A defined serum-free induction medium and induction method and application for promoting differentiation of adipose mesenchymal cells into dermal papilla cells

Abstract

The present application relates to the technical field of regenerative medicine application, and particularly relates to a serum-free induction medium with clear components for promoting differentiation of adipose-derived mesenchymal cells into dermal papilla cells, and an induction method and application. A method for promoting differentiation of adipose-derived mesenchymal cells into dermal papilla cells, which is to use a serum-free induction medium to induce culture of adipose-derived mesenchymal cells, so that the adipose-derived mesenchymal cells are differentiated into dermal papilla cells. The formula of the serum-free induction medium comprises hepatocyte growth factor, hydrocortisone, bone morphogenetic protein 2, BIO, and basic fibroblast growth factor. The present application is based on the differentiation potential of adipose-derived mesenchymal cells and the mechanism of maintaining the function of dermal papilla cells, and constructs a serum-free induction system with clear components to realize directional differentiation. The technical scheme can solve the technical problem that there is no method for inducing directional differentiation of adipose-derived mesenchymal cells into dermal papilla cells with effective hair follicle regulation function in the prior art, and has ideal application and popularization potential.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine application technology, specifically to a serum-free induction culture medium with clearly defined components that promotes the differentiation of adipose mesenchymal cells into dermal papillary cells, as well as the induction method and its application. Background Technology

[0002] Dermal papilla (DP) cells are a specialized group of mesenchymal cells located at the base of hair follicles, playing a central regulatory role in hair follicle development and hair regeneration. They have been shown to regulate hair follicle formation and growth cycles, and can also synergize with epidermal cells to promote the generation of new hair follicles. Crucially, implanting DP cells alone in the dermal-epidermal space can directly induce new hair follicle growth at the implantation site. Based on this unique function, DP cells are widely recognized as an ideal candidate cell therapy for treating hair loss.

[0003] However, the clinical translation of DP cells faces multiple practical bottlenecks: First, the number of DP cells in the human body is inherently scarce, and their expansion capacity under in vitro culture conditions is limited; second, the extraction process of DP cells causes irreversible and complete damage to the original hair follicles, which limits the cell source and raises practical and ethical concerns; third, and more importantly, the key function of DP cells in inducing hair follicle formation is significantly diminished during in vitro culture, making it difficult to obtain sufficient quantities of fully functional DP cells to meet clinical needs. These factors collectively hinder the progress of DP cell clinical application.

[0004] To overcome these challenges, scientists have focused their research on the transdifferentiation of proliferating mesenchymal cells into functional DP cells, attempting to solve the core issues of cell source and functional maintenance through this strategy. Related research has made some progress. In 2010, the team of South Korean scientist Jung-Keug Park achieved a breakthrough, successfully inducing umbilical cord mesenchymal cells (UCMSCs) to differentiate into cell spheroids with DP cell characteristics (DP-like cell spheroids), and confirming their ability to induce hair growth through nude mouse experiments. However, UCMSCs themselves have inherent drawbacks such as difficulty in storage and limited availability, greatly restricting the widespread adoption of this technology. In contrast, dermal mesenchymal cells, due to their ease of acquisition and strong proliferative capacity, have become the ideal starting cells for transdifferentiation into DP cells. Subsequently, in 2019, the team of Chinese scientist Shuang Liu further advanced the research, successfully inducing fibroblasts derived from human fetuses and adult foreskin into DP-like cells, and verifying some of their functions through in vivo experiments. However, gene expression profiling analysis showed that the purity of the obtained DP-like cells was low; and in animal experiments, their hair regrowth effect was significantly lower than that of the positive control group, suggesting that the efficiency of this induction protocol and the maintenance of cell function still need further optimization. In 2025, the team of Chinese scientist Zhongjie Liu reported new research results: using a single molecule to induce the transdifferentiation of human foreskin and scalp fibroblasts into DP-like cells. However, this protocol still has significant shortcomings: the staining intensity of alkaline phosphatase (AKP / ALP, the functional marker enzyme of DP cells) in the induced DP-like cells did not increase significantly, and the expression levels of key functional marker genes were only slightly different from those of the initiating fibroblasts; animal transplantation experiments further confirmed that the function of these DP-like cells was weak, and the amount of hair growth induced was much lower than that of the positive control group, indicating that the efficiency of cell function induction still needs to be improved.

[0005] Adipose-derived mesenchymal stem cells (ADMSCs) are a type of adult mesenchymal stem cells isolated from adipose tissue and are important members of the mesenchymal stem cell (MSC) family. Compared with other MSC sources such as bone marrow mesenchymal stem cells (BMSCs) and umbilical cord mesenchymal stem cells (UCMSCs), ADMSCs have unique advantages such as extremely abundant source, strong in vitro expansion capacity, and low immunogenicity, making them a research hotspot and core candidate cell in regenerative medicine, cell therapy, and tissue engineering. However, current research has insufficient development and utilization of ADMSCs, and existing technologies have not yet established a mature protocol for the efficient differentiation of ADMSCs into DP cells.

[0006] Therefore, there is an urgent need to develop a highly efficient induction technology system adapted to the characteristics of ADMSCs. This system should fully leverage the advantages of ADMSCs, such as their strong in vitro proliferation capacity and low immunogenicity, overcoming the technical bottleneck of the lack of mature differentiation protocols. It also needs to address the common problems in existing DP cell induction, such as unclear induction environment components (e.g., serum-containing cells), complex procedures (e.g., sorting dependence), low cell purity, and incomplete functionalization of induced cells. This will promote the directed differentiation of ADMSCs into DP-like cells that highly express DP cell marker proteins and possess complete hair follicle regulatory functions, fully releasing the application potential of ADMSCs, providing a higher-quality cell source for cell therapy of hair loss, and breaking through the current bottlenecks in the source and function of DP cells for clinical translation. Summary of the Invention

[0007] The present invention aims to provide a method for promoting the differentiation of adipose mesenchymal cells into dermal papillary cells, in order to solve the technical problem of the lack of a method in the prior art for inducing ADMSCs to differentiate into DP-like cells with effective hair follicle regulation function.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for promoting the differentiation of adipose-derived mesenchymal cells into dermal papillary cells involves inducing and culturing adipose-derived mesenchymal cells using a serum-free induction medium to differentiate them into dermal papillary cells.

[0010] The serum-free induction culture medium is formulated as follows: containing L Alanine L DMEM / F of glutamine dipeptide 12. Culture medium is used as the basal medium, which contains: 10% by volume. 20% KnockOut serum substitute, 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement, concentration 10 ng / mL 100 ng / mL of hepatocyte growth factor, with a concentration of 5 ng / mL Hydrocortisone at 100 ng / mL, with a content of 50 ng / mL Bone morphogenetic protein 2 at 400 ng / mL, BIO (a GSK-3 inhibitor) at 0-2.5 μM, and basic fibroblast growth factor at 0-40 ng / mL.

