A method for identifying and isolating human hair follicle dermal stem cells

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a method for identifying and isolating human hair follicle dermal stem cells. In a first aspect of the present invention, a marker for human hair follicle dermal stem cells is provided, the marker comprising one or more of COL11A1, RBP4, MMP11, and SOX2. By directly obtaining human hair follicle samples, combined with spatial transcriptome and single-cell RNA sequencing technology, the gene expression profile of the human hair follicle dermis is comprehensively captured, thereby identifying the characteristic gene expression of hHFDSC. At the same time, the spatial transcriptome is used to locate the structural position information of hHFDSC and analyze the spatial distribution characteristics of hHFDSC. By combining these two technologies, based on the identified characteristic genes, combined with their spatial localization and differentiation ability, hHFDSC is clearly defined, achieving efficient and accurate analysis of the characteristics and functions of hHFDSC.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a method for identifying and isolating human hair follicle dermal stem cells. Background Art

[0002] The hair follicle (HF) is a microscopic organ in the human body with remarkable regenerative potential. Its structure is composed of epithelial and dermal cells. Adult hair follicles regenerate through a cyclical cycle of growth, catagen, and telogen, continuously renewing cells within the follicle and thus achieving continuous hair regeneration and renewal. This cyclical regeneration of hair follicles relies on a population of adult stem cells located in a specific niche. These cells proliferate to achieve self-renewal and further differentiate into progeny cells, collectively maintaining the integrity of the hair follicle's structure. Within the dermis of the hair follicle, cells closely adjacent to the epithelium can be divided into dermal papilla (DP) cells and dermal sheath (DS) cells. Research has shown that DP cells function as a signaling center within the hair follicle microenvironment, regulating the proliferation and differentiation of epithelial cells. DS cells, enveloping the outer surface of the hair follicle, play a crucial role in regulating the hair follicle's regenerative cycle.

[0003] Studies have revealed the presence of a hair follicle dermal stem cell (HFDSC) at the base of mouse hair follicles. These cells remarkably overexpress Cdk1, Hic1, S100a4, and Itga8, are capable of self-renewal and differentiation into DP and DS cells, supporting the renewal of the dermal portion of the hair follicle. HFDSCs play a critical role in hair follicle regeneration. If HFDSC proliferation and differentiation are impaired, renewal of the dermal portion of the hair follicle stagnates, and hair follicle regeneration is incomplete. However, due to species differences between humans and mice, mouse HFDSC signature genes cannot be localized to cells in the same follicular region in human hair follicles, nor can cells with similar functions be identified. Furthermore, due to the lack of systematic studies of human hair follicle dermal stem cells (hHFDSCs), the existence of dermal stem cells in human hair follicles remains unknown.

[0004] Furthermore, the study discovered that DP precursor cells, dermal sheath cup cells (DSCC), exist in the lower half of the dermal sheath of human hair follicles, near the hair bulb. While the spatial location of DSCCs is consistent with that of HFDSCs, their characteristic genes are significantly different from those of HFDSCs. Furthermore, DSCCs only support their own cell renewal and differentiation into DP cells, and are unable to differentiate into DS cells like HFDSCs do. This makes it difficult for DSCCs to fully regenerate the dermis of the hair follicle, indicating that they cannot fulfill the role of hHFDSCs. Therefore, further research is necessary to identify the characteristic genes of hHFDSCs and develop a method for their efficient isolation. Summary of the Invention

[0005] The present invention provides a method for identifying and isolating human hair follicle dermal stem cells. The characteristic genes relied upon by the identification method can be used to effectively identify hHFDSCs, and the separation method can be used to efficiently isolate hHFDSCs.

[0006] In a first aspect, the present invention provides a marker for identifying human hair follicle dermal stem cells, wherein the marker comprises two or more of COL11A1, RBP4, MMP11 and SOX2.

[0007] In some embodiments of the present invention, the markers further include at least one of COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17, and TSPAN8.

[0008] In a second aspect, the present invention provides a marker detection kit, which comprises a molecule that specifically recognizes the marker.

[0009] In some embodiments of the present invention, the molecule comprises at least one of an antibody, a primer, and a probe.

