Composition for promoting hair follicle neogenesis in scalp comprising dermal papilla cell culture medium extract

Abstract

The purpose of the present invention is to provide a dermal papilla cell extract which overcomes the limitations of a conventionally known dermal papilla cell culture medium and has excellent efficacy in the regeneration of human tissues, such as hair loss prevention, hair growth, and wound treatment, by promoting hair follicle neogenesis.

Description

Composition for promoting scalp hair follicle regeneration comprising a culture medium extract of human hair papilla cells

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2024-0073783, filed with the Korean Intellectual Property Office on June 5, 2024, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for promoting scalp hair follicle regeneration comprising a culture extract of human hair papilla cells.

[0003] Hair is produced by the hair follicle, located beneath the skin. The matrix cells surrounding the hair root break down to form cortical cells, which become the material for the hair fiber. The hair shaft, which protrudes outside the skin, is composed of hair shaft fibers, the inner root sheath, and the outer root sheath. The central hair shaft fibers are connected to the inner root sheath within the hair follicle. The hair follicle acts as a reservoir of stem cells that can generate all the cell lines necessary to reconstruct the hair follicle itself, the epithelium, and the sebaceous glands. The hair follicle contains the hair shaft along with dermal papilla cells and dermal sheath cells derived from the mesenchyme. Dermal papilla cells are the key cells responsible for hair development and growth, and are located at the very base of the hair root, which is the subcutaneous root of the hair. When dermal papilla cells are collected from the scalp, cultured, and transplanted, new hair grows.

[0004] Hair has a hair cycle consisting of growth (anagen), decline (catagen), and resting (telogen) phases. The period during which hair follicle cells surrounding the hair papilla divide and proliferate is called the anagen phase. Hair growth stops and the hair detaches from the hair papilla during the catagen phase, and then the hair follicle cells stop growing and fall out after a certain period of time during the resting phase.

[0005] Generally, hair loss is a natural phenomenon in which hair falls out from the scalp through the hair cycle. Causes of hair loss include genetics, stress, diet, and the use of scalp products, and hair growth and hair loss generally occur in cycles over approximately 3-5 years. However, in the case of alopecia, a phenomenon in which hair falls out faster than the normal hair growth cycle due to various reasons other than normal hair loss, external stimuli such as ultraviolet rays, stress, and nutritional deficiencies cause the hair to pass through the catagen phase early and enter the resting phase, resulting in hair loss.

[0006] Treatments and solutions for alopecia have evolved significantly over time. Wigs, partial hairpieces, and hair extensions can conceal bald patches but do not promote new hair growth. Topical treatments such as minoxidil, finasteride, and dutasteride can slow the progression of alopecia, but they have side effects associated with hormonal administration and struggle to induce and stimulate new hair follicles. Hair transplants, while free of hormonal side effects, are expensive and have several limitations on the number of transplants they can perform.

[0007] A treatment for alopecia, which has recently gained attention, involves culturing hair papilla cells in vitro and using the cultured extract. However, when isolated and cultured in vitro, hair papilla cells lose their hair follicle-inducing ability over time, preventing the hair follicle-activating components in the cultured extract from exerting their effects.

[0008] The present invention aims to overcome the limitations of previously known culture solutions of hair papilla cells and to provide a hair papilla cell extract that has excellent efficacy in preventing hair loss, promoting hair growth, and regenerating human tissues, such as wound healing, by promoting hair follicle regeneration.

[0009] In order to achieve the above object, the present invention provides a composition for promoting scalp hair follicle regeneration, which comprises, as an active ingredient, a dermal papilla cell extract containing a complex protein associated with the hair growth process and expressing hair growth markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0010] In addition, the present invention provides a pharmaceutical composition for preventing or treating hair loss, which comprises as an active ingredient an extract of hair papilla cells containing a complex protein associated with the hair growth process and expressing hair growth markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0011] In addition, the present invention provides a composition for preventing or treating hair loss, which comprises as an active ingredient an exosome isolated from a dermal papilla cell extract that contains a complex protein associated with the hair growth process and expresses hair growth markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0012] The composition for promoting scalp hair follicle regeneration comprising a hair papilla cell extract according to the present invention contains complex proteins related to the hair growth process and expresses hair growth factors, and thus has superior hair follicle regeneration promotion efficacy compared to existing human-derived hair papilla cells, and is therefore effective in preventing and treating hair loss and promoting hair growth.

[0013] Figure 1 shows the protocol for generating hair papilla cells from human-derived induced pluripotent stem cells (hiPSCs), their morphological changes, and the expression patterns of related markers.

[0014] Figure 1a is a schematic diagram showing a protocol for differentiation of human-derived induced pluripotent stem cells (hiPSCs) into dermal papilla cells (iPSC-DPs).

[0015] Figure 1b is a diagram showing the morphological changes during the iPSC-DP differentiation process.

[0016] Figure 1c shows the qRT-PCR results analyzing the pluripotency markers Nanog and Oct4, which decrease 8 days after the initiation of differentiation.

[0017] Figure 1d shows the qRT-PCR results analyzing the expression of MSC markers CD73, CD44, and CD105 8 days after the initiation of differentiation.

[0018] Figure 1e shows the results of flow cytometry analysis according to CD73, CD44, and CD90 expression in primary hDP, immortalized hDP, and hiPSC-DP.

[0019] Figure 1f is a diagram showing the results of immunostaining of MSC markers Vimentin and Nestin in primary hDP and hiPSC-DP.

[0020] Figure 1g is a diagram showing the results of analysis of similar expression patterns of Vimentin and α-SMA.

[0021] Figure 1h is a diagram showing the results of analysis of Versican, a growth-phase hair papilla cell marker, between primary hDP and hiPSC-DP.

[0022] Figure 2 shows the results of expression marker analysis of hiPSC-derived mammary papilla cells (hiPSC-DP).

[0023] Figure 2a shows the ALP staining results of primary-hDP, immortalized-hDP, and hiPSC-DP.

[0024] Figure 2b shows the results of ALP activity verification by ALP assays.

[0025] Figure 2c shows the qRT-PCR results analyzing functional DP markers CD133, beta-catein, ALPL, SOX2, SOX9, LEF1, and BMP4 in iPSC-DP.

[0026] Figure 2d is an immunostaining image of CD133 and Versican, functional markers of iPSC-DP.

[0027] Figure 2e shows the results of flow cytometry analysis of CD133 and p75 expression at various passages of primary-hDP and hiPSC-DP.

[0028] Figure 2f is an image showing the results of hiPSC-DP double-stained with CD133 and beta-catenin forming 3D spheroids.

[0029] Figure 2g is an immunostaining image showing the expression of CD49 and beta-catenin in hiPSC-DP.

[0030] Figure 2h is a representative immunostaining image of SOX2 and Nestin, neural stem cell markers, in hiPSC-DP.

[0031] Figure 2i shows the qRT-PCR results analyzing Nestin in hiPSC-DP.

[0032] Figure 2j shows the qRT-PCR results analyzing SOX2, SOX9, and PAX3 in hiPSC-DP.

[0033] Figure 2k shows the qRT-PCR results analyzing the similar expression patterns of SKP markers Twist, slug, and Snail between primary-hDP and hiPSC-DP.

[0034] Figure 3 shows the results of promoting hair growth on the back of mice by subcutaneously injecting hiPSC-DP into mice.

[0035] Figure 3a is an image showing the results of observation and quantification of hair coverage.

[0036] Figure 3b is a graph showing the quantification results of hair coverage (*p<0.05 compared to PBS group).