[0011] Furthermore, the serum-free induction culture medium is formulated as follows: containing L... Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (v / v) and 1% insulin (v / v). transferrin Selenium culture medium supplement, hepatocyte growth factor at a concentration of 50 ng / mL, hydrocortisone at a concentration of 100 ng / mL, bone morphogenetic protein 2 at a concentration of 200-400 ng / mL, GSK-3 inhibitor BIO at a concentration of 2.5 μM, and basic fibroblast growth factor at a concentration of 5-10 ng / mL.

[0012] Furthermore, the adipose-derived mesenchymal cells are cultured in a culture vessel coated with vitrin;

[0013] The coating method is as follows: use DMEM / F12 medium containing dissolved porphyrin to contact the cell growth side of the culture vessel and incubate, then remove the DMEM / F12 medium containing dissolved porphyrin to complete the coating of porphyrin.

[0014] Furthermore, the serum-free induction medium was used to induce and culture adipose-derived mesenchymal cells for 7 days; the serum-free induction medium was changed daily during the culture process.

[0015] When the adipose-derived mesenchymal cells reach a confluence of 60%-85%, serum-free induction medium is used to induce the adipose-derived mesenchymal cells to culture.

[0016] Furthermore, the obtained dermal papillary cells were cultured in maintenance medium to obtain dermal papillary cell microspheres;

[0017] The maintenance medium formulation is as follows: in DMEM / F 12. Add 10% by volume to the basal culture medium. 20% KnockOut serum substitute, and 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement.

[0018] Furthermore, adipose-derived mesenchymal cells were obtained by the following method:

[0019] After the adipose tissue was allowed to stand, the middle milky yellow layer was taken and transferred to physiological saline. After mixing and centrifugation, the supernatant and the lower red precipitate were discarded to obtain clean adipose tissue. The fascia in the clean adipose tissue was picked out and cut into small pieces, and then treated with type I collagenase. After enzymatic hydrolysis, culture medium was added, mixed, filtered, and centrifuged to remove the oil layer and supernatant, and cell pellet was obtained. After washing the cell pellet with physiological saline, adipose mesenchymal cells were obtained.

[0020] This technical solution also provides a composition for promoting the differentiation of adipose mesenchymal cells into dermal papillary cells, comprising a content of 10 ng / mL 100 ng / mL of hepatocyte growth factor, with a concentration of 5 ng / mL Hydrocortisone at 100 ng / mL, with a content of 50 ng / mL Bone morphogenetic protein 2 at 400 ng / mL, BIO (a GSK-3 inhibitor) at 0-2.5 μM, and basic fibroblast growth factor at 0-40 ng / mL.

[0021] Furthermore, the composition contains 10% by volume. 20% KnockOut serum substitute, 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement containing L Alanine L DMEM / F of glutamine dipeptide In the environment of 12 culture medium.

[0022] This technical solution also provides a serum-free induction culture medium with well-defined components to promote the differentiation of adipose mesenchymal cells into dermal papillary cells. The formulation is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Alanine L DMEM / F of glutamine dipeptide 12. Culture medium is used as the basal medium, which contains: 10% by volume. 20% KnockOut serum substitute, 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement, concentration 10 ng / mL 100 ng / mL of hepatocyte growth factor, with a concentration of 5 ng / mL Hydrocortisone at 100 ng / mL, with a content of 50 ng / mL Bone morphogenetic protein 2 at 400 ng / mL, BIO (a GSK-3 inhibitor) at 0-2.5 μM, and basic fibroblast growth factor at 0-400 ng / mL.

[0023] This technical solution also provides a method for promoting the differentiation of adipose mesenchymal cells into dermal papillary cells, and the application of the dermal papillary cells obtained in the preparation of preparations with hair growth effects.

[0024] In summary, the principle of this technical solution is as follows:

[0025] This invention, based on the differentiation potential of adipose-derived mesenchymal cells (ADMSCs) and the mechanism of maintaining the function of dermal papilla (DP) cells, constructs a serum-free induction system with well-defined components to achieve the directed differentiation of ADMSCs into functional DP cells. The specific principle is as follows:

[0026] Basic culture environment construction: DMEM / F-12 containing L-alanyl-L-glutamine dipeptide was used as the basic culture medium to ensure the basic metabolic needs of cells. KnockOut serum substitute and insulin-transferrin-selenium medium supplement were added to simulate the physiological nutrient microenvironment under serum-free conditions, avoid interference from batch-to-batch serum differences, and provide ADMSCs with essential amino acids, trace elements and signaling molecules for growth and differentiation, maintain cell homeostasis and lay the foundation for differentiation initiation.

[0027] Targeted regulation of key signaling pathways: Synergistic activation of DP cell differentiation through precise formulation of multi-component inducing factors.

[0028] Hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) synergistically promote ADMSC proliferation and activate their specific differentiation into the mesenchymal lineage. Hydrocortisone (Hc) regulates cell metabolism and differentiation direction, inhibiting ADMSC differentiation into unrelated lineages such as adipocytes and enhancing DP cell differentiation fate. Bone morphogenetic protein 2 (BMP2) activates the BMP signaling pathway, regulating the expression of DP cell differentiation-related genes. The GSK-3 inhibitor BIO specifically inhibits GSK-3 kinase activity, relieving its inhibition of the Wnt / β-catenin pathway, activating the transcription of DP cell functional marker genes, and inducing ADMSCs to acquire the core functional phenotype of DP cells.

[0029] Differentiation microenvironment optimization: A porphyrin-coated culture vessel was used. The specific binding of porphyrin to integrins on the surface of ADMSCs promotes cell adhesion and morphological polarization, mimicking the in vivo growth matrix microenvironment of DP cells and further enhancing the efficiency of differentiation signal transmission. Induction was initiated when the confluence of ADMSCs reached 60%-85%, at which point the cells were in a state of active proliferation and high differentiation potential, thus improving differentiation efficiency. The induction period was set at 7 days with daily medium changes to ensure stable inducing factor concentrations and continuously drive ADMSC differentiation into DP cells.

[0030] Postdifferentiation function maintenance: The induced DP cells were transferred into maintenance medium and dermal papillary cell spheres (DPLTs) were formed through three-dimensional spheroidization culture. The three-dimensional structure similar to that of DP cells in vivo was reconstructed by intercellular tight junctions, maintaining the high expression of stemness markers of DP cells and the secretion of hair follicle-induced related signaling molecules, thus ensuring the functional integrity of differentiated cells.

[0031] The beneficial effects of this technology are as follows:

[0032] (1) Solving the bottleneck of DP cell source and clinical translation: In view of the problems of scarce DP cell source, damaged hair follicle extraction and functional decay in in vitro expansion in the existing technology, the present invention uses ADMSC as the starting cell. ADMSC has extremely rich source, strong in vitro expansion capacity and low immunogenicity. Combined with targeted induction technology, functional DP cells can be prepared on a large scale, breaking through the source limitation of DP cell clinical application; at the same time, it avoids the defects of difficult storage of umbilical cord mesenchymal cells (UCMSC) and low purity of foreskin fibroblast induction, and broadens the raw material channels for DP cell preparation.