[0010] In some embodiments of the invention, the molecule is conjugated to a detectable label.

[0011] In some embodiments of the present invention, the detectable marker comprises at least one of a fluorescent marker, a chemiluminescent marker, a radioactive marker, an enzyme, biotin, and digoxigenin.

[0012] A third aspect of the present invention provides a method for identifying human hair follicle dermal stem cells, the method comprising detecting whether the aforementioned markers are expressed in the cells to be identified.

[0013] A fourth aspect of the present invention provides a method for screening human hair follicle dermal stem cells, the method comprising screening cells in a cell population that express the aforementioned markers.

[0014] In some embodiments of the present invention, the screening is performed using the aforementioned detection kit.

[0015] In some embodiments of the invention, the cell population is derived from skin.

[0016] In some embodiments of the invention, the cell population is derived from hair follicles.

[0017] A fifth aspect of the present invention provides the use of the aforementioned detection kit in the preparation of products for identifying, screening, and preparing human hair follicle dermal stem cells.

[0018] A sixth aspect of the present invention provides a method for isolating human hair follicle dermal stem cells, the method comprising the following steps:

[0019] Separate the dermis of the hair bulb from the hair follicle;

[0020] digesting the dermis with type I collagenase to obtain a digestion product;

[0021] The digestion product is subjected to a first centrifugation to obtain a supernatant containing the human hair follicle dermal stem cells, and the centrifugal force of the first centrifugation is 5 g to 30 g.

[0022] In some embodiments of the present invention, the method further comprises subjecting the supernatant to a second centrifugation to collect the precipitate, which is the human hair follicle dermal stem cells, and the centrifugal force of the second centrifugation is 200-400 g.

[0023] In some embodiments of the present invention, the supernatant further comprises cell filtration before the second centrifugation.

[0024] In some embodiments of the present invention, the working concentration of the type I collagenase is 0.8-1.5 mg / mL.

[0025] In a seventh aspect, the present invention provides a method for producing human hair follicle dermal stem cells, which comprises obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them.

[0026] In some embodiments of the present invention, the obtained human hair follicle dermal stem cells are genetically engineered to reduce immunogenicity.

[0027] In an eighth aspect, the present invention provides a method for preparing cell secretions, which comprises obtaining human hair follicle dermal stem cells according to the aforementioned method, culturing the stem cells, and isolating the cell secretions.

[0028] In some embodiments of the present invention, the cell secretions include at least one of extracellular vesicles and cytokines.

[0029] In a ninth aspect, the present invention provides a use of human hair follicle dermal stem cells obtained by the aforementioned method, or cell secretions obtained by the aforementioned preparation method, in the preparation of products for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases.

[0030] The beneficial effects of the present invention are:

[0031] To address the challenge of identifying dermal stem cells due to species differences, researchers directly obtained human hair follicle samples and combined spatial transcriptomics and single-cell RNA sequencing technologies to comprehensively capture the gene expression profile of the dermis of human hair follicles, thereby identifying the characteristic gene expression of hHFDSCs. Simultaneously, spatial transcriptomics was used to locate the structural position of hHFDSCs and analyze their spatial distribution characteristics. By combining these two technologies, hHFDSCs were clearly defined based on the identified characteristic genes, combined with their spatial localization and differentiation capabilities, enabling efficient and accurate analysis of their characteristics and functions.

[0032] Furthermore, the present invention has developed a highly efficient method for isolating hHFDSCs. Activity and functional assessments of the isolated cells confirmed that they retained relatively intact biological functions. This isolation method exhibits high specificity and sensitivity, improving cell purity and activity and optimizing the isolation process for human hair follicle dermal stem cells.

[0033] At the same time, the isolated hHFDSC or its extract can be further used as lyophilized powder, exosomes, cell transplantation, culture medium treatment, or combined with delivery systems for hair loss treatment, providing patients with hair loss with a new, safe and effective treatment method, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a bubble chart of the expression levels of characteristically expressed genes of hHFDSC in Example 1 of the present invention, wherein the average expression level represents the average expression level of a specific gene in hHFDSC / DP / DS cells, and the expression percentage represents the proportion of cells expressing a specific gene in hHFDSC / DP / DS cells in this type of cells.