[0037] Figure 3c shows the results of observing hair follicle length in the H&E-stained section of mouse dorsal skin.

[0038] Figure 3d is a graph showing the results of quantitative analysis of the H&E stained section of mouse dorsal skin.

[0039] Figure 3e shows the qRT-PCR results analyzing hair induction markers.

[0040] Figure 4a shows the results of hair shaft growth by hiPSC-DP derived conditioned media.

[0041] Figure 4b is a graph showing the results of hair shaft growth by hiPSC-DP derived conditioned media (length of hair extended in μm).

[0042] Figure 5a shows the results of hair shaft growth induced by hiPSC-DP derived exosomes.

[0043] Figure 5b is a graph showing the results of hair shaft growth by hiPSC-DP derived exosomes (length of hair extended in μm).

[0044] A composition for promoting scalp hair follicle regeneration, comprising as an active ingredient an extract of hair papilla cells containing a complex protein involved in the hair growth process and expressing hair growth factor markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0045] Hereinafter, the present invention will be described in detail with reference to the contents described in the attached drawings.

[0046] One aspect of the present invention relates to a composition for promoting scalp hair follicle regeneration, comprising as an active ingredient an extract of hair papilla cells containing a complex protein associated with the hair growth process and expressing hair growth markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0047] In the present invention, the 'dermal papilla cells (DP)' for producing the composition are differentiated from stem cells, and the differentiated stem cells are not limited in type, and may preferably be dermal papilla cells differentiated from human-derived induced pluripotent stem cells (hiPSC).

[0048] In the present invention, 'dermal papilla cells (DP)' are cells present at the base of the hair follicle, and are a subpopulation of specialized fibroblasts in the hair bulb. They are known to play an important role in the hair growth cycle and androgenetic alopecia through cell proliferation (Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Identification of Androgen-inducible TGF-betal Derived from Dermal Papilla Cells as a Key Mediator in Androgenetic Alopecia. J Investing Dermatol Symp Proc. 2003;8(1):69-71.). In particular, inhibition of growth and apoptosis of dermal papilla cells is necessary for maintaining the hair growth phase, and plays an important role in the activation of hair follicle stem cells required for a new hair growth cycle at the end of the resting phase (telogen). Furthermore, after the growth phase (anagen) begins, dermal papilla cells in the cell cycle continue to provide inductive signals that induce the proliferation and differentiation of hair matrix cells, which form the multilayered hair shaft and inner root sheath. Therefore, promoting the proliferation of dermal papilla cells induces hair follicle formation, promotes hair growth, and prevents hair loss.

[0049] In the present invention, "human-derived induced pluripotent stem cells (hiPSCs)" refer to pluripotent differentiated cells created by dedifferentiation from human somatic cells. hiPSCs are formed by transforming somatic cells into a state very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear transfer, and overexpression of pluripotency regulatory factors.

[0050] In the present invention, 'hair growth marker' refers to a general term for cytokine and growth factor proteins involved in the regulation of human hair morphogenesis and hair growth cycle.

[0051] CD133 was first described as a marker for human hematopoietic stem cells (Yin et al., 1997) and has recently been proposed as a universal marker for tissue stem / progenitor cells (Weigmann et al., 1997; Uchida et al., 2000; Richardson et al., 2004; Bussolati et al., 2005). CD133+ dermal papilla cells isolated from embryonic or adult dermal papilla cells have the ability to induce new hair follicles in vivo. Further studies using an in vitro 3D hydrogel culture system and a skin reconstitution assay showed that CD133+ DP cells contributed to the establishment of dermal papilla cells in both primary and secondary hair follicles (Driskell, R. R et al., 2012).

[0052] In the present invention, 'β-catenin' plays an important role in the Wnt signaling system by binding to transcription factors and directing numerous growth processes, inducing target gene expression by binding to T cell factor / lymphoid enhancing factor (TCF / LEF), and connecting the actin cytoskeleton and cadherin in cell-to-cell adhesion. The Wnt / β-catenin signaling pathway has a significant impact on hair follicle embryogenesis and hair growth.

[0053] In the present invention, 'ALP (alkaline phosphatase)' is known as an indicator of angiogenesis in the hair growth cycle, and is used as an indicator of hair growth because its enzyme activity increases when hair is induced into the growth phase.

[0054] In the present invention, 'LEF1 (lymphoid enhancer-binding factor 1)' is one of the major families belonging to the HMG (high mobility group) family, and regulates the expression of the T-cell receptor-alpha gene and is one of the key regulatory molecules of epithelial-mesenchymal interactions. LEF1 is known to actively regulate the Wnt / beta-catenin signaling pathway and is involved in cell proliferation, differentiation, and apoptosis. In particular, in various epithelial tissues such as hair follicles, mammary glands, and teeth, LEF1 promotes normal development by regulating the interaction between epithelial cells and mesenchymal cells, and is closely related to the self-renewal of hair follicles.

[0055] In the present invention, 'BMP4 (bone morphogenetic protein 4)' is a member of the BMP family and functions to regulate the development, cell proliferation, differentiation, and apoptosis of skin and hair follicles. In particular, BMP4, produced by dermal papilla fibroblasts during the telogen phase, regulates the development, cell proliferation, differentiation, and apoptosis of skin and hair follicles.

[0056] In the present invention, 'IGF1 (insulin-like growth factor-1)' is known to play an important role as a growth factor that stimulates hair growth, and plays a role in regulating cell proliferation and migration during hair follicle development (Peus D et al., 1996). Androgen stimulates the production of IGF1, thereby promoting the proliferation of follicular epithelial cells co-cultured with beard dermal papilla cells.

[0057] In the present invention, the 'hair growth process' refers to a series of biological processes including the occurrence, development, growth, and maintenance of hair follicles. Specifically, it includes the entire hair cycle, starting from the activation of hair follicle stem cells and going through the neogenesis of hair follicles, the growth phase (anagen), the regression phase (catagen), and the resting phase (telogen). The hair growth process is achieved through interactions between various cells such as dermal papilla cells (DP), hair follicle stem cells, and keratinocytes, as well as the activation of related signaling pathways, and in particular, the Wnt / β-catenin, BMP, and IGF signaling pathways play an important role.

[0058] In the present invention, "association" means that the complex protein performs a biological function by directly or indirectly participating in the hair growth process. Specifically, it includes that the complex protein contributes to the hair growth process by activating signaling pathways related to hair follicle formation and hair growth, regulating the expression of hair growth factor markers, or promoting the differentiation and proliferation of hair follicle stem cells. This association can be confirmed through in vitro or in vivo experiments, and can be proven by effects such as increased expression of hair growth factor markers, promotion of hair follicle neogenesis, and improved hair growth in the presence of the complex protein.

[0059] In the present invention, the 'complex protein' refers to a protein complex extracted from a culture medium of differentiated dermal papilla cells derived from hiPSCs, and is composed of proteins expressed at a relatively high level compared to a culture medium of human-derived dermal papilla cells, and is different from the protein composition included in a culture medium of human-derived dermal papilla cells composed only of growth factors. The complex protein is composed of proteins that play a role in promoting hair follicle formation and hair growth. Therefore, in the present invention, the 'complex protein associated with the hair growth process' refers to a protein complex extracted from a culture medium of differentiated dermal papilla cells derived from hiPSCs, and refers to a combination of various proteins that promote hair follicle formation and hair growth. The combination of proteins is related to the expression of hair growth factor markers such as CD133, beta-catenin, ALP, LEF1, BMP4, and IGF1, and has the characteristic that these proteins interact and exhibit a synergistic effect. In particular, it is composed of proteins expressed at a relatively high level compared to a culture medium of human-derived dermal papilla cells, and is different from the protein composition included in a culture medium of human-derived dermal papilla cells composed only of growth factors. These complex proteins effectively promote scalp hair follicle regeneration and hair growth by activating signaling pathways essential for hair follicle development, growth, and maintenance.

