deficiencies, or hormonal imbalances; smokers; pregnant and lactating women; male patients with a Norwood scale of 5 or higher; and female patients with a Ludwig scale of 3 or higher were excluded from this study.

[0107] Once a patient was selected, photographs were taken before the first treatment; see Figures 6A-6G. Treatments were administered weekly, typically for a total of six treatments. A second set of photographs was taken during and / or upon completion of treatment; see Figures 6A-6G.

[0108] The protocol for each treatment was as follows: · Scalp disinfection with 0.5% chlorhexidine. 2.5 ml of CALECIM® Advanced Hair System, formulated from conditioned medium derived from cultures of red deer umbilical cord mesenchymal stem cells, was applied to the treatment site and needled using a derma stamp with microneedles of 1 mm, 1.2 mm, and 1.5 mm in length. After treatment with the Dermastamp, apply an additional 2.5ml and gently rub into the scalp. Patients were advised not to wash their hair for at least 24 hours, and optimally 48 hours, after the procedure.

[0109] result As shown in Figures 6A-6G, all patients experienced objective improvements in hair density and coverage in the areas treated with the CALECIM® Advanced Hair System, as assessed by the treating physician. Similarly, all patients reported experiencing improvements in hair quality and coverage. Patients underwent 3- and 6-month follow-up to assess further progress.

[0110] Example 7: Conditioned medium derived from cultures of RD-MLSCs stimulates improved hair growth in patients with androgenetic alopecia (AGA) background Androgenetic alopecia (AGA), also known as male and female pattern hair loss, is the most common cause of hair loss worldwide today. It can affect up to 50% of Caucasian men. AGA has many causes and is widely recognized as multifactorial, including genetic, environmental, dietary, and hormonal influences. In fact, the hair follicle growth cycle shortens in time, and the hair produced in that cycle becomes shorter and thinner. Over time, the hair follicles become completely inactive within the scalp. The purpose of this study is to evaluate the ability of CALECIM® product, formulated from conditioned medium derived from cultures of mesenchymal stem cells from red deer umbilical cords as shown in Example 2, to stimulate improved hair growth from inactive or dormant hair follicles in men with AGA.

[0111] method A total of five male patients with androgenetic alopecia (AGA), rated as a 3-4 on the Norwood scale of hair loss, were selected. All participants were tested for vitamin deficiencies, iron deficiencies, and thyroid function. Patients with abnormal results were excluded from the study. Smokers were also excluded from the study. Once patients were selected, photographs were taken before the first treatment. Treatments were performed weekly for a total of six treatments. A second set of photographs was taken upon completion of six weekly treatments; see Figure 5.

[0112] The protocol for each treatment was as follows: · Scalp disinfection with 0.5% chlorhexidine. 2.5 ml of a composition containing 80% conditioned medium and 20% aqueous hyaluronic acid (CALECIM® Advanced Hair System), formulated from conditioned medium derived from cultures of mesenchymal stem cells from red deer umbilical cord, was applied to the treatment area and needled using a dermastamp with 2 mm-long microneedles. After treatment with the Dermastamp, apply an additional 2.5ml and gently rub into the scalp. Patients were advised not to wash their hair for at least 24 hours, and optimally 48 hours, after the procedure.

[0113] result As shown in Figure 5, at week 6, all patients experienced objective improvements in hair density and coverage in the areas treated with the conditioned medium composition, as assessed by the treating physician. Similarly, all patients reported experiencing improvements in hair quality and coverage. Patients were followed up at 3 and 6 months to assess further progress.

[0114] Example 8: Case Study - Conditioned medium derived from cultures of RD-MLSCs stimulates improved hair growth in patients with COVID-19-induced scalp alopecia background COVID-19-induced scalp alopecia can induce telogen effluvium (TE), a condition that refers to diffuse scalp alopecia. A patient with severe, chronic TE, whose hair loss had not spontaneously resolved after 6 months and was unresponsive to conventional treatment with minoxidil and LED light therapy (Hairmax), was treated with a composition containing conditioned medium derived from cultures of umbilical cord mesenchymal stem cells (CALECIM® Advanced Hair System).

[0115] Methods and Results The patient's scalp was treated in stages; after scrubbing with an alcohol-based swab, 1 ml of a composition containing conditioned medium derived from umbilical cord mesenchymal stem cell culture was applied and massaged until dry. The patient was advised to wait until the evening before washing her hair. Five treatments with conditioned medium derived from umbilical cord mesenchymal stem cell culture were administered at monthly intervals. The patient experienced a gradual improvement in hair density, with regrowth of villi hair at the frontal hairline and prefrontal area (Figure 7). Hair brittleness improved, and the hair did not break when combed.