[0033] (2) The induction system has clear components and is serum-free, with high safety and reproducibility: Existing DP cell induction technology mostly relies on serum-containing culture medium (complex composition, large batch-to-batch variation) or single-molecule induction (low efficiency). The types and concentrations of each component (including nutritional supplements and induction factors) of the induction culture medium of this invention are clear and controllable, and the entire process is serum-free. This not only eliminates the interference of unknown components in serum on cell differentiation and ensures batch-to-batch reproducibility of induction results, but also avoids the safety risks of serum carrying pathogenic microorganisms or foreign proteins. It is more in line with the standards for clinical-grade cell preparation and lays a safe foundation for subsequent cell therapy applications.

[0034] (3) High induction efficiency and complete differentiation cell function: Compared with the problems of low activity, weak expression of key functional genes, and poor hair follicle induction ability of DP-like cells induced by existing technologies, this invention can induce ADMSCs to differentiate into DP cells efficiently through the synergistic regulation of multiple signaling pathways. The differentiated cells highly express DP cell functional markers. Moreover, by maintaining the DPLTs formed by culture, the hair follicle induction ability can be stably preserved, solving the core problem of functional decline of DP cells in vitro, and providing functional cell raw materials for hair loss treatment.

[0035] (4) Simple operation and conducive to large-scale application: The induction method of this invention does not require complicated cell sorting or gene editing steps. The directional differentiation of ADMSCs into DP cells can be achieved simply by optimizing the culture medium formula and culture conditions. The operation process is simple and highly controllable. At the same time, ADMSCs have strong in vitro expansion capabilities and can be combined with this induction technology to achieve integrated "expansion-induction" large-scale production, reduce production costs, facilitate industrialization, and provide a scalable technical solution for cell therapy of hair loss.

[0036] (5) Broad application prospects and promotion of regenerative medicine: The DP cells (and DPLTs) obtained by this invention can be used to prepare hair growth agents and directly applied to cell therapy for diseases such as androgenetic alopecia and alopecia areata; at the same time, this induction system provides a precise regulatory paradigm for the directional differentiation of mesenchymal cells into specific functional cells, which can provide technical reference for cell preparation in other tissue engineering fields (such as skin repair) and promote the application of regenerative medicine in clinical treatment. Attached Figure Description

[0037] Figure 1 This is a typical microscopic image of adipose-derived mesenchymal cells (ADMCS) obtained from primary mesenchymal stem cells extracted from fat in Example 1.

[0038] Figure 2 This is a schematic diagram of the induction and culture process of dermal papillary cells (ADMSC-iDPC) obtained by adipose mesenchymal cells in Example 2, along with typical microscopic images of different stages.

[0039] Figure 3 The results of alkaline phosphatase activity detection in dermal papillary cells (ADMSC-iDPC) obtained by adipose mesenchymal cell induction in Example 2 are shown.

[0040] Figure 4 The immunofluorescence identification results of dermal papillary cells (ADMSC-iDPC) obtained by adipose mesenchymal cells in Example 2 are shown (Versican and α-smooth muscle actin (α-SMA) show green fluorescence; 4',6-diamidinyl-2-phenylindole (DAPI) shows blue fluorescence to display the cell nucleus).

[0041] Figure 5 This is the result of the study on the effect of different coating proteins on the induced culture of dermal papillary cells in Experiment Example 1.

[0042] Figure 6 The results of cell differentiation induction experiments in Example 2 at different Hc and HGF concentrations are shown below (first row: cell spheroidization state on day 8; second row: AKP detection results of ADMSC-iDPC on day 7; third row: immunofluorescence identification results of pluripotent proteoglycan (Versican) of ADMSC-iDPC on day 7; fourth row: immunofluorescence identification results of α-smooth muscle actin (α-SMA) of ADMSC-iDPC on day 7).

[0043] Figure 7 The results of the study on the effects of BMP2, BIO, and bFGF on the induction effect in Experiment Example 3 are shown in the following images: (First row: ADMSC-iDPC status on day 7; Second row: Cell spheroidization status on day 8; Third row: AKP detection results of ADMSC-iDPC on day 7).

[0044] Figure 8 The results of Example 3 on the effect of different concentrations of BIO and bFGF on the induction effect on the basis of IHH (first row of images: ADMSC-iDPC status on day 7; second row of images: cell spheroidization status on day 8; third row of images: AKP detection results of ADMSC-iDPC on day 7).

[0045] Figure 9 The results of the study on the effect of different concentrations of BMP2 on the induction effect on the basis of IHH in Example 3 are as follows (A: First row image: ADMSC-iDPC status on day 7; Second row image: Cell spheroidization status on day 8; Third row image: AKP detection results of ADMSC-iDPC on day 7; B: Immunofluorescence identification results of pluripotent proteoglycan (Versican) and α-smooth muscle actin (α-SMA) of ADMSC-iDPC on day 7).

[0046] Figure 10 The images show the results of cell differentiation induction under different induction time conditions in Experiment Example 4 (first row: AKP detection results, pluripotent proteoglycan (Versican) and α-smooth muscle actin (α-SMA) immunofluorescence identification results of ADMSC-iDPC on day 7; second row: AKP detection results, pluripotent proteoglycan (Versican) and α-smooth muscle actin (α-SMA) immunofluorescence identification results of ADMSC-iDPC on day 10).

[0047] Figure 11 The results of the RT-qPCR identification experiment of ADMSC-iDP cells in Example 5 (AE represent...) Noggin , Hey1 , Akp2 , Rspo3 and Rspo2 Gene).

[0048] Figure 12 The results of the hair regrowth experiment using a mixture of ADMSC-iDP cells and epidermal cells in Example 6 are shown.

[0049] Figure 13 The results of the animal experiment on the promotion of hair growth by ADMSC-iDP cells in Example 7 are shown in Figure 7 (A: Hair growth in the control group and ADMSC-iDP experimental group on day 13; B: Statistical analysis of the difference in hair length in the same area on the back of mice). Detailed Implementation

[0050] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0051] The following are explanations of some key terms:

[0052] Dermal papilla cells induced by adipose-derived mesenchymal cells (ADMSC-induced Dermal Papilla Cell, abbreviated as ADMSC-iDPC): These are dermal papilla cells obtained by induced differentiation of adipose-derived mesenchymal cells (ADMSCs), possessing the expression of characteristic markers and related functions of dermal papilla cells.

[0053] Dermal papilla cell granules (DPLTs) are spherical structures formed by the self-aggregation of dermal papilla cells (including induced ADMSC-iDPCs) under specific conditions. They are an important morphological indicator reflecting the cell's self-aggregation ability.

[0054] Dermal papilla (DP) cells: These are cells derived from the dermal papilla and possess properties such as inducing hair follicle formation and self-aggregation. They are key cells regulating hair follicle development and regeneration. Dermal papilla cells are abbreviated as DPC.

[0055] Alkaline phosphatase (AKP): Its activity is positively correlated with the ability of dermal papilla (DP) cells to induce hair follicle formation. It can be used as one of the important indicators for in vitro assessment of DP cell functional status and can be used to evaluate the induction differentiation effect of ADMSCs into DP cells.

[0056] Versican is a characteristic marker of dermal papillary (DP) cells. Its expression level is closely related to the function of DP cells and can be used to evaluate the induction of ADMSCs into DP cells.