[0035] Figure 2 It is the spatial position of hHFDSC in the spatial transcriptome data in Example 1 of the present invention.

[0036] Figure 3 This is a fluorescence staining image of hHFDSC-specific antibodies in Example 1 of the present invention.

[0037] Figure 4 These are the pseudo-time series results and RNA velocity analysis results of human hair follicle dermal lineage cells in Example 1 of the present invention. A is the Dynamo analysis result, and B is the Monocle3 analysis result.

[0038] Figure 5 This is the result of obtaining and isolating hHFDSCs in Example 2 of the present invention. A represents the follicular unit obtained from the hospital, B represents the bulb at the end of the excised hair follicle, C represents the remaining dermis after removing the pigment and keratin from the bulb, and D represents the compacted DP and discrete hHFDSC cell suspension after enzyme digestion.

[0039] Figure 6 These are the microscopic examination results of primary and passaged hHFDSC cells in Examples 2 and 3 of the present invention. Table A shows the cell morphology and activity of primary hHFDSCs cultured in culture medium for three days, and table B shows the cell morphology and activity of hHFDSCs cultured once in culture medium for three days.

[0040] Figure 7 The expression of the marker genes verified in Example 3 of the present invention in isolated hHFDSC cells is shown in Figure 3. A shows the co-staining result of MMP11 and COL3A1 (20X), B shows the co-staining result of MMP11 and RBP4 (20X), C shows the co-staining result of COL11A1 and COL1A1 (20X), and D shows the co-staining result of SOX2 and RBP4 (20X).

[0041] Figure 8 These are the experimental results of hHFDSC differentiation into DP cells in Example 3 of the present invention. A shows the cell morphology and activity (10X) of hHFDSCs cultured for three days in culture medium at passage 1; B shows the cell morphology and activity (10X) of DP cells differentiated from hHFDSC; and C shows the co-staining results for RBP4 and AQP1 (20X).

[0042] Figure 9 These are the experimental results of hHFDSC differentiation into DS cells in Example 3 of the present invention. A shows the cell morphology and activity (10X) of hHFDSC cultured for three days at passage 1, B shows the cell morphology and activity (10X) of DS cells differentiated from hHFDSC, and C shows the SMA staining result (20X). DETAILED DESCRIPTION

[0043] In the description of the present invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include at least one of such features. In the description of the present invention, "multiple" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0044] In a first aspect, the present invention provides a marker for identifying human hair follicle dermal stem cells, comprising multiple markers selected from COL11A1, RBP4, MMP11 and SOX2.

[0045] The term "hair follicle" refers to the sheath-like structure surrounding the hair root. It consists of two layers: the inner layer, a stratified epithelium continuous with the epidermis called the epithelial sheath (including the inner and outer root sheaths), and the outer layer, a connective tissue continuous with the dermis called the connective tissue sheath. The terminal end of the hair follicle swells to form the hair bulb. The base of the hair bulb is concave, forming a depression containing connective tissue, blood vessels, and nerve endings, forming a papilla called the dermal papilla. Hair follicle dermal stem cells (HFDSCs) are stem cells that support the renewal of the dermal portion of the hair follicle. They are capable of self-renewal and have the ability to differentiate into dermal papilla (DP) and dermal sheath (DS) cells. HFDSCs are typically located at the base of the hair bulb, near the dermal papilla. Accordingly, human hair follicle dermal stem cells refer to hair follicle dermal stem cells derived from humans, including those directly isolated from human hair follicles, as well as cells that have been cultured and passaged (e.g., P1 to P50, such as P2, P3, P4, P5, P10, P20, P30, P40, and P50). Furthermore, human hair follicle dermal stem cells also include cells differentiated from stem cells that meet their morphological characteristics, differentiation potential, and express the same markers. The stem cells used to differentiate into human hair follicle dermal stem cells can be non-embryonic stem cells, such as induced pluripotent stem cells (iPSCs). It is understood that human hair follicle dermal stem cells can also be cells obtained by genetically engineering or other treatment methods (such as pretreatment with growth factors or small molecule compounds, such as a stepwise regulation method using co-treatment with bFGF and the BMP inhibitor LDN-193189, see Nature 582, 399-404 (2020)) to obtain cells with the same morphological characteristics, differentiation potential, and expression of the same markers as the human hair follicle dermal stem cells described above. The purpose of the modification or treatment can be, for example, to enhance cell proliferation, differentiation, and hair follicle regeneration. In some embodiments, the human hair follicle dermal stem cells are adult stem cells, i.e., hair follicle dermal stem cells derived from adults aged 18 years or older.