[0060] According to one embodiment of the present invention, the complex protein may be PWP1, HDAC3, JAG1, NFKB1, FGF7, IL1R1, PDGFC, MTOR, FADD, NACC1, HDAC2, SFRP2, KAP3, and DCN. A description of each of the complex proteins is disclosed in Table 3 of the present invention.

[0061] According to one embodiment of the present invention, the dermal papilla cells may express dermal papilla cell markers Versican and Corin; MSC markers Vimentin and α-SMA (α-Smooth muscle actin); and SKP (Skin-Derived Precursor cell) markers Wnt5, SOX2, Nestin, Twist, slug, and Snail.

[0062] In the present invention, 'MSC' is called mesenchymal stem cell or mesenchymal stromal cell, and is a multipotent cell of embryonic mesoderm origin with a fibroblast-like morphology. These cells can differentiate into other cell types, such as adipocytes, osteoblasts, chondroblasts, neural cells, and muscle cells, depending on stimulation and culture conditions.

[0063] In the present invention, 'SKP (Skin-Derived Precursor Cell)' has characteristics similar to neural crest stem cells (NCSC) and also differentiates into peripheral neural cells including mesenchymal-derived smooth muscle cells, adipocytes, Schwann cells, and catecholaminergic neurons. SKP is a representative embryonic neural crest-related precursor cell produced in the skin during embryogenesis, and is maintained in small numbers in adults (Fernandes KJ et al., 2004).

[0064] According to one embodiment of the present invention, the mammary papilla cells may be differentiated from human-derived induced pluripotent stem cells through a manufacturing method comprising the following steps:

[0065] a) A step of culturing human-derived induced pluripotent stem cells in a neural system-restricted medium;

[0066] b) a step of culturing the cells cultured in step a) in a neural crest cell induction medium to differentiate them into neural crest cells; and

[0067] c) A step of culturing the cells differentiated in step b) in a medium for differentiation of mammary papilla cells to differentiate them into mammary papilla cells.

[0068] The above step a) is a step for differentiating human-derived induced pluripotent stem cells into the neural ectoderm stage by blocking the SMAD signal transduction pathway.

[0069] According to one embodiment of the present invention, the step a) may be culturing human-derived induced pluripotent stem cells in DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing 20% ​​KSR (KnockOut Serum Replacement), LDN193189, SB431542, and Y-27632.

[0070] The concentration of LDN193189 in step a) may be 0.1 to 5 μM, 1 to 3 μM. For example, the concentration of LDN193189 may be 2 μM.

[0071] The concentration of the SB431542 in step a) may be 1 to 50 μM, 5 to 20 μM. For example, the concentration of the SB431542 may be 10 μM.

[0072] The concentration of Y-27632 in step a) may be 1 to 20 μM, 5 to 20 μM. For example, the concentration of Y-27632 may be 10 μM.

[0073] According to one embodiment of the present invention, step a) may be culturing human-derived induced pluripotent stem cells for 12 to 48 hours, or 16 to 32 hours. For example, step a) may be culturing human-derived induced pluripotent stem cells for 24 hours.

[0074] In the present invention, human-derived induced pluripotent stem cells were differentiated into the nervous system by blocking the SMAD signaling pathway using a well-known cocktail of LDN193189, SB431542, and Y-27632 (Fig. 1a).

[0075] The above step b) is a step for inducing differentiation of cells at the neural ectoderm stage into neural crest cells (neural stem cells), and through the above step, migration, proliferation, and cell survival rate into neural stem cells can be improved.

[0076] According to one embodiment of the present invention, the neural crest cell induction medium may be an induction medium containing 1% MEM Non-Essential amino acids solution (MEM-NEAA), 1X GlutaMAX, N2 supplement, EGF, bFGF, SB431542, and Y-27632.

[0077] The neural crest cell induction medium may be a commercially available known neural crest cell or neural stem cell induction medium. For example, the neural crest cell induction medium may be Neurobasal. ™ Medium (Gibco TM , 21103049).

[0078] In the present invention, 'MEM Non-Essential amino acids solution (MEM-NEAA)' is a non-essential amino acid cell culture supplement commercially available from many manufacturers such as Thermo Fisher Scientific or Cyagen, and is generally provided as a stock solution containing various types of non-essential amino acids such as glycine, L-alanine, L-asparagine, L-glutamic acid, L-aspartic acid, L-serine, or L-proline. It is used as a supplement to cell culture media for optimized cell growth.

[0079] In the present invention, the 'N2 supplement' is Gibco TM Supplied as a liquid concentrate containing 10,000 mg / L human transferrin, 500 mg / L human recombinant insulin, 0.63 mg / L progesterone, 1611 mg / L putrescine and 0.52 mg / L selenite by PAA Laboratories.

[0080] The concentration of the EGF in step b) may be 1 to 50 ng / mL, 10 to 30 ng / mL. For example, the concentration of the EGF in step b) may be 20 ng / mL.

[0081] The concentration of the bFGF in step b) may be 1 to 50 ng / mL, 10 to 30 ng / mL. For example, the concentration of the bFGF in step b) may be 20 ng / mL.

[0082] The concentration of SB431542 in step b) may be 1 to 50 μM, 5 to 20 μM. For example, the concentration of SB431542 may be 10 μM.

[0083] The concentration of Y-27632 in step b) may be 1 to 20 μM, 5 to 20 μM. For example, the concentration of Y-27632 may be 10 μM.

[0084] Step b) according to one embodiment of the present invention may be culturing in a neural crest cell induction medium for 1 to 5 days, or 2 to 4 days. For example, step b) may be culturing in a neural crest cell induction medium for 3 days.

[0085] The above step c) is a step for differentiating differentiated neural crest cells into dermal papilla cells. Since dermal papilla cells are at a stage where neural crest cells are not completely differentiated into mesenchymal stem cells (MSCs), in step c), neural crest cells are differentiated for a shorter period of time than the period for directly differentiating into mesenchymal stem cells. In the present invention, differentiation was performed over a short period of 4 days by adding 10% FBS and bFGF to a general MSC differentiation medium (Figs. 1a and 1b).

[0086] According to one embodiment of the present invention, the medium for differentiation of mammary papilla cells in step c) may be a low-concentration glucose DMEM medium containing 10% FBS, bFGF, and Y-27632.

[0087] According to one embodiment of the present invention, the medium for differentiation of mammary papilla cells in step c) may be a low-concentration glucose DMEM medium containing 5% HPL (human platelet lysate), bFGF, and Y-27632.

[0088] The concentration of the bFGF in step c) may be 1 to 50 ng / mL, 10 to 30 ng / mL. For example, the concentration of the bFGF in step b) may be 20 ng / mL.

[0089] The concentration of Y-27632 in step c) may be 1 to 20 μM, 5 to 20 μM. For example, the concentration of Y-27632 may be 10 μM.

[0090] According to one embodiment of the present invention, step c) may be culturing in a medium for differentiation of mammary papilla cells for 2 to 8 days, or 3 to 6 days. For example, step b) may be culturing in a medium for differentiation of mammary papilla cells for 4 days.