[0057] α-smooth muscle actin (α-SMA) is a protein commonly expressed by cultured dermal papilla (DP) cells. It can be used as one of the characteristic markers of DP cells to evaluate the induction of ADMSCs into DP cells.

[0058] VTN, or vitronectin, is an extracellular matrix protein that may be involved in biological processes such as cell adhesion, migration, and signal transduction, and may affect cell growth and differentiation in cell culture.

[0059] Laminin-511 (LM511): also known as laminin 511, is a member of the laminin family and an extracellular matrix component. It may be involved in the construction of the extracellular matrix, cell adhesion, and signal transduction. It is abbreviated as LM511.

[0060] BMP2: Bone Morphogenetic Protein 2; BIO: GSK-3 inhibitor; bFGF: basic fibroblast growth factor; Hc: hydrocortisone; HGF: hepatocyte growth factor. All of the above substances are additives in the induction medium of this protocol.

[0061] Example 1: Method for primary extraction of ADMSCs

[0062] Observe the state of the adipose tissue: Fresh fat is usually milky yellow and soft. Let it stand until clear layers are formed: the upper layer is yellow oil, the middle layer is mainly milky yellow adipose tissue, and the lower layer is red or pink bloody fluid.

[0063] Prepare a sufficient number of 50ml sterile centrifuge tubes based on the estimated adipose tissue volume. Add 20ml of pre-warmed 37°C sterile saline to each 50ml centrifuge tube. Using a sterile 25ml pipette, carefully insert the tip of the pipette into the middle layer of adipose tissue (milky yellow layer). Slowly aspirate the adipose tissue into the pipette. Gently transfer the aspirated adipose tissue to a 50ml centrifuge tube containing 20ml of pre-warmed saline. Avoid vigorous agitation that may create air bubbles or damage the tissue. Repeat the above aspiration steps until most of the target adipose layer has been transferred. After all the fat has been transferred, perform an initial mixing: invert the centrifuge tube 10-15 times to ensure thorough contact and mixing between the saline and adipose tissue. After inversion, you should see the adipose tissue dispersed into particles of varying sizes suspended in the saline, and the liquid should appear pale pink.

[0064] Centrifuge the fat-saline mixture at 400g for 6 minutes. Remove the lower precipitate and supernatant. Carefully aspirate the lower red precipitate and most of the supernatant using a sterile pipette, retaining the fatty tissue at the bottom of the tube (clean fatty tissue). Repeat this step 3-4 times.

[0065] Place a 50ml centrifuge tube containing relatively clean adipose tissue in a biosafety cabinet. Remove the fascia. Mince the adipose tissue. Add type I collagenase solution (Sigma; catalog number: C-0130) preheated to 37°C until the total volume reaches 30ml. Tightly cap the centrifuge tube and shake vigorously by hand for 10-20 seconds to ensure thorough mixing of the fat fragments and collagenase solution. The tube should contain a cloudy emulsion. Securely fix the sealed centrifuge tube on a preheated 37°C shaker for digestion.

[0066] After digestion, remove the centrifuge tubes from the shaker, sterilize them, and place them in a biosafety cabinet. Add an equal volume (30 ml) of pre-warmed serum-free mesenchymal stem cell culture medium (Yocon Biotechnology, culture medium catalog number: NC0107; matching additive catalog number: NC0106.S; add according to the instructions, e.g., 100:1 volume ratio) to the digestion solution to terminate the digestion reaction. Tightly cap the tubes and invert 10 times to mix. Filter using a sterile 100μm cell filter to remove undigested tissue. Centrifuge to remove the lipid layer and supernatant. Add 20-30 ml of sterile physiological saline pre-warmed to 37°C to the cell pellet. Gently and repeatedly pipette the pellet from the bottom of the tube to fully disperse the cell clumps and form a single-cell suspension. Centrifuge again and collect the cell pellet. This washing step usually needs to be repeated 2-3 times until the supernatant is completely clear.

[0067] Resuspend the cell pellet in serum-free mesenchymal stem cell culture medium (Yocon Biotechnology, medium catalog number: NC0107; matching additive catalog number: NC0106.S; added according to the instructions, e.g., volume ratio 100:1) pre-warmed to 37°C. Seed the pellet into T175 culture flasks, adding 25 ml of culture medium to ensure the medium covers the entire bottom surface of the flask. Incubate in a CO2 incubator. ADMSC morphology: long spindle-shaped or fusiform with slender protrusions at both ends, resembling a spindle. Growth pattern: cells grow in whorls, radially, or parallel bundles. As cell density increases, they connect with each other, forming an orderly arrangement. Nucleus: clearly defined, round or oval, located in the center of the cell; nucleoli are prominent in healthy cells. Cytoplasm: uniform, translucent, with good refractive properties and clear edges (ADMSC morphology as shown). Figure 1 (As shown). This technical solution only involves the process of obtaining adipose-derived mesenchymal cells (ADMSCs) from adipose tissue, and does not involve the process of obtaining adipose tissue.

[0068] Example 2: Method for inducing adipose-derived mesenchymal cells (ADMSCs) into dermal papillary (DP) cells

[0069] (1) Preparation of coating solution and coating of culture flasks: Thaw Vitronectin (VTN) at room temperature (15-25℃). Take a 50mL centrifuge tube, add 15mL of DMEM / F12 medium, and add 1mg of thawed VTN to 15mL of DMEM / F12 medium. Gently invert to mix and ensure sufficient dilution. Immediately use the diluted VTN solution for coating. Add VTN solution to the culture flask at a ratio of 15mL diluted VTN solution / T175 cell culture flask, and gently shake the flask to evenly cover the entire bottom surface of the flask. After standing at room temperature (15-25℃) for at least 1 hour, aspirate the supernatant. The coating is now complete. ADMSCs can be coated at 8000 / cm³. 2 The inoculum was inoculated into pre-coated culture flasks and maintained in an incubator at 37°C and 5% CO2. Every 3 days, an appropriate amount of serum-free mesenchymal stem cell culture medium containing additives was replaced to culture and expand the cells until the cell confluence reached 60%-85%, and then subsequent culture induction was performed.

[0070] (2) Induction phase

[0071] For a detailed flowchart of the induction process, please refer to [link / reference]. Figure 2 .

[0072] Day 0: Microscopic observation was used to confirm the growth status and cell confluence of ADMSCs. The culture medium was removed from the cells, and 0.1 mL / 1 cm⁻¹ was used to treat the cells. 2Wash with a sufficient amount of DPBS (Duborough phosphate buffer) to remove dead cells and residual culture medium. Add 0.2 mL / cm³ of DPBS. 2 Apply a specific amount of serum-free induction medium containing well-defined components that promote the differentiation of ADMSCs into DP cells, spread it evenly in a culture dish, place it in an incubator, and incubate for 24 hours.