[0116] Example 9: Growth-promoting effect of conditioned medium on cultured human dermal papilla cells (HFDPC) Testing Protocols and Procedures Hair growth is controlled by a unique, repetitive cycle consisting of anagen, catagen, and telogen phases. Dermal papilla cells (DPCs), a specialized group of fibroblasts within the hair follicle bulb, play an essential role in regulating hair growth not only during the normal hair cycle but also in the pathogenesis of certain pathologies, such as androgenetic alopecia. Therefore, factors affecting DPC function during hair loss are of great therapeutic importance. HFDPC proliferation was assessed by measuring metabolic activity using the highly water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8). HFDPCs were treated with either different concentrations of minodixil (0.0125–200 μg / mL; see left column of Table 3, which serves as a reference) or different dilutions of the present conditioned medium (PC, dilution range 10–1280; see third column of Table 3). Negative control (NC) refers to HFDPCs incubated with basal medium only.

[0117] Analysis results The results are shown in Table 3 and illustrated in Figure 9. HFDPCs treated with crude extracts have approximately 10% more cells than the control at a concentration of 0.048 mg / mL. HFDPCs treated with minoxidil (standard) have approximately 20% more cells than the negative control at a 320-fold dilution.

[0118] Table 3. Cell counts of HFDPCs treated with conditioned medium (right), minoxidil (left, as a reference compound), and negative control (NC). TIFF2025538460000008.tif81136

[0119] statistical analysis All data are presented as mean ± standard deviation (SD). Student's t-test was used to compare different treatments between two groups. p<0.05 is considered significant, p<0.01 is usually considered highly significant, and p<0.001 is considered extremely significant. After calculation, p is 0.03.

[0120] conclusion When samples treated with the conditioned medium of the present invention were diluted 160-fold and 320-fold, approximately 10% and 20% more cells were obtained compared to negative control-treated cells (see also Figure 8); the proliferation-promoting effect in this model experiment was similar to that of the reference compound minoxidil, whose optimal concentration was 0.2-2 μg / mL. In other words, this experiment demonstrated that the conditioned medium of the present invention increased dermal papilla cell proliferation by up to approximately 24%, comparable to the most effective concentration of minoxidil. It should be noted that minoxidil is known to have serious undesirable side effects, including, but not limited to, itching and skin rash, scalp burning, inflammation and pain of hair roots, redness of the skin, 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 with the conditioned medium of the present invention to date. Furthermore, while minoxidil has been reported to be effective in treating hair loss in only 40% of patients, the conditioned medium of the present application was found to be effective in inducing, stimulating, and / or promoting hair growth and hair regrowth in up to 95% of patients participating in this study, without any side effects (data not shown). Thus, this example demonstrates that the conditioned medium of the present invention represents a clear improvement over minoxidil.

[0121] Example 10: Anti-inflammatory activity of conditioned medium (CALECIM) on UV-induced primary normal human epidermal keratinocytes (NHEK) Test Procedure The anti-inflammatory activity of the samples was tested against UVB-induced NHEK cells in 6-well culture plates. First, NHEK cells were seeded in 6-well plates at a density of 100,000 cells / well. After 24 hours, the cells were washed once with warm PBS and then irradiated with UVB (40 mJ / cm). 3) and then induce the cells with PBS. The PBS is then removed and replaced with fresh medium containing the sample or vitamin D (4 μM). The cells are incubated for an additional 24 hours at 37°C and 5% CO2. The cell culture supernatant is collected and stored at -80°C before use. Sample preparation: Prepare the test sample by adding 1 ml of B to 49 ml of A. The sample was diluted 2, 4, 8, 16, 32, and 64 times using NHEK culture medium.

[0122] Measurement of TNF-α a. Label removable 8-well strips for the experiment. Wash the microwell strips twice with approximately 400 μl of wash buffer per well, thoroughly aspirating the contents of the microwells between washes. 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. c. Cover the wells and incubate at room temperature with gentle shaking for 2 hours. d. Discard the solution and wash four times with 1x wash solution. Use a multichannel pipette to fill each well with wash buffer (400 μl). Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by aspiration or decantation. Invert the plate and blot it against a clean paper towel. e. Add 100 μl of the prepared streptavidin solution to each well. Incubate at room temperature with gentle shaking for 1 hour. f. Discard the solution. Repeat the washing procedure as in step d. g. Add 100 μl of TMB substrate solution to each well. Incubate at room temperature for 30 minutes in the dark. h. Add 100 μl of stop solution to each well. Read immediately at 450 nm.