[0046] In addition, human hair follicle dermal stem cells can be one or more of autologous cells and allogeneic cells. Among them, autologous and allogeneic refer to the individual on whom the human hair follicle dermal stem cells are used. Autologous cells are human hair follicle dermal stem cells derived from the individual itself, and allogeneic cells are human hair follicle dermal stem cells derived from individuals other than the individual. When autologous cells are used, immune rejection reactions can be completely avoided during the use process. In some embodiments, when the human hair follicle dermal stem cells are allogeneic cells, the cells can be modified in advance before use to reduce their immunogenicity, for example, using gene editing technology to modify the allogeneic cells, or subjecting the individual to local or systemic immunosuppression.

[0047] The term "marker" refers to a biochemical marker that can specifically label the structure and function of a system, organ, tissue, cell, or subcellular structure, as well as changes or potential changes in structure and function. Specifically, in this application, it refers to a marker that can specifically label human hair follicle dermal stem cells, thereby distinguishing them from other cells. Markers can be one or more of DNA, RNA, and protein.

[0048] The markers include multiple markers selected from COL11A1, RBP4, MMP11, and SOX2. For example, the markers may include two, three, or four of the markers. Specifically, the markers may include COL11A1 and RBP4, the markers include COL11A1 and MMP11, the markers include COL11A1 and SOX2, the markers include RBP4 and MMP11, the markers include RBP4 and SOX2, the markers include MMP11 and SOX2, the markers include COL11A1, RBP4, and MMP11, the markers include COL11A1, RBP4, and SOX2, the markers include COL11A1, MMP11, and SOX2, the markers include RBP4, MMP11, and SOX2, or the markers include COL11A1, RBP4, MMP11, and SOX2. It is understood that the markers include at least one of the above genes and the proteins encoded by the above genes. Thus, in some embodiments, the marker comprises genes COL11A1 and RBP4, the marker comprises genes COL11A1 and MMP11, the marker comprises genes COL11A1 and SOX2, the marker comprises genes RBP4 and MMP11, the marker comprises genes RBP4 and SOX2, the marker comprises genes MMP11 and SOX2, the marker comprises genes COL11A1, RBP4, and MMP11, the marker comprises genes COL11A1, RBP4, and SOX2, the marker comprises genes COL11A1, MMP11, and SOX2, the marker comprises genes RBP4, MMP11, and SOX2, or the marker comprises genes COL11A1, RBP4, MMP11, and SOX2. In other embodiments, the markers include COL11A1 protein and RBP4 protein, the markers include COL11A1 protein and MMP11 protein, the markers include COL11A1 protein and SOX2 protein, the markers include RBP4 protein and MMP11 protein, the markers include RBP4 protein and SOX2 protein, the markers include MMP11 protein and SOX2 protein, the markers include COL11A1 protein, RBP4 protein and MMP11 protein, the markers include COL11A1 protein, RBP4 protein and SOX2 protein, the markers include COL11A1 protein, MMP11 protein and SOX2 protein, the markers include RBP4 protein, MMP11 protein and SOX2 protein, or the markers include COL11A1 protein, RBP4 protein, MMP11 protein and SOX2 protein.

[0049] In some embodiments, the markers further comprise at least one of Set A, which is a set consisting of COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17, and TSPAN8. In some embodiments, the markers further comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, or all twenty-two of Set A.