[0073] The serum-free induction culture medium contains the following components: [amount of medium containing L...] Alanine L DMEM / F of glutamine dipeptide 12 culture medium as basal medium (commercially available, of which L Alanine L The content of glutamine dipeptide is 0.542 mg / mL, and it contains 10% by volume of added ingredients. 20% KnockOut serum substitute (KSR), 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement (ITS), concentration 10 ng / mL 100 ng / mL hepatocyte growth factor (HGF), with a concentration of 5 ng / mL 100 ng / mL hydrocortisone (Hc, CAS: 50-23-7), with a content of 50 ng / mL Bone morphogenetic protein 2 (BMP2, CAS: P12643, preferably 200 ng / mL-400 ng / mL) at 400 ng / mL, a GSK-3 inhibitor (BIO, CAS: 667463-62-9, preferably 1-2.5 μM) at 0-2.5 μM, and basic fibroblast growth factor (bFGF, CAS: 152051-61-1, preferably 5-40 ng / mL) at 0-40 ng / mL. KSR is a commercially available additive with a well-defined chemical composition used to replace fetal bovine serum (FBS). ITS is a commercially available additive commonly used in cell culture that can replace various growth factors and hormones in fetal bovine serum (FBS). It is commonly used in serum-free or low-serum culture media to support cell growth and proliferation.

[0074] Preferably, the serum-free induction culture medium has the following composition: containing L... Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin The medium contains selenium supplement (ITS), hepatocyte growth factor (HGF) at a concentration of 50 ng / mL, hydrocortisone (Hc) at a concentration of 100 ng / mL, bone morphogenetic protein 2 (BMP2) at a concentration of 400 ng / mL, GSK-3 inhibitor (BIO) at a concentration of 2.5 μM, and basic fibroblast growth factor (bFGF) at a concentration of 5-10 ng / mL (more preferably 5 ng / mL).

[0075] Day 1-7: Continue culturing cells for 7 days in a serum-free induction medium with well-defined components to promote ADMSC differentiation into DP cells. Microscopic observation was used to confirm cell growth and confluence. The medium was changed daily at a volume of 0.2 mL / cm². 2 .

[0076] Day 7: Microscopic observation was used to confirm cell status and cell density. Dermal papillary cells (ADMSC-iDPC) induced from adipose-derived mesenchymal cells were obtained through induction culture. On day 7, the following tests were performed:

[0077] ADMSC-iDPC alkaline phosphatase activity assay

[0078] Alkaline phosphatase (AKP) activity in DP cells is positively correlated with their ability to induce hair follicle formation. Therefore, in vitro detection of AKP activity in DP cells can serve as one of the indicators for assessing their functional status. Following the instructions of the alkaline phosphatase assay kit, AKP activity was detected in ADMSCs and ADMSC-iDPCs obtained through induced differentiation. The results are as follows: Figure 3 The results shown (using the optimal serum-free induction medium) are ADMSC-iDP cells from three different donors, demonstrating their state before and after AKP staining. All three ADMSC-iDP cells from different donors showed extremely strong AKP activity (proportion of alkaline phosphatase-active cells = number of alkaline phosphatase-active cells under 4x magnification / total number of cells under 4x magnification, a total of 98, 753, and 368 cells were counted; the AKP activity mentioned below are statistical results for a large number of cells and will not be elaborated further).

[0079] ADMSC-iDPC Immunofluorescence Identification

[0080] Versican is a characteristic biomarker of DP cells, and its expression level is correlated with DP cell function. Furthermore, cultured DP cells typically express α-smooth muscle actin (α-SMA). To assess the induction of differentiation, we performed immunofluorescence detection of Versican and α-SMA 7 days after ADMSC induction into DP cells. The results are as follows: Figure 4 As shown in the experimental results (using the optimal serum-free induction medium), ADMSC-iDP cells from three different donors all showed positive expression of Versican and α-SMA. The expression of these markers suggests that ADMSCs have been successfully induced to differentiate into DP cells.

[0081] Next, the culture medium was removed from the cells, and 0.1 mL / 1 cm⁻¹ was used. 2 Wash twice with 0.1 mL / 1 cm of DPBS to remove dead cells. 2 Cells were treated with a controlled amount of TryplE (digestive enzyme) for 6 min, then digestion was stopped with culture medium. Cells were collected by centrifugation at 950 rpm for 5 min, and the supernatant was removed to eliminate the digestive fluid. Cells were resuspended in DP cell maintenance medium. Cells were collected in centrifuge tubes and gently pipetted the cell suspension until single-celled status was achieved (a portion was observed under a microscope for confirmation). Cell counts were performed, and the cells were diluted to 2 × 10⁶ cells / mL with DP cell maintenance medium. 5 50 μL / well was seeded into a 96-well U-bottom ultra-low adsorption plate with 1 mL of cells per well. The plate was then incubated for 24 h.

[0082] The specific components of the DP cell maintenance culture medium are as follows: in DMEM / F... 12. Basic culture medium (the basic culture medium does not contain L) Alanine L Add 10% by volume of glutamine dipeptide. 20% KnockOut serum substitute (KSR), and 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement (ITS).

[0083] Preferably, the composition of the DP cell maintenance culture medium is as follows: in DMEM / F 12% KnockOut serum substitute (KSR) and 1% insulin were added to the basal culture medium. transferrin Selenium culture medium supplement (ITS).

[0084] Day 8: Microscopic observation was used to confirm the aggregation of DP cells, the state of DP spheres, and the size of DP spheres in the 96-well plate. 50 μL of DP cell maintenance medium was added to each well. DP cells have a strong self-aggregation ability; the cells obtained by induction will self-aggregate into spherical shapes (dermal papillary cell spheres, DPLTs) within 6-24 hours.

[0085] Experimental Example 1: Screening of Extracellular Matrix Protein Coating Gels

[0086] To provide cells with an environment mimicking the in vivo extracellular matrix during induction, this protocol screened various extracellular matrix protein coating gels. VTN and Laminin-511 (LM511) are used as examples. Following the method in Example 2, culture flasks were coated with VTN and LM511, with a control group not coated. Other operations were performed as in Example 2 (induction was performed according to the preferred method of Example 2, but the induction time was extended to 16 days).

[0087] The induction culture medium consists of: a medium containing L... Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin Selenium culture medium supplement (ITS), hepatocyte growth factor (HGF) at a concentration of 20 ng / mL, and hydrocortisone (HC) at a concentration of 20 ng / mL.

[0088] Experimental results are as follows Figure 5 As shown, the results indicate that both the uncoated control group and the LM511-coated experimental group exhibited curling and detachment on day 5 of induction. The optimal NTV matrix protein, used as a coating protein to promote ADMSC differentiation into DP cells, extended the induction time to 16 days without exhibiting curling or detachment. Its effect is significantly superior to other existing coating proteins.

[0089] Experimental Example 2: Screening of Hc and HGF Concentrations

[0090] Two small molecule compounds, Hc and HGF, which promote DP differentiation, were added to DP culture medium to induce ADMSC differentiation. This combination was found to promote the differentiation of ADMSCs into DP cells with spherical morphology and the expression of their markers. The concentrations of each factor were increased to identify the main components promoting DP differentiation.