[0123] Analysis results Keratinocytes are the primary target of UVB and play a central role in inflammatory and immunoregulatory changes, which are induced by UV-induced release of inflammatory 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). The production of TNF-α, a representative inflammatory cytokine, was observed at 40 mJ / cm2 in NHEK cells. 2 UVB irradiation. As expected, UVB exposure causes increased release of TNF-α (1.6 pg / ml vs. 485 pg / ml). This sample showed the strongest inhibition of TNF-α production at 2x and 4x dilutions, which is comparable to that of vitamin D; see Figure 9.

[0124] conclusion In this experiment, the conditioned medium of the present invention reduces the expression / production of a key inflammatory cytokine (TNF-α) by 30-fold, comparable to vitamin D.

[0125] Example 11: Conditioned medium derived from cultures of RD-MLSCs is effective in improving hair growth in both male and female patients with androgenetic alopecia (AGA) participants Study participants (n=5 women, n=5 men) with a mean age of 34.1±14.2 years were recruited and enrolled at a single site in Vienna, Austria. Patients with symptoms and subjective perception of hair thinning / hair loss were recruited primarily via social media and study recruitment platforms.

[0126] On-site screening was performed to identify eligible subjects according to the exclusion and inclusion criteria shown in Table 4; the latter included the Hamilton-Norwood and Ludwig scales for male and female hair loss patients, respectively. All subjects consented to the use of demographic and study outcome data for scientific and marketing purposes.

[0127] (Table 4) Inclusion and Exclusion Criteria TIFF2025538460000009.tif36146

[0128] Treatment Protocol Each of the 10 patients had their scalps disinfected with 0.5% chlorhexidine before application. Treatment sites were subjectively selected based on the patient's area of ​​concern and varied slightly from patient to patient. 2.5 ml of the conditioned medium composition of the present invention (containing 80% conditioned medium and 20% aqueous hyaluronic acid solution) was applied to the treatment area. Each zone was then treated with microneedling using a 0.5 mm needle-length dermastamp, passing it three times in alternating directions, and an additional 2.5 ml of conditioned medium composition was applied and gently rubbed into the scalp. Patients were advised not to wash their hair for at least 24 hours, and optimally 48 hours, after treatment.

[0129] This treatment cycle was repeated weekly for a total of 12 weeks. Patients continued this weekly treatment at home, were given a Dermastamp and weekly products to use, and returned for on-site evaluations at weeks 4, 6, 7, 10, and 12. Evaluations included subjectively reported outcomes such as psychological questionnaires and objective measurements of hair growth using Canfield Scientific's HairMetrix® software.

[0130] measurement Phototrichograms to assess hair changes during treatment were recorded for all patients by two trained raters using the HairMetrix® D200-evo system with HairMetrix® software from Canfield Scientific (4 Wood Hollow Road, Parsippany, NJ 07054, USA). Canfield's non-invasive hair consultation tool provides immediate results during the consultation without cutting hair. HairMetrix® ensures objective data and allows study personnel to take photographs, eliminating the need for additional laboratory evaluation.

[0131] Standardized questionnaires Standardized questionnaires were used to assess patients' quality of life and subjective procedural experience (questionnaires not shown).

[0132] result Ten patients (mean age 34.1±14.2 years, 50% female, as shown in Table 5) received conditioned medium according to the treatment protocol described in the Methods section. Of note, male patients with Norwood types 4, 5, and 6 alopecia (i.e., severe hair loss) participated in this study.

[0133] Table 5. Basic demographic information of hair loss patients (n=10) receiving conditioned medium on the scalp (capillitium) for 12 weeks according to the treatment protocol described in the Methods section. TIFF2025538460000010.tif86152

[0134] After 12 weeks of treatment, a significant increase (P<0.05) in follicular units per square centimeter (sq cm) and a corresponding decrease (P<0.05) in intrafollicular distance were detected, as shown in Figures 17A and 17B. Similarly, as shown in Table 6 and Figures 17C and 17D, the total number of hairs per sq cm increased (P<0.05), and therefore the total hair diameter per sq cm also increased (P<0.05). Notably, there was no change in the number of hairs per FU or the mean hair diameter. No adverse effects were reported. Based on patient subjectivity, standardized questionnaires indicated significant improvement in hair growth, decreased need for concealment, and improved quality of life related to hair loss, as shown in Table 7 and Figures 18A–18C. Nine out of ten patients would recommend the conditioned medium product, indicating that the conditioned medium is effective in treating severe hair loss, such as Norwood types 4, 5, and 6.