[0091] In the present invention, hiPSC-derived and differentiated hair papilla cells (hiPSC-DP) showed significantly higher expression of MSC markers such as CD73, CD44, and CD105 compared to hiPSC and hiPSC-NC, while cells lost the expression of pluripotency markers such as Nanog and OCT4 (Figures 1c and 1d).

[0092] In the present invention, after step c), the following steps may be additionally included:

[0093] d) A step of collecting the culture solution of the differentiated mammary papilla cells in step c) to obtain a mammary papilla cell extract.

[0094] The above step d) may be to centrifuge at 300 Xg, 25°C for 10 minutes, then separate the supernatant, and centrifuge the separated supernatant at 2000 Xg, 4°C for 4 minutes to collect the cell extract of the supernatant.

[0095] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating hair loss, comprising as an active ingredient an extract of hair papilla cells containing a complex protein involved in the hair growth process and expressing hair growth factors CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0096] In the present invention, 'hair loss' may include, but is not limited to, androgenetic alopecia, telogen effluvium, drug-induced alopecia, mechanical alopecia, traumatic alopecia, compression alopecia, anagen alopecia, pityriasis versicolor, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, congenital alopecia, alopecia areata, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex and alopecia universalis.

[0097] The pharmaceutical composition of the present invention may be in a dosage form selected from the group including, but not limited to, tablets, capsules, injections, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas.

[0098] The pharmaceutical composition of the present invention may additionally include pharmaceutically acceptable carriers, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. commonly used in formulation.

[0099] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, eye drop administration, topical administration, etc., but is not limited thereto.

[0100] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, body weight, sex, pathological condition, food intake, administration time, administration route, excretion rate, and response sensitivity. Preferably, the dosage of the pharmaceutical composition of the present invention may be 0.0001-100 mg / kg (body weight) for adults, but is not limited thereto.

[0101] Since the pharmaceutical composition of the present invention includes the composition for promoting scalp hair follicle regeneration, the description of the overlapping contents of the two compositions is omitted to avoid excessive complexity of the present specification due to the description of the overlapping contents.

[0102] In addition, another aspect of the present invention relates to a composition for preventing or treating hair loss, comprising as an active ingredient an exosome isolated from a hair papilla cell extract containing a complex protein involved in the hair growth process and expressing hair growth factors CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

[0103] In the present invention, 'exosomes' are membrane vesicles with a lipid bilayer membrane structure that are secreted by cells or exist within cells. Exosomes have a diameter of approximately 30-1000 nm and are released from cells when multivesicular bodies fuse with the cell membrane or are released directly from the cell membrane. It is well known in the art that exosomes play a role in transporting intracellular biomolecules such as proteins, bioactive lipids, and RNA (miRNA) to perform functional roles in mediating coagulation, cell-to-cell communication, and cellular immunity.

[0104] In the present invention, exosomes (hiPSC-DP-Exo) produced from hiPSC-derived hair papilla cells (hiPSC-DP) contain complex proteins associated with the hair growth process derived from hair papilla cells. The complex proteins contained in the exosomes are described in Table 3 below.

[0105] The exosomes may have a particle size of 125 to 150 nm. For example, the exosomes may have a particle size of 135 to 140 nm.

[0106] The above exosomes are 2.75 x 10 11 particles / mL to 2.95x10 11 It may have a concentration of particles / mL. For example, the exosomes may have a concentration of 2.82 x 10 11 particles / mL to 2.92 x 10 11 It may have a concentration of particles / mL.

[0107] Hair follicle neogenesis by complex proteins may be efficiently achieved within the above exosome particle size and concentration range.

[0108] Since the composition of the present invention includes the composition for promoting scalp hair follicle regeneration, the description of the overlapping contents of the two compositions is omitted to avoid excessive complexity of the present specification due to the description of the overlapping contents.

[0109] In addition, another aspect of the present invention relates to a method for producing a hair papilla cell extract containing a complex protein involved in the hair growth process and expressing hair growth factors CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1), which comprises the following steps:

[0110] a) A step of culturing human-derived induced pluripotent stem cells in a neural system-restricted medium;

[0111] b) a step of culturing the cells cultured in step a) in a neural crest cell induction medium to differentiate them into neural crest cells;

[0112] c) a step of culturing the cells differentiated in step b) in a medium for differentiation of mammary papilla cells to differentiate them into mammary papilla cells; and

[0113] d) A step of collecting the culture solution of the differentiated mammary papilla cells in step c) to obtain a mammary papilla cell extract.

[0114] Since the manufacturing method of the present invention is included in the composition for promoting scalp hair follicle growth, the overlapping contents of the manufacturing method and the composition for promoting scalp hair follicle growth are omitted to avoid excessive complexity of the present specification due to the description of overlapping contents.

[0115] Hereinafter, the present invention will be described in detail with reference to the following examples. However, these examples are intended to more specifically explain the present invention, and the scope of the present invention is not limited to these examples.

[0116] Example 1. Experimental materials and methods

[0117] 1-1. Experimental materials

[0118] Stem cells: Cryopreserved human induced pluripotent stem cells (iPSCs)

[0119] Protein: Vitronectin (RHVTN-N (RUO)) (Gibco TM ,A14700), Matrigel (Corning®, 354230), Gelatin (Sigma, G1393),

[0120] Enzyme: STEMPRO Accutase (Gibco) TM , A11105-1)

[0121] Badge: TeSR TM -E8 TM (STEMCELL Technologies, 05990), Low glucose DMEM (Gibco TM ,11885-084) DMEM / F12 (Gibco TM ,11330-032), Neurobasal TM Medium (Gibco TM , 21103049)

[0122] Supplements: Antibiotic-Antimycotic (100X) (Gibco TM , 15240062), KnockOut TM Serum Replacement (Gibco TM , 10828028), Fetal bovine serum (FBS) (ExCell, FSP500), MEM Non-Essential Amino Acids Solution (NEAA) (Gibco TM , 11140050), GlutaMAX TM Supplement (Gibco TM ,35050061), N-2 Supplement (100X) (Gibco TM ,17502048)

[0123] Growth factors: Recombinant human fibroblast growth factor (FGF) (GenScript, Z03166) Recombinant human epidermal growth factor (EGF) (R&D System, 236-EG)

[0124] Small Molecules: Y-27632 (SelleckChem, S1049), SB431542 (SelleckChem, S1067), LDN193189 (SelleckChem, S7507)

[0125] Other additives: DPBS (calcium and magnesium free) (Gibco TM , 14190144)

[0126] 1-2. Experimental method using the C57BL / 6 mouse model

[0127] The mouse model used in the experiment was a 7-week-old male C57BL / 6 mouse, which is an appropriate model for studying hair regeneration because it enters the catagen stage after hair removal on the dorsal skin. For the experiment, some hair was first removed with a clipper, and hair removal cream was applied to completely remove the hair so that the pink skin was exposed. After that, the mice were randomly divided into three groups (n=6) and hair regeneration was observed. As a positive control group, 5% minoxidil was applied topically (on the left side of the dorsal skin) daily from day 0 to day 9. Dermal papilla cells differentiated from human induced pluripotent stem cells were recombined in PBS-added ROCK inhibitor (ROCKi) to improve cell survival before injection into the mouse model. After that, 0.25 x 10 6 After re-incubating cells in 200 μL of PBS + ROCKi, they were injected subcutaneously at 10 sites (20 μL per site) on the left flank of the dorsal skin. In the negative control group, 200 μL of PBS + ROCKi was injected subcutaneously at 10 sites (20 μL per site) on the left flank of the dorsal skin. Images of each mouse model were taken on days 0, 5, 10, and 15.