[0050] In some embodiments, the marker also includes COL3A1. In some embodiments, the marker also includes ASPN. In some embodiments, the marker also includes LEF1. In some embodiments, the marker also includes HAPLN1. In some embodiments, the marker also includes POSTN. In some embodiments, the marker also includes MME. In some embodiments, the marker also includes TNMD. In some embodiments, the marker also includes DPEP1. In some embodiments, the marker also includes DKK3. In some embodiments, the marker also includes TMEM119. In some embodiments, the marker also includes CPXM2. In some embodiments, the marker also includes SFRP1. In some embodiments, the marker also includes LTBP2. In some embodiments, the marker also includes SMOC2. In some embodiments, the marker also includes COL8A2. In some embodiments, the marker also includes FGFBP2. In some embodiments, the marker also includes LAMC3. In some embodiments, the marker also includes FAM180A. In some embodiments, the marker also includes COL15A1. In some embodiments, the marker also includes F2RL2. In some embodiments, the marker also includes LRRC17. In some embodiments, the marker further comprises TSPAN 8. It is understood that the markers in the above set A may also be at least one of genes or proteins.

[0051] The second aspect of the present invention provides a marker detection kit, which includes a molecule that specifically recognizes the marker.

[0052] In some embodiments, the molecules are detected by immunohistochemistry, enzyme-linked immunosorbent assay, radioimmunoassay, immunoprecipitation, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, complement fixation analysis, flow cytometry fluorescence resolution, single molecule detection technology, Western blotting, Northern blotting, amplification (such as polymerase chain reaction PCR, and isothermal amplification such as loop-mediated isothermal amplification LAMP, recombinase polymerase amplification RPA, rolling circle amplification RCA, cross primer amplification CPA, strand displacement amplification The marker can be detected using one or more of the following techniques: SDA, helicase-dependent amplification (HDA), biochip method, nucleic acid sequencing method, amino acid sequencing method, high performance liquid chromatography, capillary gel electrophoresis method, near-infrared spectroscopy, mass spectrometry (such as electron impact mass spectrometry, chemical ionization mass spectrometry, photoionization mass spectrometry, threshold ionization mass spectrometry, double-focusing mass spectrometry, quadrupole mass spectrometry, time-of-flight mass spectrometry, ion trap mass spectrometry, Fourier transform mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry), surface plasmon resonance technology, immuno-PCR technology, biotin-avidin technology, etc. It is understood that the type of molecule specifically recognized may also vary depending on the specific type of marker. For example, in some embodiments, the molecule of each marker in the specific recognition marker can be any one of an antibody that can specifically bind to the marker, a ligand protein or polypeptide that specifically binds to the marker, a non-protein compound that specifically recognizes the marker, a probe that specifically binds to the marker, a nucleic acid aptamer that specifically recognizes the marker, etc.

[0053] In some embodiments, the antibody further comprises an antigen-binding fragment. Specifically, the antibody can be at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

[0054] In some embodiments, the molecule is coupled to a detectable label. In some embodiments, the detectable label comprises at least one of a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme, biotin, and digoxin. In some embodiments, the fluorescent label comprises a fluorescent dye, such as a cyanine (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Alexa Fluor 555, etc.), a rhodamine (e.g., Rhodamine 101, Rhodamine 6G, Tetramethylrhodamine, Rhodamine B, X-rhodamine, Texas Red, Alexa Fluor 488, Alexa Fluor 594, etc.), a fluorescein (e.g., fluorescein, fluorescein isothiocyanate, 5 (6)-carboxyhexachlorofluorescein, 5 (6)-carboxynaphthylfluorescein, Oregon Green, Pennsylvania Green, etc.), a coumarin (e.g., 7-AMCA, Alexa Fluor 350, Coumarin 343, etc.), fluorescent proteins (e.g., green fluorescent protein, phycoerythrin, phycocyanin, allophycocyanin), quantum dots (Group IV quantum dots, such as C, Si, Ge; Group III-V quantum dots, such as InAs, InP, GaSb, GaN, GaAs, InGaAs, AlGaAs, InAlAs, InGaN; Group II-VI quantum dots, such as BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe; Group IV-IV quantum dots, such as SiC, SiGe; Group IV-VI quantum dots, such as PbS, PbSe), nanocrystals, etc. In some embodiments, chemiluminescent labels include acridinium esters, ruthenium pyridine, luminol and its derivatives (e.g., isoluminol), etc. In some embodiments, radioactive labels include tritium, carbon-14, phosphorus-32, sulfur-35, iodine-125, iodine-131, etc. In some embodiments, the enzyme includes horseradish peroxidase (HRP), alkaline phosphatase (ALP), and the like.