[0135] Example 12: Encapsulation of conditioned medium from the culture of RD-MLSCs into a liposomal formulation and use of this formulation to improve hair growth in patients with androgenetic alopecia (AGA) Pro-Lipo™ Neo, a mixture of phospholipids available from Lucas Meyer Cosmetics, was used to encapsulate conditioned medium derived from the culture of RD-MLSCs to form bilayer liposomes suitable for topical administration. This liposome formulation was then used to treat AGA patients. In this study, the conditioned medium was used together with other compounds reported to prevent and stop hair loss and / or simultaneously stimulate hair growth. Compounds reported to stimulate hair growth included Capixyl™, a biomimetic peptide (acetyl tetrapeptide-3) available from Lucas Meyer Cosmetics, ribose, menthol, and caffeine. The encapsulated hair serum produced in this experiment had the following composition: Encapsulated Hair Serum a. Conditioned medium: 50% (v / v) b. Capixyl™ (acetyl tetrapeptide-3): 2% (reference value, v / v) c. Riboxyl (ribose): 0.5% (reference value, v / v) d. Pro-Lipo Neo (liposome mixture) - delivery system: 20% (v / v) e. Menthol: 0.1% (reference value, v / v) f. Caffeine: 2% (reference value, v / v) The remainder is excipients.

[0136] The encapsulated hair serum was formulated as follows: Step 1: Encapsulating the active ingredient in liposomes 1. Thaw conditioned medium (CM); 2. Mix CM, Capixyl™, Riboxyl, caffeine and PLN under low shear with stirring; 3. Stir for 30 minutes until homogenous. Step 2: Blend menthol into the serum base 1. Dissolve menthol in the excipient base; 2. Remove from heat and allow the excipient base to cool. Step 3: Blend liposome-encapsulated active ingredients into a serum base 1. Mix the liposome-encapsulated active ingredient, excipient base, and preservative under low shear stirring until homogeneous; 2. Ensure the pH of the final product is 7.

[0137] Because this liposomal formulation should improve dermal absorption of the conditioned medium after topical application to the scalp, the hair serum thus produced was applied topically to the scalp of AGA patients without the use of microneedling or a dermastamp. During treatment with this liposomal formulation of conditioned medium, a significant improvement in hair growth was observed (data not shown), demonstrating the effectiveness of this formulation for inducing and promoting hair growth.

[0138] The present invention is also characterized by the following items.