[0128] 1-3. Hair growth score

[0129] Hair growth score was quantified and evaluated according to the hair regeneration score (S.1) based on pigmentation and hair induction, a common method used to evaluate hair regeneration in mouse models (Phil-June Park et al. Life Science, 2012).

[0130] Score description1Skin pink, no hair2Skin thick, pigmented, no hair3Skin thick, highly pigmented, no hair4Skin thick, highly pigmented, scattered hair5Hair 1-10% of body6Hair 10-25% of body7Hair 25-50% of body8Hair 50-75% of body9Hair > 75% of body10Hair 100% of body

[0131] 1-4. Skin tissue evaluation

[0132] On the 15th day of the experiment, mice were euthanized, and the entire dorsal skin was collected. The skin tissue was fixed in 4% paraformaldehyde overnight for histochemistry, and the fixed samples were precipitated in 30% sucrose and embedded in organic cation carrier (OCT) compound (Sakura Finetek USA Inc., Torrance, CA, USA). 10 μm-thick sections were prepared using a cryostat, and H&E staining was performed according to the protocol. This method is a common method used to analyze the structure and morphology of skin tissue in mouse models. After preparing skin tissue through the fixation, embedding, and sectioning processes, H&E staining can be used to observe the cellular structure and inflammatory response of the skin tissue, which can be used to evaluate changes in skin tissue related to hair regeneration.

[0133] 1-5. Ex Vivo Experimental Design

[0134] Hair follicles isolated from mouse whiskers were randomly divided into five groups (eight hair follicles per group), including basal medium (DMEM, low glucose) as a control and 5% minoxidil as a positive control, and a group containing 50% cell culture medium extracted from two other types of human dermal papilla cells (human primary-DP, human immortalized-DP) and hiPSC-derived dermal papilla cells (hiPSC-DP). Since hair follicles can survive for up to 7 days under basic in vitro culture conditions, the hair shaft status of the experimental group was imaged on days 0, 3, and 7 after hair follicle isolation treatment. A total of 40 hair shaft lengths were measured after 0, 3, and 7 days of culture.

[0135] For exosome treatment, mouse whisker hair follicles were randomly divided into four groups (n = 9 per group), including a control group receiving basal medium (DMEM, low glucose), and groups containing exosomes isolated from two other types of human dermal papilla cells (primary human DP, immortalized human DP) and hiPSC-derived dermal papilla cells. Exosomes derived from the above groups were treated at a final concentration of 0.0005 ug / ul, and the hair shaft status of the experimental group was imaged on days 0, 3, and 7 after treatment. A total of 36 hair shaft lengths were measured after 0, 3, and 7 days of culture.

[0136] 1-6. Quantitative real-time PCR

[0137] Skin tissue was collected on the 10th day, and RNA was isolated according to an established protocol. The cDNA was converted to cDNA using reverse transcriptase, and qPCR reactions were performed using Power SYBR green dye on a StepOnePlus™ Real-Time PCR system (Applied Biosystems, Foster City, CA). qPCR primers for β-catenin (ctnnb1), VCAN, CD133, Alpl, Bmp4, Wnt5a, FGF7, IGF1, LEF1, Sox2, Nexin, Corin, and Fgf10 were purchased from Thermo Fisher, and all qPCR data were compared relative to the 18s expression value.

[0138] 1-7. Preparation of conditioned medium and exosome isolation

[0139] Differentiated hiPSC-derived dermal papilla cells were cultured to 90% confluence. The culture medium was then removed, washed twice with PBS, and cultured in FBS-free medium for 48 h. To isolate exosomes, conditioned media (CM) was concentrated at a ratio of 1:200. The CM was centrifuged at 4,000 g for 20 min at 4°C, 15 mL / time, and 90 mL of CM was concentrated to 500 μL using a 100-KDa Amicon® Ultra-15 Centrifugal Filter Unit. Exosomes were eluted using the 70 nm qEVORIGINAL GEN 2 kit according to the instructions, and 1.6 mL of exosomes from each group were concentrated to 90 μL by centrifugation at 7,500 g for 10 minutes at 4 °C using a 30 KDa Amicon® Ultra-4 Centrifugal Filter Unit.

[0140] Example 2. Differentiation of hiPSC-derived mammary papilla cells

[0141] 2-1. hiPSC maintenance (Day -4 ~ Day 0)

[0142] Prepare a vitronectin-coated plate, keep it for 1 hour, remove vitronectin, and then add Y-27632 to TeSR TM -E8 TM was added to make the final concentration 10 μM. The iPSCs frozen in a nitrogen tank in water at 37°C were gently shaken to thaw the cells, and then thawed without stirring the cells until a small frozen cell pellet remained. The thawed iPSCs were slowly transferred to a 15 mL conical tube containing 5-7 mL of culture medium (media) using a 1 mL pipettor. Centrifuged at 200 × g for 5 min, the supernatant was removed with a pipette, and only a small amount of culture medium was left so as not to disturb the cell pellet. The cells were gently resuspended and then seeded onto coated plates. TeSR was added daily until 80-90% confluence was reached. TM -E8 TM The badge was replaced using .

[0143] 2-2. Differentiation of mammary papilla cells (Day 0 ~ Day 1)

[0144] hiPSCs can be induced to the neuroectodermal stage by blocking the SMAD signaling pathway involving the BMP and TGF-β family.

[0145] One hour before differentiation, prepare Matrigel-coated dishes (Matrigel diluted 1:60 to 1:100 with DMEM / F12) at 37°C and prepare neural linage restriction (NLR) culture medium with the following composition:

[0146] DMEM / F12 + 20% KSR (total will be around 40 mL, then 8mL KSR) + 2 μM LDN193189 (2 mM stock, take 1000x) + 10 μM SB431542 (20 mM stock, take 2000x) + 10 μM Y27632 (10 mM stock, take 1000 x).

[0147] Using the above culture medium, prepare NLR culture medium (32 mL DMEM F12 + 8 mL KSR + 40 uL LDN + 20 μL SB431542 + 40 μL Y27632) to make a total of 40 mL.

[0148] hiPSCs were cultured in E8 medium on vitronectin-coated 60 mm dishes until 90% confluence, the medium in the hiPSC culture dishes was aspirated at room temperature, washed once with 3 mL of PBS, and 1 mL of Accutase was added to each dish. After incubating the plates in a 37°C, 5% CO2 incubator for 5 min, the hiPSCs were gently washed once with PBS without disturbing the cells, and the cells were detached using the 40 mL of NLR medium described above. All cells were collected in a 15 ml conical tube, mixed well, and the total cell number was counted using a hemocytometer.

[0149] 1.65 × 10 cells without centrifugation 4 After direct plating on Matrigel-coated plates at a high seeding density of 100 cells per 100 cm2, the plates were placed back into a 37°C, 5% CO2 incubator and incubated for 24 h.

[0150] 2-3. Neural Crest Elaboration (Day 1 to Day 4)

[0151] Subsequent differentiation of cells into neural crest stem cells (NCSCs) rather than neural progenitor cells (NPCs) requires specific factors. Specifically, growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have been shown to regulate NCSC morphogenesis and promote migration, proliferation, and survival. Furthermore, some pathway inhibitors, such as the Rho-associated protein kinase inhibitor Y27632 and the TGF-β pathway inhibitor SB431542, are also important for NCSC differentiation.