[0055] In some embodiments, the detection kit further comprises reagents for assisting in the detection of gene expression, such as at least one of a reaction reagent that can visualize the amplicons corresponding to the primers, an RNA extraction reagent, a reverse transcription reagent, a cDNA amplification reagent, a standard substance for preparing a standard curve, and a positive control. In some embodiments, the detection kit further comprises reagents for assisting in the detection of protein expression, such as at least one of a color developer, a blocking solution, an antibody diluent, a wash buffer, a color stop solution, a standard substance for preparing a standard curve, and a positive control.

[0056] A third aspect of the present invention provides a method for identifying human hair follicle dermal stem cells, the method comprising detecting whether the aforementioned markers are expressed in the cells to be identified.

[0057] In some embodiments of the present invention, the detection is performed using the aforementioned detection kit.

[0058] In some embodiments, the cells to be identified are derived from skin. In some embodiments, the cells to be identified are derived from hair follicles.

[0059] In some embodiments, the total number of cells in the cells to be identified can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 It is understandable that, depending on the detection method, one or more cells in the cells to be identified can be identified simultaneously or sequentially.

[0060] A fourth aspect of the present invention provides a method for screening human hair follicle dermal stem cells, the method comprising screening cells in a cell population that express the aforementioned markers.

[0061] In some embodiments, screening is performed using the aforementioned detection kit.

[0062] In some embodiments, the cell population is derived from skin. In some embodiments, the cell population is derived from hair follicles.

[0063] In some embodiments, the total number of cells in the cell population can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 1210 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 More than one.

[0064] In some embodiments, screening or identifying is performed by flow cytometry.

[0065] A fifth aspect of the present invention provides the use of the aforementioned detection kit in preparing a product for identifying, screening, or preparing human hair follicle dermal stem cells. In some embodiments, the product comprises any one of a chip, device, or system, for example, a chip, device, or system for identifying, screening, or preparing human hair follicle dermal stem cells.

[0066] A sixth aspect of the present invention provides a method for isolating human hair follicle dermal stem cells, the method comprising the following steps:

[0067] Separate the dermis of the hair bulb from the hair follicle;

[0068] The dermis was digested with type I collagenase to obtain the digestion product;

[0069] The digestion product is subjected to a first centrifugation to obtain a supernatant containing human hair follicle dermal stem cells.

[0070] In some embodiments, the dermis is digested in an enzyme solution containing 0.1 to 10 mg / mL of type I collagenase. The working concentration of the enzyme solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL. In some embodiments, the dermis is digested in an enzyme solution containing 0.8 to 1.5 mg / mL of type I collagenase. In some embodiments, the digestion temperature is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, the digestion temperature is 35-40°C. In some embodiments, the digestion time is 10 min to 10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, or 10 h. In some embodiments, the digestion time is 50 min to 60 min. Type I collagenase is an enzyme mixture whose main components include clostripain, neutral protease, and two collagenases, thereby degrading collagen and other intracellular substances. Human hair follicle dermal stem cells are dermal fibroblasts of the hair follicle. Compared with other collagenases or other types of enzymes, type I collagenase can effectively digest collagen in the extracellular matrix, especially type I collagen, which is the most important type of collagen in the dermis of the hair follicle, thereby achieving better digestion and separation effects during the separation process. One or more enzymes in type I collagenase can be wild-type enzymes, or products modified by genetic engineering on the basis of wild-type enzymes, such as mutant enzymes obtained by random mutation, site-directed mutation, DNA shuffling, etc. for the purpose of improving one or more of the following purposes, such as enzyme activity and thermal stability, and have similar or higher ability to effectively release cells from tissues. In some embodiments, after the hair bulb is digested with type I collagenase, the digestion product is further subjected to a first centrifugation step.