[0091] Experimental groups: control group (10 ng / mL Hc, 20 ng / mL HGF), low concentration Hc group (20 ng / mL Hc, 20 ng / mL HGF), medium concentration Hc group (50 ng / mL Hc, 20 ng / mL HGF), high concentration Hc group (100 ng / mL Hc, 20 ng / mL HGF), low concentration HGF group (10 ng / mL Hc, 50 ng / mL HGF), high concentration HGF group (10 ng / mL Hc, 100 ng / mL HGF).

[0092] In the above experimental groups, in addition to adding Hc and HGF as described above, the basic configuration of the induction medium was as follows: containing L... Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin Selenium culture medium supplement (ITS). Based on the above basic configuration, add the specified amounts of HGF and Hc according to the experimental groupings.

[0093] The induced adipose-derived mesenchymal stem cells (ADMSCs) were uniformly grouped and induced according to the optimal method of Example 2, using the corresponding culture medium for each group during induction. The induced cells were subjected to AKP activity assay (after 7 days of induction according to Example 2) and immunofluorescence assay (after 7 days of induction according to Example 2), and cell spheroidization was observed on day 8 according to Example 2.

[0094] Experimental results are as follows Figure 6 As shown, the results indicate that increasing Hc concentration significantly increases AKP expression after ADMSC differentiation, thus concluding that high Hc concentration promotes ADMSC induction into DP cells. Increasing HGF concentration promotes DP cell spheroidization, while decreasing AKP expression with increasing concentration. No significant differences were observed among the Versican and α-SMA groups. Therefore, 100 ng / mL Hc and 50 ng / mL HGF are preferred for promoting ADMSC differentiation into DP cells.

[0095] Experiment Example 3: Screening for cytokines and small molecule compounds that promote the differentiation of ADMSCs into DP cells

[0096] Three factors that promote DP differentiation—bone morphogenetic protein 2 (BMP2), GSK-3 inhibitor BIO, and basic fibroblast growth factor (bFGF)—were added to the culture medium for ADMSC differentiation. The concentrations and combinations of each factor were explored to identify the main components that promote induction.

[0097] Each experimental group was supplemented with the following basic configuration: containing L Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin Selenium culture medium supplement (ITS). The experimental groupings are as follows:

[0098] IHH group (with the following additional components added to the basic configuration: 100 ng / mL Hc, 50 ng / mL HGF);

[0099] BBb group (with the following additional components added to the basic configuration: 200 ng / mL BMP2, 2.5 μM BIO, and 10 ng / mL bFGF);

[0100] IHH+BBb group (with the following additional components added to the basic configuration: 100 ng / mL Hc, 50 ng / mL HGF, 200 ng / mL BMP2, 2.5 μM BIO, and 10 ng / mL bFGF).

[0101] IHH+BIO+BMP2 group (with the following additional components added to the basic configuration: 100 ng / mL Hc, 50 ng / mL HGF, 200 ng / mL BMP2, and 2.5 μM BIO; bFGF not added).

[0102] IHH+bFGF+BIO (additional components to the basic configuration: 100 ng / mL Hc, 50 ng / mL HGF, 2.5 μM BIO, 10 ng / mL bFGF; BMP2 not added).

[0103] IHH+BMP2+bFGF (additional components to the basic configuration: 100ng / mL Hc, 50ng / mL HGF, 200ng / mL BMP2, 10ng / mL bFGF; BIO not added).

[0104] ADMSCs were induced and cultured according to the optimal method of Example 2, using serum-free induction medium for each experimental group. AKP activity was measured in the induced cells after 7 days of induction as described in Example 2, and immunofluorescence was also performed (after 7 days of induction as described in Example 2). Cell spheroidization was observed on day 8 as described in Example 2.

[0105] Based on the IHH group (see the previous section on induction medium preparation for the IHH group), a BMP2, BIO, and bFGF concentration gradient experiment was set up to screen suitable BMP2, BIO, and bFGF concentrations for induction to obtain ADMSC-iDP. The specific induction medium for the IHH group was: containing L... Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin Selenium culture medium supplement (ITS), 100 ng / mL Hc, and 50 ng / mL HGF. The experimental group arrangements are as follows:

[0106] Low concentration bFGF group (5 ng / mL bFGF added to the IHH group), medium concentration bFGF group (10 ng / mL bFGF added to the IHH group), high concentration bFGF group (40 ng / mL bFGF added to the IHH group).

[0107] Low concentration BIO group (1 μM BIO added to the IHH group), medium concentration BIO group (2.5 μM BIO added to the IHH group), high concentration BIO group (5 μM BIO added to the IHH group);

[0108] Low concentration BMP2 group (50 ng / mL BMP2 added to the IHH group), medium concentration BMP2 group (200 ng / mL BMP2 added to the IHH group), high concentration BMP2 group (400 ng / mL BMP2 added to the IHH group).

[0109] ADMSCs were induced and cultured according to the optimal method of Example 2, using serum-free induction medium for each experimental group. AKP activity was measured in the induced cells after 7 days of induction as described in Example 2, and immunofluorescence was also performed (after 7 days of induction as described in Example 2). Cell spheroidization was observed on day 8 as described in Example 2.

[0110] right Figure 7 , 8 The experimental results for 9 are explained in detail below:

[0111] (1) For the conventional method of adding only Hc and HGF

[0112] Adding only Hc and HGF to the culture medium will result in an excessively low AKP positivity rate, poor cell spheroidization, and relatively loose and small cell spheroids (see [link to article]). Figure 7 (IHH group). AKP activity in DP cells is positively correlated with their ability to induce hair follicle formation, and detecting AKP activity in DP cells can serve as one of the indicators for assessing their functional status. Therefore, for the induction culture of ADMSCs, it is not feasible to simply add Hc and HGF as in the existing techniques for inducing ordinary pluripotent stem cells.

[0113] (2) Further add the effect of BMP2

[0114] To improve the culture medium, the inventors experimented with various additives. When different concentrations of BMP2 were added to the existing Hc and HGF regimen, they found that the AKP positivity rate of ADMSCs induced to form DP cells was significantly increased. (See details...) Figure 9 Experimental data showed that BMP2 at concentrations of 200 ng / mL to 400 ng / mL effectively promoted the increase of AKP positivity rate. In addition, the DP cell markers Versican and α-SMA were also detected, and BMP2 at concentrations of 200 ng / mL to 400 ng / mL effectively promoted the expression of both markers.