[0152] Prepare a neural crest stem cell (NC) culture medium using the following composition:

[0153] Neural basal media + 1% NEAA + 1X GlutaMAX + 1X N-2 Supplement + 20 ng / mL EGF + 20 ng / mL bFGF + 20 μM SB431542 + 10 μM Y27632.

[0154] Using the above culture medium, prepare NC culture medium (47 mL Neural basal media + 500 μL NEAA + 500 uL GlutaMAX (stock 100x) + 500 uL N-2 Supplement (100 x stock) + 500 μL EGF (2 μg / mL stock) + 1 mL bFGF (1 μg / mL stock) + 50 μl SB431542 (20 mM stock) + 50 μL Y27632 (10 mM stock)) to make a total volume of 50 mL.

[0155] The medium was aspirated and replaced with the NC culture medium at room temperature, and replaced with fresh medium every day until Day 4.

[0156] 2-4. Differentiation of mammary papilla cells (Day 4 to Day 8)

[0157] Dermal papilla cells are a group of cells with specific characteristics of mesenchymal stem cells (MSCs), but although they are specifically derived from NCs, they have not yet fully differentiated into MSCs. Therefore, we induced NCs into MSCs over a short period (4 days) using MSC media containing 20 ng / mL bFGF. hiPSC-DPs are in an intermediate state exhibiting MSC characteristics by strongly expressing MSC markers and ALP, thereby revealing that NCs share similar characteristics with dermal papilla cells.

[0158] One hour before differentiation, prepare gelatin-coated dishes (0.2% in PBS) at 37°C and prepare the medium for differentiation of dermal papilla cells (DP) with the following composition:

[0159] Low glucose DMEM + 10% FBS or 5% HPL (human platelet lysate) + 20 ng / mL bFGF + 10 uM Y27632 (10 mM stock, take 1000 x, only required for Day 4).

[0160] The medium in the NC culture dishes was aspirated and washed once with 3 mL of PBS. 1 mL of Accutase was added to each dish at room temperature. The plates were then incubated for 5 minutes in a 37°C, 5% CO2 incubator. The cells were separated with complete DP medium, collected in a 15 mL conical tube, and mixed well. The cells were centrifuged at 200 g for 5 minutes at room temperature, and the total cell number was counted using a hemocytometer.

[0161] Afterwards, the cell gelatin-coated dish was placed on the plate and cultured in a 37°C, 5% CO2 incubator until Day 8, replacing it with fresh room temperature DP medium every two days.

[0162] 2-5. Analysis of differentiated mammary papilla cells (Figure 1)

[0163] The origin of dermal papilla varies depending on the site of origin in the human body, and scalp dermal papilla has unique characteristics derived from neural cell differentiation. Dermal papilla cells isolated from the human body lose both neural properties and hair induction capacity after culture. Considering this, we established a step-by-step protocol to generate dermal papilla cells from human induced pluripotent stem cells (iPSCs), resulting in differentiated dermal papilla cells possessing both neural and papilla characteristics. To achieve this, iPSCs were first restricted to the nervous system by blocking the SMAD signaling pathway using a well-known cocktail of LDN193189, SB431542, and Y27632 (Fig. 1a). After 24 hours, cells in the neuroectodermal stage were further differentiated into neural crest stem cells (NCs) by adding epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). This process helped to enhance migration, proliferation, and cell survival of NCs, and the differentiation period was 3 days. Given that the papilla cells are a cell population of mesenchymal stem cells (MSCs) with specific properties and that the remaining NCs were not fully differentiated into MSCs, the general MSC medium supplemented with 10% FBS and 20 ng / mL bFGF was used for a short period of 4 days to directly differentiate NCs into MSCs (Figs. 1a and 1b).

[0164] At this stage, hiPSC-derived papilla cells lost the expression of pluripotency markers such as Nanog and OCT4, while acquiring significantly higher expression of MSC markers such as CD73, CD44, and CD105 compared to hiPSCs and hiPSC-NCs (Figures 1c and 1d). To further verify the MSC characteristics of hiPSC-derived papilla cells, flow cytometry (FACS) and immunofluorescence staining (ICC) targeting surface and structural markers of MSCs were additionally performed. Consistent with the qPCR results, the MSC surface proteins CD44, CD73, and CD105 were strongly detected in more than 97% of the cell population of hiPSC-derived papilla cells in the FACS analysis, which was similar to the other two human-derived papilla cells (Figure 1e). ICC showed that on day 8, cells expressed vimentin and smooth muscle actin (SMA), cytoskeletal proteins expressed in MSCs, providing further evidence for the differentiation of NCs into the MSC lineage (Fig. 1f). Furthermore, nestin, considered a marker of neural stem / progenitor cells, was expressed in both human-derived papilla cells and hiPSC-derived papilla cells, despite its characteristic MSC markers. qPCR verified that the expression of SMA, vimentin, and nestin was higher in hiPSC-derived papilla cells than in human-derived papilla cells, hBM-MSCs, and other cell types (Fig. 1g). As an extracellular matrix, Versican is specifically expressed in the anagen phase of hair growth and has been studied as a specific dermal papilla cell marker. Therefore, we further investigated the expression of Versican in hiPSC-derived dermal papilla cells, and confirmed strong expression similar to conventional human-derived dermal papilla cells compared to other types of cells (Fig. 1h).

[0165] In summary, the above data confirmed the efficient generation of hiPSC-derived papilla cells within an 8-day differentiation period, and that these cells expressed MSC and NC markers similar to those of conventional human-derived papilla cells. In addition, the expression of Versican in hiPSC-derived papilla cells was confirmed, confirming that hiPSC-derived papilla cells were efficiently generated through the above differentiation protocol within an 8-day differentiation period.

[0166] Example 3. Expression marker analysis of hiPSC-derived dermal papilla cells (hiPSC-DP) (Figure 2)

[0167] Conventional human-derived dermal papilla cells lose their hair-inducing potential when isolated from hair follicles using conventional cell culture methods. Therefore, although conventional human-derived dermal papilla cells and hiPSC-DP are similar in terms of the expression of MSC and NC markers, it is still necessary to evaluate whether differentiated cells express markers related to hair induction. Alkaline phosphatase (ALP) is considered an important indicator and functional marker of dermal papilla cells, and overexpressed ALP enhances the hair-inducing potential of cultured dermal papilla cells.

[0168] In this example, we performed an ALP staining assay, in which ALP-positive cells were stained deep purple within 30 minutes. Two different sources of human-derived dermal papilla cells were used as controls, and the comparison confirmed that hiPSC-DP had a significantly higher number of ALP-positive cells (Fig. 2a). To minimize the interference of cell size and density on the ALP signal, we further investigated ALP activity by normalizing protein amounts. hiPSC-DP exhibited approximately twice the ALP activity compared to other human-derived dermal papilla cells and human dermal fibroblasts (hDF) (Fig. 2b).

[0169] For further validation, a panel of hair-inducing genes, including CD133, β-catenin, ALPL, LEF1, and BMP4, were analyzed by qPCR, and high expression was observed in hiPSC-DP (Fig. 2c). CD133 / prominin-1 has been reported as a specific surface marker of dermal papilla cells during the early anagen stage of hair growth and morphogenesis, and CD133-positive cells isolated from the skin can regenerate hair follicles in nude mice. Furthermore, Versican, a type of extracellular matrix (ECM), has also been shown to play a role in identifying active dermal papilla cells. ICC targeting these two markers confirmed strong expression in hiPSC-DP, but not in other human-derived dermal papilla cells (Fig. 2d).