[0139] 1. A method for inducing, stimulating, and / or promoting hair growth and / or hair regrowth, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. 2. A method for alleviating and / or reducing hair loss and / or thinning, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. 3. The method of item 1 or 2, wherein the conditioned medium is obtained or obtainable by culturing mesenchymal stem cells of the umbilical cord in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). 4. The method of item 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 item 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 of items 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 culture medium comprises: (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml; or (ii) the culture medium contains EGF at a final concentration of 10 ng / ml; or (iii) the culture medium contains insulin at a final concentration of 1 μg / ml to 10 μg / ml; or (iv) the culture medium contains insulin at a final concentration of 5 μg / ml; or (v) the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3); or (vi) The culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), and optionally, the culture medium contains adenine at a final concentration of 0.01 to 0.1 μg / ml, hydrocortisone at a final concentration of 0.1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml. 7. The method of any one of items 3 to 6, further comprising: 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. 9. The method of any of items 1 to 8, wherein the conditioned medium is derived from a culture of a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, a culture of a mesenchymal stem cell population from Wharton's jelly (WJ) of the umbilical cord, a culture of a perivascular (PV) mesenchymal stem cell population, or a culture of a mixed mesenchymal stem cell population (MC) from the umbilical cord. 10. The method of any of items 1 to 9, wherein the conditioned medium is applied topically to the skin, particularly the scalp, of the subject. 11. The method of any of items 1 to 10, wherein the conditioned medium is applied to the subject's hairline. 12. The method of any of items 1-11, wherein the conditioned medium is administered about once, twice, or three times per week for a period of 3, 4, 5, 6, 7, 8, 10, or more weeks. 13. The method of any 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. 14. The method of any of items 1 to 13, wherein the subject suffers from a disease or condition related to hair loss / thinning, in particular alopecia, such as androgenetic alopecia, alopecia areata, alopecia totalis, alopecia universalis, or COVID-19-induced alopecia. 15. The following steps: (a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum); (b) removing the umbilical cord mesenchymal stem cells from the culture medium; (c) optionally culturing the umbilical cord mesenchymal stem cells in a further culture medium, wherein the cell culture medium optionally comprises a water-soluble antioxidant. Including, The conditioned medium is obtained by collecting the cell culture medium. Methods for generating conditioned medium. 16. The method of item 15, wherein the cell culture medium does not contain growth factors and / or serum and / or the cells are cultured at a concentration of about 1 million cells per ml. 17. The method of item 16, wherein the umbilical cord mesenchymal stem cells are red deer umbilical cord mesenchymal stem cells. 18. The method of items 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 regrowth, and / or to alleviate and / or reduce hair loss and / or thinning in a subject. 19. The method of any of items 16 to 18, wherein the conditioned medium is diluted with a control medium. 20. The method of any of items 1 to 14, wherein the conditioned medium is obtained or obtainable by the method of any of items 15 to 19. 21. A conditioned medium derived from umbilical cord mesenchymal stem cells, obtained or obtainable by the method of any of items 15 to 19. 22. A composition comprising the conditioned medium of item 21. 23. Use of conditioned medium derived from a culture of umbilical cord mesenchymal stem cells to induce, stimulate, and / or promote hair growth and / or hair regrowth, and / or to alleviate and / or reduce hair loss and / or thinning in a subject. 24. Use according to item 23, wherein the conditioned medium derived from the culture of umbilical cord mesenchymal stem cells is obtained or obtainable by the method according to any of items 15 to 19. 25. Use of conditioned medium derived from a culture of umbilical cord mesenchymal stem cells for the manufacture of a medicament for inducing, stimulating, and / or promoting hair growth and / or hair regrowth and / or for alleviating and / or reducing hair loss and / or thinning in a subject. 26. Use of the conditioned medium of item 25, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells. 27. Use of item 25 or 26, wherein the conditioned medium is obtained or obtainable by culturing mesenchymal stem cells of the umbilical cord in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). 28. Use of 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). 29. Use of 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). 30. Use of any of items 26 to 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). 31. The culture medium comprises: (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml; or (ii) the culture medium contains EGF at a final concentration of 10 ng / ml; or (iii) the culture medium contains insulin at a final concentration of 1 μg / ml to 10 μg / ml; or (iv) the culture medium contains insulin at a final concentration of 5 μg / ml; or (v) the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3); or (vi) The culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), and optionally, the culture medium contains adenine at a final concentration of 0.01 to 0.1 μg / ml, hydrocortisone at a final concentration of 0.1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml. Use of any of items 26 to 30, further including: 32. The use of any of items 25 to 31, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells. 33. Use of any of items 25 to 32, wherein the conditioned medium is derived from culturing a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, culturing a mesenchymal stem cell population from Wharton's jelly (WJ) of the umbilical cord, culturing a mesenchymal stem cell population from perivascular (PV), or culturing a mixed mesenchymal stem cell population (MC) from the umbilical cord. 34. Use of any of items 25 to 33, comprising topically applying the conditioned medium to the skin, particularly the scalp, of a subject. 35. Use of any of items 25 to 34, comprising applying the conditioned medium to the hairline of a subject. 36. The use of any of items 25-35, wherein the conditioned medium is administered about once, twice, or three times per week for a period of 3, 4, 5, 6, 7, 8, 10, or more weeks. 37. Use of any of items 25 to 36, wherein the subject suffers from hair loss / thinning, in particular inflammation-induced hair loss / thinning, such as chronic inflammation-induced hair loss. 38. The use of any of items 25 to 37, wherein the subject suffers from a disease or condition related to hair loss / thinning, in particular alopecia, such as androgenetic alopecia, alopecia areata, alopecia totalis, alopecia universalis, or COVID-19-induced alopecia.