[0115] However, the experiment also revealed a key problem: while adding BMP2 to the culture medium effectively enhanced the AKP activity of DP cells (an important indicator of DP cells' potential for hair follicle induction), the self-aggregation ability of the induced DP cells was not improved simultaneously. Specifically, the volume of the self-aggregated cell spheres (DPLTs) was generally small, with their diameter maintained in the range of 200–280 μm according to microscopic measurements, far below the ideal size standard for DP cell spheres (usually requiring more than 300 μm to ensure efficient intercellular signal transduction). More importantly, the surface of the cell spheres was accompanied by some incompletely aggregated discrete cells. These discrete cells not only could not participate in the construction of the signal network inside the cell sphere, but their free state may also affect the overall structural stability of the cell sphere, resulting in the sphere formation effect not meeting the requirements of subsequent experiments and applications. The unsatisfactory spheroidization effect of cells is not simply a structural problem; it is directly related to the functional realization of DP cells. This means that DP cells cannot form tight contact connections, which are a key prerequisite for maintaining stemness and activating autocrine signaling pathways. Simultaneously, the small spheroid volume and the presence of discrete cells prevent DP cells from mimicking the three-dimensional microenvironment of the dermal papilla in vivo, potentially restricting their subsequent interaction with hair follicle epithelial cells. Even if the core functional indicator of AKP activity is improved, the defect in spheroidization will still become a bottleneck limiting the overall hair follicle induction function of DP cells. Therefore, current experimental results clearly point to a core need: the urgent need to develop an optimized culture medium formulation or strategy that, while retaining the BMP2-promoting effect on AKP activity in DP cells, further improves the self-aggregation ability of DP cells, achieving a dual guarantee of high AKP activity and excellent spheroidization effect. This would yield functional DP cells with both high hair follicle induction potential and stable three-dimensional structure, laying the foundation for subsequent in vitro research and clinical translational applications (such as cell transplantation in hair loss treatment).

[0116] (3) Attempts to enhance the self-aggregation ability of DP cells

[0117] To improve cell spheroidization, the inventors experimented with adding agents that promote cell formation to BMP2. For example, they further added different concentrations of bFGF and BIO to Hc and HGF, as detailed below. Figure 8The experimental results showed that all concentration gradients of bFGF addition promoted cell proliferation, forming cell spheroids with a diameter of approximately 320–380 μm. These spheroids were nearly round, with smooth edges, dense interiors, and no vacuoles, but the AKP positivity rate was significantly reduced in this group. In contrast, the concentration gradients of BIO addition resulted in poor cell spheroidization, loose spheroid structure, numerous discrete cells, and virtually no AKP expression. Therefore, bFGF has the potential to be used as a further addition to BMP2 to enhance the self-aggregation ability of DP cells.

[0118] Based on the above experimental results, the inventors combined bFGF, which promotes spheroidization, with BMP2, which promotes AKP activity. Detailed experimental results can be found in [link to experimental results]. Figure 7 The IHH+BMP2+bFGF group was tested. To the inventors' surprise, while the addition of bFGF improved the spheroidization of DP cells to some extent, it significantly reduced the AKP activity that had been enhanced by BMP2 (only 2% AKP positivity rate). In addition, the inventors also tried adding only BIO to BMP2, but the spheroidization effect was still not ideal; the cell spheroids were relatively loose and small in size.

[0119] When the inventors tried adding BIO to the culture medium containing BMP2 and bFGF, they unexpectedly discovered that while adding BMP2 could increase the AKP activity of DP cells, adding bFGF would completely reverse this trend. Further addition of BIO eliminated this reversal, and the AKP activity of DP cells returned to a relatively high level. Figure 7 The IHH+BBb group). Furthermore, the combined use of BMP2, bFGF, and BIO ensures effective DP cell spheroidization, resulting in cell spheroids with a diameter of approximately 320–380 μm, exhibiting a nearly round shape, smooth edges, dense interior, and no vacuoles. BIO itself does not enhance AKP activity or promote DP cell spheroidization. Figure 8 Therefore, the effect of adding BIO to BMP2 and bFGF was unexpected by the inventors. That is, a substance that does not have the ability to promote AKP activity or promote spheroidization can promote the AKP activity of DP cells and ensure the self-aggregation ability of DP cells in specific application scenarios.

[0120] (4) Synergistic effect of Hc, HGF, BMP2, bFGF and BIO

[0121] Adding only Hc and HGF to the culture medium will result in an excessively low AKP positivity rate (50%), poor cell spheroidization, relatively loose cell spheroids, and smaller cell size (see [link to relevant documentation]). Figure 7(IHH group). Adding only BMP2, bFGF, and BIO to the culture medium also resulted in an excessively low AKP positivity rate (40%) and unsatisfactory cell spheroidization (see [link to relevant documentation]). Figure 7 (BBb group). However, the combined use of the above substances effectively improved the spheroidization of DP cells: the formed cell spheroids were approximately 320–380 μm in diameter, nearly round, with smooth edges, dense interiors, and no vacuoles. Furthermore, the combined use of these substances also effectively ensured an AKP positivity rate of over 90%. Therefore, Hc, HGF, BMP2, bFGF, and BIO can work synergistically to promote the formation of high-quality DP cells after ADMSC induction culture, facilitating further application of these cells.

[0122] In summary, BMP2 alone significantly improves AKP expression, but results in poor spheroidization. While the combination of BMP2 and bFGF effectively improves spheroidization, it significantly inhibits AKP expression. BIO alone leads to poor AKP expression and spheroidization. However, adding BIO to the combination of BMP2 and bFGF effectively mitigates the shortcomings of using either factor. Therefore, 2.5 μM BIO, 5 ng / mL bFGF, and 400 ng / mL BMP2 are the optimal induction medium combination for promoting ADMSC differentiation into iDPCs.

[0123] Experiment Example 4: Screening of Induction Time

[0124] The experiment was divided into two groups: a 7-day induction group and a 10-day induction group. The induction medium used was: [a medium containing L...]. Alanine L DMEM / F of glutamine dipeptide 12. Culture medium was used as the basal medium, which contained: 15% KnockOut serum substitute (KSR) and 1% insulin. transferrin Selenium culture medium supplement (ITS), 100 ng / mL Hc, 50 ng / mL HGF, 400 ng / mL BMP2, 2.5 μM BIO, 10 ng / mL bFGF.

[0125] The adipose-derived mesenchymal stem cells (ADMSCs) were uniformly grouped and induced using the induction medium of this example as in Example 2. After 7 and 10 days of induction, AKP activity was detected and immunofluorescence was performed.

[0126] Experimental results are as follows Figure 10As shown, the results indicate that compared to the group induced for 7 days, the cell count after 10 days of induction was significantly higher, with more severe cell stacking. However, AKP activity decreased significantly with prolonged induction time, while the expression levels of Versican and α-SMA decreased slightly. Therefore, 7 days of induction was the preferred induction time for ADMSCs.

[0127] Experimental Example 5: Detection of ADMSC-iDP Cell Fluorescent RT-qPCR

[0128] Key genes of ADMSC-iDP cells obtained according to the optimal method in Example 1 were detected by RT-qPCR, including: Noggin (Noggin Protein), Hey1 (Hes Related Family bHLHTranscription Factor With YRPW Motif 1), Akp2 (Alkaline Phosphatase 2), Rspo3 (R-Spondin 3), and Rspo2 (R-Spondin 2). These genes are involved in regulating the function of DP cells during hair follicle regeneration. The experimental results are as follows: Figure 11 As shown, the expression levels of the aforementioned genes in ADMSC-iDP cells were significantly higher than those in undifferentiated ADMSCs. This result suggests that ADMSC-iDP cells possess the functional potential to promote hair regeneration.