[0170] FACS analysis revealed that while other human-derived dermal papilla cells showed a low CD133(+) cell percentage of 3.54% and 0.55%, respectively, in both early and late passages, hiPSC-DPs contained more than 15% CD133(+) cells (Fig. 2e), and this positive cell population was maintained at 14.86% after passage. Based on the fact that the hair-inducing potential of dermal papilla cells can be restored through 3D aggregate formation, hiPSC-DPs were cultured in ultra-low attachment dishes, and these cells generated condensed 3D spheres with strong expression of CD133 (green) and β-catenin (red) (Fig. 2f). Although various growth factors and signaling pathways have shown promising effects on hair growth, activation of Wnt / β-catenin signaling plays a leading role in hair follicle morphogenesis and regeneration. Therefore, we re-examined hiPSC-DP maintained in two dimensions and confirmed high expression of β-catenin in hiPSC-DP compared to other human-derived mammary papilla cells (Fig. 2g).

[0171] Example 4. Hair regeneration efficacy of hiPSC-derived hair papilla cells (hiPSC-DP) (Figure 3)

[0172] Minoxidil, a standard treatment for hair loss, was used as a positive control. Since cultured dermal papilla cells have been reported to gradually lose their hair-inducing capacity over time, research on infinite cell resources capable of maintaining hair-inducing capacity has become increasingly important. In this study, we analyzed the hair regeneration efficacy in vivo using hiPSC-derived dermal papilla cells (hiPSC-DP), which exhibited functionally superior dermal papilla cell characteristics, to determine their capacity for hair-inducing capacity.

[0173] C57BL / 6 mice were randomly divided into three groups (six mice per group) treated with PBS (negative control), minoxidil (positive control), and hiPSC-DP. Hair recovery status in mice with advanced hair loss was imaged on days 0, 10, and 15 after treatment, and the hair growth area was analyzed through morphological observation (Fig. 3a).

[0174] Analysis results showed that black pigmentation began to appear in the minoxidil and cell-treated groups on the 5th day after treatment, and all mice in the hiPSC-DP-injected group had darker skin than the other groups. On the 10th day after treatment, the PBS and minoxidil groups showed hair coverage rates of 5% and 10%, respectively, while the hiPSC-DP-injected group showed an average fur coverage rate of 20%. On the 15th day after treatment, the cell-treated group showed a significantly higher hair regeneration score than the other groups, indicating complete recovery of hair growth (Fig. 3b, *p<0.05 vs. PBS group). Hematoxylin and eosin (H&E) staining of hair on the 15th day after treatment showed that the group injected with dermal papilla cells differentiated from human-induced pluripotent stem cells induced longer and larger hair follicles than the other groups (Figs. 3c and 3d). As the hair follicles in the resting phase transitioned to the growth phase, the size of the hair follicles increased, while in the control group, the hair follicles were smaller and the collagen layer was thinner.

[0175] Since the group injected with hiPSC-DP showed significantly stronger hair induction ability than the other groups from day 0, to elucidate the molecular mechanism, the gene levels of hair induction markers β-catenin, ALP, BMP4, FGF7, LEF1, and IGF1; anagen-phase dermal papilla cell markers Versican and Corin; MSC markers Vimentin and α-SMA (α-smooth muscle actin); and SKP markers Wnt5, SOX2, and Nexin were evaluated in the dorsal skin on day 10 after treatment. qPCR analysis showed that the expression of all target markers was significantly upregulated in the hiPSC-DP group, and in particular, the expression of β-catenin, BMP4, LEF1, SMA, Wnt5, and Nexin1 in the cell therapy group was significantly higher than that in the minoxidil group (Fig. 3e). The WNT / β-catenin and BMP pathways are known to play important roles in regulating the hair cycle by inducing hair growth, LEF-1-deficient mice lack body hair and whiskers, and Nexin1 has been reported to be associated with the ability of dermal papilla cells to support hair growth.

[0176] These results suggest that hiPSC-derived papilla cells can induce hair development by upregulating the expression of functional genes in papilla cells, suggesting that hiPSC-derived papilla cells could be a potential strategy for improving hair loss treatment.

[0177] Example 5. Hair growth promotion effect of hiPSC-derived hair papilla cell (hiPSC-DP) culture medium extract (Figure 4)

[0178] While cell-based therapies are a necessary step in creating hair follicles, utilizing cellular components as therapeutic catalysts rather than cells as therapeutic agents would result in more convenient therapeutic products, and would eliminate the need for cell carriers as therapeutic agents. Since exosomes and growth factors can contribute to the hair growth process, we investigated the effect of hiPSC-derived dermal papilla cell cultures on hair length extension.

[0179] First, hair follicles isolated from the whiskers of mice were randomly divided into five groups (eight hair follicles per group), and treated with basal medium (DMFM, low glucose) as a negative control, 5% minoxidil as a positive control, and 20% cell culture medium extracts derived from two different types of human-derived dermal papilla cells and hiPSC-derived dermal papilla cells (hiPSC-DP). Since hair follicles can survive for up to 7 days under basic in vitro culture conditions, the length extension of 40 hairs was applied to each experimental group, and the status of hair length extension was measured by imaging on days 0, 3, and 7 (Fig. 4a).

[0180] As a result, the group treated with the extract of human-induced pluripotent stem cell-derived culture medium showed a significant increase in hair length extension compared to the other groups from day 3 after treatment, and hair length extension was continuously induced until day 7 after treatment. However, the other groups showed no effect even after 7 days of treatment (Fig. 4b). These results suggest that the hiPSC-DP culture medium contains various proteins, including potential exosomes and growth factors, that can promote hair development in vitro.

[0181] Example 6. Confirmation of the hair growth promotion effect of exosomes isolated from hiPSC-derived hair papilla cell (hiPSC-DP) culture medium (Figure 5)

[0182] It is well known that exosomes, growth factors, and various proteins can contribute to hair development. Since the conditions of hiPSC-derived dermal papilla cell culture media showed potential effects on hair length extension, the effect of exosomes produced from hiPSC-derived dermal papilla cells (hiPSC-DP) on hair length extension was further investigated.

[0183] First, the conditioned medium (CM) was centrifuged at 300 g for 10 min to remove dead cell pellets, and then centrifuged at 2000 g for 10 min to remove cell debris. The supernatant was then centrifuged at 3000 g for 20 min and transferred to a 100 Kda Centricon Plus-70 ultrafilter (Millipore) to produce concentrated CM. Subsequently, 500 μL of concentrated CM containing 3.6 mL of DPBS buffer was loaded onto a qEV 70 nm column (IZON) for elution, and 1.6 mL of purified exosome fraction was collected and further concentrated at 7500 g for 10 min on a 30 Kda Amicon Ultra-15 filter (Millipore) to obtain exosomes, and the nanoparticle analysis results are shown in Table 2 below.

[0184] hiPSC-DP-ExohDP2-Exo(immotalized-DP-Exo)hDP1-ExoAverage size (nm)137.5142.3127.1Concentration (particles / mL)2.87X10 11 8.81X10 10 1.49X10 10

[0185] Hair follicles isolated from mouse whiskers were randomly divided into four groups (nine hair follicles per group) and treated with basal medium (DMFM, low glucose) as a negative control, two types of human-derived dermal papilla cells, and exosomes derived from hiPSC-DP culture medium at a final concentration of 0.0005 μg / μL. In the experimental group, hair length status was imaged on days 0, 3, and 7 after treatment, and the length extension of 36 hairs was measured on days 0, 3, and 7 after culture (Fig. 5a).