[0140] It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0141] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0142] The invention illustratively described herein can suitably be practiced in the absence of one or more elements, or one or more limitations, not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be interpreted expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description, rather than terms of limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described, or portions thereof, recognizing that various modifications are possible within the scope of the claimed invention. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it will be understood that those skilled in the art can make modifications and variations of the invention disclosed and embodied therein, and that such modifications and variations are considered to be within the scope of the present invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings included in the generic disclosure also constitutes part of the invention. This includes the generic description of inventions with a proviso or negative limitation excluding any item from the genus, whether or not the excluded item is specifically described herein. Furthermore, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member of the Markush group or subgroup thereof. Further aspects 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, comprising treating a subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

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

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

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

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

8. 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. further comprising the conditioned medium is obtained by collecting the further cell culture medium. The method according to any one of claims 3 to 7.

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

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

11. 11. The method of claim 10, wherein the basal 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. 12. The method of any one of claims 8 to 11, wherein the conditioned medium is diluted with a carrier vehicle, wherein the final concentration of the conditioned medium is preferably from about 10% to about 90% (v / v), 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% of the total volume of the composition containing the conditioned medium.

13. The method according to any one of claims 1 to 12, wherein the umbilical cord mesenchymal stem cells are human or red deer umbilical cord mesenchymal stem cells.

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

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

16. 16. The method of any one of claims 1 to 15, wherein the treating step comprises applying the conditioned medium to the subject's hairline.

17. 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 3, 4, 5, 6, 7, 8, 10, or more weeks.

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

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

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

21. 19. The method of claim 18, wherein the hair loss caused by drug treatment is hair loss caused by cancer chemotherapy.

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

23. 23. The method of claim 22, wherein the alopecia areata is alopecia areata monolocularis, alopecia areata multiplex, alopecia serpiginosa, or alopecia universalis.

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

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

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

27. The following steps: (a) culturing umbilical cord mesenchymal stem cells in a culture medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum); (b) removing the umbilical cord mesenchymal stem cells from the culture medium; and (c) culturing the umbilical cord mesenchymal stem cells in an additional culture medium, wherein the additional culture medium comprises a water-soluble antioxidant. Including, the conditioned medium is obtained by collecting the further cell culture medium; Methods for generating conditioned medium.

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

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

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

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

33. 33. The method of claim 32, wherein the culture medium comprises DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v).

34. 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. 35. The method of any one of claims 27 to 34, wherein the conditioned medium is diluted with a carrier vehicle, wherein the final concentration of the conditioned medium is preferably from about 10% to about 90% (v / v), 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% of the total volume of the composition containing the conditioned medium.

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

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

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

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

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

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

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

46. Use of conditioned medium derived from the culture of umbilical cord mesenchymal stem cells for the manufacture 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 thinning in a subject.

47. 47. The use of claim 46, comprising treating the subject's hair with conditioned medium derived from a culture of umbilical cord mesenchymal stem cells.

48. 48. The use of claim 46 or 47, wherein the conditioned medium is obtained or obtainable by culturing mesenchymal stem cells of the umbilical cord in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).

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

50. 50. The use of 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. 51. The use according to any one of claims 46 to 50, 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 culture medium comprises: (i) epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml; or (ii) the culture medium contains EGF at a final concentration of 10 ng / ml; or (iii) the culture medium contains insulin at a final concentration of 1 μg / ml to 10 μg / ml; or (iv) the culture medium contains insulin at a final concentration of 5 μg / ml; or (v) the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3); or (vi) the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3), and optionally the culture medium contains adenine at a final concentration of 0.01 to 0.1 μg / ml, hydrocortisone at a final concentration of 0.1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

52. The use according to any one of claims 46 to 51, further comprising:

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

55. 55. The use according to any one of claims 44 to 54, comprising topically applying the conditioned medium to the skin, in particular the scalp, of a subject.

56. 56. The use of any one of claims 44 to 55, comprising applying the conditioned medium to the hairline of a subject.

57. 57. The use of any one of claims 44-56, wherein the conditioned medium is administered about once, twice, or three times per week for a period of 3, 4, 5, 6, 7, 8, 10, or more weeks.

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

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

60. 60. The use of claim 59, wherein the hormone-related hair loss / thinning is menopause-related hair loss or postpartum-related hair loss.

61. 59. The use of claim 58, wherein the hair loss due to drug treatment is hair loss due to cancer chemotherapy.

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

61. 61. The use of claim 60, wherein the alopecia areata is alopecia areata monolocularis, alopecia areata multiplex, alopecia serpiginosa, alopecia totalis, or alopecia universalis.

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

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

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

Images