[0129] Experimental Example 6: Hair regeneration from a mixture of ADMSC-iDP and neonatal mouse epidermal cells

[0130] The "Patch" transplantation model is a classic in vivo experiment for evaluating the ability of dermal papilla cells (DP cells) to induce hair growth. For example... Figure 12 As shown: 4.0×10 6 One newborn mouse epidermal cell and 4.0 × 10 6 ADMSC-iDP cells prepared in the optimal manner in Example 2 were mixed in DMEM / F12 medium (experimental group) for later use. Nude mice (thymus-deficient type) were anesthetized. The above-mentioned mixed cell suspension was injected subcutaneously into the nude mice. Positive control group: 4.0 × 10⁻⁶ cells were injected. 6 One newborn mouse epidermal cell and 4.0 × 10 6 A mixed suspension of 100 newborn mouse dermal cells. Negative control group: only 4.0 × 10⁶ cells were injected. 6 One newborn mouse epidermal cell.

[0131] The experimental results are as follows: 21 days post-transplantation, both the ADMSC-iDP cell group and the positive control group successfully induced significant hair regeneration, and the amount of hair growth was comparable in both groups. In contrast, the negative control group only showed localized melanin deposition without significant hair growth. These results confirm that ADMSC-iDP cells have the function of inducing hair regrowth.

[0132] Experiment Example 7: ADMSC-iDP bulb hair growth promotion experiment

[0133] Previous studies have reported that transplanting primary DP cell spheres into the hair-removing area of ​​C57BL / 6 mice can shorten the telogen phase of hair follicles and promote faster hair growth. (C57BL / 6 JGpt-Rag2 mice were anesthetized.) - / - l2rg - / - Immunodeficient black mice (C57BL / 6J mice with the Recombination Activating Gene 2 and Interleukin-2 Receptor Gamma Chain knocked out; Gpt is the strain identifier of the mouse supplier). The fur on the back of the mice was shaved. The skin in the hair removal area was cleaned and dried. Talcum powder was used to remove residual moisture and oil from the skin. Hair removal wax was applied evenly to the target area. The strip of wax was applied along the direction of hair growth, left for 3 minutes, and then quickly torn off against the direction of hair follicle growth to synchronize the hair cycle.

[0134] Then, cell transplantation is performed, as follows:

[0135] Experimental group: Subcutaneous injection of 1×10⁻⁶ ppm of [a specific substance] was administered to the hair removal area. 6 DMEM medium for ADMSC-iDP cell spheres prepared in the optimal manner in Example 2.

[0136] Control group: An equal volume of DMEM culture medium was injected subcutaneously into the hair removal area.

[0137] Experimental results are as follows Figure 13 As shown, the hair density and length in the experimental group (injected with ADMSC-iDP cell spheres) were significantly higher than those in the control group (injected with culture medium only). This result confirms in an in vivo model that ADMSC-iDP cells have the ability to shorten the resting phase of hair follicles and promote hair growth.

[0138] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method of promoting differentiation of adipose mesenchymal cells into dermal papilla cells, characterized by: The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The serum-free induction culture medium is formulated as follows: containing L Alanine L DMEM / F of glutamine dipeptide 12. Culture medium is used as the basal medium, which contains: 10% by volume. 20% KnockOut serum substitute, 0.5% by volume. 5% insulin transferrin Selenium culture medium supplement, concentration 10 ng / mL 100 ng / mL of hepatocyte growth factor, with a concentration of 5 ng / mL Hydrocortisone at 100 ng / mL, with a content of 50 ng / mL Bone morphogenetic protein 2 at 400 ng / mL, BIO, a GSK-3 inhibitor at 1-2.5 μM, and basic fibroblast growth factor at 5-40 ng / mL.

2. The method of claim 1, wherein the method promotes differentiation of adipose mesenchymal cells into dermal papilla cells. The serum-free induction medium has the following formulation: DMEM / F Alanyl L Glutamine dipeptide DMEM / F 12 medium as basal medium, with the addition of: KnockOut Serum Replacement at 15% v / v, insulin Transferrin Selenium medium supplement, Hepatocyte Growth Factor at 50 ng / mL, Hydrocortisone at 100 ng / mL, Bone Morphogenetic Protein 2 at 200-400 ng / mL, GSK-3 inhibitor BIO at 2.5 μM, Basic Fibroblast Growth Factor at 5-10 ng / mL.

3. The method of claim 1, wherein the method comprises: a) culturing the adipose tissue-derived mesenchymal stem cells in a medium comprising a TGF-β superfamily member; and b) culturing the adipose tissue-derived mesenchymal stem cells in a medium comprising a Wnt signaling pathway activator. The adipose-derived mesenchymal cells are cultured in a culture container coated with vitronectin; The coating method is as follows: the DMEM / F12 medium containing dissolved vitronectin is contacted with the cell growth surface of the culture container and incubated, and then the DMEM / F12 medium containing dissolved vitronectin is removed, so that the vitronectin coating is completed.

4. The method for promoting the differentiation of adipose-derived mesenchymal cells into dermal papillary cells according to claim 1, characterized in that: The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; 5. The method of claim 1, wherein the method is for promoting differentiation of adipose mesenchymal cells into dermal papilla cells. The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The formulation of the maintenance medium is: DMEM / F12 medium with 10% KnockOut serum replacement, 0.5% insulin, 0.1% transferrin, 0.02% sodium selenite. 12The formulation of the maintenance medium is: DMEM / F12 medium with 10% KnockOut serum replacement, 0.5% insulin, 0.1% transferrin, 0.02% sodium selenite. 20% of KnockOut serum replacement, and 0.5% 5% of insulin transferrin sodium selenite.

6. The method of claim 1, wherein the method is for promoting differentiation of adipose mesenchymal cells into dermal papilla cells. The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; 7. A composition for promoting differentiation of adipose mesenchymal cells into dermal papilla cells, characterized by: including 10 ng / mL 100 ng / mL of hepatocyte growth factor, 5 ng / mL of 100 ng / mL of hydrocortisone, 50 ng / mL of 400 ng / mL of bone morphogenetic protein 2, 1-2.5 μM of GSK-3 inhibitor BIO, 5-40 ng / mL of basic fibroblast growth factor.

8. The composition for promoting differentiation of adipose mesenchymal cells into dermal papilla cells according to claim 7, characterized by: The composition is in a medium containing 10% 20% KnockOut serum replacement, 0.5% 5% insulin Transferrin L-selenomethionine Alanyl L Glutamine dipeptide DMEM / F 12 medium.

9. A defined serum-free induction medium that promotes differentiation of adipose mesenchymal cells into dermal papilla cells, characterized in that: The formulation is: L Alanyl L Glutamine dipeptide DMEM / F 12 medium as basal medium, wherein are added: 10% 20% KnockOut serum replacement, 0.5% 5% insulin Transferrin Selenium medium supplement, 10 ng / mL 100 ng / mL Hepatocyte Growth Factor, 5 ng / mL 100 ng / mL Hydrocortisone, 50 ng / mL 400 ng / mL Bone Morphogenetic Protein 2, 1-2.5 μM GSK-3 inhibitor BIO, 5-40 ng / mL Basic Fibroblast Growth Factor. The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal cells are induced to differentiate into dermal papilla cells by using a serum-free induction medium; The adipose-derived mesenchymal

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