[0186] As a result, hair length extension was significantly greater in hiPSC-DP culture-derived exosomes (hiPSC-DP-Exo) compared to the other groups from day 3 after treatment, and hair length extension was continuously induced until day 7 after treatment. In contrast, the other groups did not show any significant effect even after 7 days of treatment. Therefore, it was confirmed that exosomes isolated from hiPSC-DP culture (hiPSC-DP-Exo) can be a powerful tool for promoting hair regeneration.

[0187] Example 7. Identification of hair growth-related complex proteins contained in hiPSC-derived hair papilla cell (hiPSC-DP) culture medium extracts.

[0188] Mass spectrometry (MS) was performed at BGI in data-independent acquisition mode (DIA), and the two other types of dermal papilla cell cultures mentioned above and the culture extracts isolated from hiPSC-derived dermal papilla cells (hiPSC-DP) were analyzed using a Q-Exactive HF X instrument (Thermo Fisher Scientific, San Jose, CA). The cultures were separated by centrifugation at 300 × g for 10 min at 25 °C, and the supernatant was separated. The supernatant was collected by a second centrifugation at 2000 × g for 4 min at 4 °C and stored at -80 °C.

[0189] A total of 6,986 proteins were quantified through logarithmic (base 10) transformation of peak intensity areas, of which 5,935 proteins were identified in hiPSC-DP cultures, and 3,560 and 4,994 proteins in the other two types of dermal papilla cell cultures, respectively. Data analysis revealed that 14 complex proteins, PWP1, HDAC3, JAG1, NFKB1, FGF7, IL1R1, PDGFC, MTOR, FADD, NACC1, HDAC2, SFRP2, KAP3, and DCN, were expressed at significantly higher levels in hiPSC-DP cultures compared to the other two types of dermal papilla cell cultures (Table 3).

[0190] Gene TermProteinReferenceSummaryPWP1Periodic tryptophan protein 1 homologDevelopmental Cell (IF13.5)2017PWP1 mediates nutrient-dependent growth control of Drosophila wing hair via mTOR signalingHDAC3Histone deacetylase 3Genes & Development (IF12.89)2020HDAC3 ensures stepwise epidermal stratification via NCoR / SMRT-reliant mechanisms independent of its histone deacetylase activityJAG1Protein jagged-1Cell (IF66.85)2017Treg cells in regulating the proliferation and differentiation of hair follicle stem cells (HFSCs) by activating the Jag1-Notch signaling pathwayNFKB1Nuclear factor NF-kappa-B p105 subunitJournal of Investigative Dermatology (IF8.551)2018NF-κB plays a poteintial role in HF stem / progenitor cell activation during anagen induction and involved in hair fiber morphogenesis during HF cyclingFGF7Fibroblast growth factor 7Inflammation & Regeneration (IF10.426)2020FGF7 is an established DP biomarker which promotes the proliferation of hair germ / matrix cells and initiates a new hair cycleIL1R1Interleukin-1 receptor type 1Inflammation & Regeneration(IF10.426)2023Injury-induced interleukin-1 alpha promotes Lgr5 hair follicle stem cells de novo regeneration and proliferation via regulating regenerative microenvironment in micePDGFCPlatelet-derived growth factor CNPJ Regen Med (IF14.404)2017Platelet-derived growth factor signaling modulates adult hair follicle dermal stem cell maintenance and self-renewalMTORSerine / threonine-protein kinase mTORJMCB (IF8.185)2015mTOR signaling may promote stem cell activation via counterbalancing BMP-mediated suppression during hair regenerationFADDFAS-associated death domain proteinPNAS (IF11.1)2010Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis through TNF signaling pathwayNACC1Nucleus accumbens-associated protein 1Transcriptional corepressor in neuronal cells through recruitment of HDAC3 and HDAC4HDAC2Histone deacetylase 2Nature Communications (IF16.6)2023Hdac1 and Hdac2 in the DP promote the survival of DP cells throughout the hair cycleSFRP2Secreted frizzled-related protein 2KAMJE Synapse (IF2.17)2014Expression of Sfrp2 Is Increased in Catagen of Hair Follicles and Inhibits Keratinocyte ProliferationKAP3Human Hair Keratin-Associated Protein3A major component of the hair fiber, and play crucial roles in forming a strong hair shaft through a cross-linked network with keratin intermediate filaments (KIF), which are produced from hair keratinsDCNDecorinExperimental Dermatology(IF3.6)2018Decorin promotes proliferation and migration of ORS keratinocytes and maintains hair anagen in mice.

[0191] The 14 complex proteins listed in Table 3 differ from the components of cell culture media consisting solely of growth factors, and these complex proteins are known to play a crucial role in hair growth and differentiation. These results suggest that hiPSC-DP culture media plays a crucial role in promoting hair growth, suggesting the potential for developing novel therapeutics that promote hair growth.

[0192] While the present invention has been described in detail through representative examples above, those skilled in the art will understand that various modifications can be made to the above-described embodiments without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined by all changes or modifications derived from the claims and equivalent concepts.

[0193] The present invention aims to overcome the limitations of previously known culture solutions of hair papilla cells and to provide a hair papilla cell extract that has excellent efficacy in preventing hair loss, promoting hair growth, and regenerating human tissues, such as wound healing, by promoting hair follicle regeneration.

Claims

1. A composition for promoting scalp hair follicle regeneration, comprising as an active ingredient an extract of hair papilla cells containing complex proteins related to the hair growth process and expressing hair growth factor markers CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4) and IGF1 (insulin-like growth factor 1).

2. In paragraph 1, A composition wherein the above complex proteins are PWP1, HDAC3, JAG1, NFKB1, FGF7, IL1R1, PDGFC, MTOR, FADD, NACC1, HDAC2, SFRP2, KAP3 and DCN.

3. In paragraph 1, A composition wherein the above-mentioned hair papilla cells express hair papilla cell markers Versican and Corin; MSC markers Vimentin and α-SMA (α-Smooth muscle actin); and SKP (Skin-Derived Precursor cell) markers Wnt5, SOX2, Nestin, Twist, slug, and Snail.

4. In paragraph 1, The above-mentioned mammary papilla cells are differentiated from human-derived induced pluripotent stem cells through a manufacturing method including the following steps: a) A step of culturing human-derived induced pluripotent stem cells in a neural system-restricted medium; b) a step of culturing the cells cultured in step a) in a neural crest cell induction medium to differentiate them into neural crest cells; and c) A step of culturing the cells differentiated in step b) in a medium for differentiation of mammary papilla cells to differentiate them into mammary papilla cells.

5. In paragraph 4, A composition wherein the medium for differentiation of mammary papilla cells in the above step c) is a low glucose DMEM medium containing 10% FBS, bFGF, and Y-27632.

6. In paragraph 4, A composition wherein the medium for differentiation of mammary papilla cells in the above step c) is a low glucose DMEM medium containing 5% HPL (human platelet lysate), 1% MEM-non-essential amino acids (MEM-NEAA), 1X GlutaMAX, bFGF, and Y-27632.

7. A pharmaceutical composition for preventing or treating hair loss, comprising as an active ingredient an extract of hair papilla cells containing complex proteins related to the hair growth process and expressing hair growth factors CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4), and IGF1 (insulin-like growth factor 1).

8. A composition for preventing or treating hair loss, comprising as an active ingredient an exosome isolated from a hair papilla cell extract containing a complex protein involved in the hair growth process and expressing hair growth factors CD133, β-catenin, ALP (alkaline phosphatase), LEF1 (lymphoid enhancer-binding factor 1), BMP4 (bone morphogenetic protein 4), and IGF1 (insulin-like growth factor 1).

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