[0071] In some embodiments, the centrifugal force during the first centrifugation is greater than 5 g, specifically 5 to 30 g, for example, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 18 g, 20 g, 25 g, 30 g, etc. If the centrifugal force during the first centrifugation is too low, the separation effect is unsatisfactory, making it difficult to clearly distinguish between the supernatant containing HFDSC and the pellet containing DP cell mammospheres. Excessive centrifugal force facilitates separation but may damage the pellet of DP cell mammospheres. In some embodiments, the first centrifugation duration is 1 to 5 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes.

[0072] In some embodiments, the supernatant containing human hair follicle dermal stem cells is further filtered. In some embodiments, after the first centrifugation, impurities and large cell clumps are removed by filtration using a filter with a pore size of 20 to 60 μm, such as 20 μm, 30 μm, 40 μm, 50 μm, or 60 μm. In some embodiments, the filter is a cell sieve with a 40 μm pore size.

[0073] In some embodiments, the supernatant is subjected to a second centrifugation to collect the precipitate, which is the human hair follicle dermal stem cells. In some embodiments, the centrifugal force of the second centrifugation is 200 g or greater, specifically 200-400 g, for example, 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, or 400 g. In some embodiments, the second centrifugation time is 2-10 min, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0074] In some embodiments, the bulb of a hair follicle is the portion 0.1 to 2 mm from the distal end of the hair follicle, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm. In some embodiments, the pigment and keratin in the bulb are removed to obtain the dermis of the bulb. In some embodiments, the pigment and keratin in the bulb are removed by mechanical extrusion.

[0075] In a seventh aspect, the present invention provides a method for producing human hair follicle dermal stem cells, which comprises obtaining human hair follicle dermal stem cells according to the aforementioned method and culturing them.

[0076] The term "culture" refers to the process of cell growth and proliferation under certain conditions, thereby producing a large number of cells. In some embodiments, culture can be 2D or 3D culture. In some embodiments, 3D culture methods include scaffold-based or scaffold-free 3D culture. In some embodiments, scaffold-free 3D culture methods include at least one of the low-adhesion planar method, the hanging drop method, the stirring method, and a 3D culture dish. In some embodiments, the scaffold material for 3D culture can be one or more of bioceramics, bioglass, metal, natural polymers (such as alginate, silk, hyaluronic acid, chitosan, fibroin, fibrin, collagen, and pluronic acid), and synthetic polymers (such as polylactic acid, polyglycolic acid, polycaprolactone, polyacrylonitrile, polyethylene glycol, polyimide, and polyvinyl alcohol). In some embodiments, the culture duration is 10 to 60 days, for example, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days. In some embodiments, the culture is performed in at least one of a culture dish, a culture vessel, a cell factory, a microfluidic chip, and the like.

[0077] In some embodiments, the obtained human hair follicle dermal stem cells are genetically engineered to reduce immunogenicity.

[0078] In an eighth aspect, the present invention provides a method for preparing cell secretions, comprising obtaining human hair follicle dermal stem cells according to the aforementioned method, culturing them, and isolating the cell secretions. In some embodiments, the cell secretions include at least one of extracellular vesicles (e.g., exosomes, microvesicles, etc.) and cytokines. In some embodiments, the method for isolating the extracellular vesicles includes one or more of centrifugation (e.g., ultracentrifugation), ultrafiltration, size exclusion chromatography, precipitation, immunoaffinity, and microfluidics. In some embodiments, isolating the cytokines includes collecting the supernatant of the culture product.

[0079] A ninth aspect of the present invention provides the use of human hair follicle dermal stem cells obtained by the aforementioned method, or the cell secretions obtained by the aforementioned preparation method, in the preparation of products for skin or hair follicle reconstruction, hair growth promotion, and skin disease treatment. Due to the differentiation potential of human hair follicle dermal stem cells, DP cells function as signaling centers within the hair follicle microenvironment, regulating epithelial cell proliferation and differentiation, while DS cells encapsulate the outer surface of the hair follicle and play a crucial role in regulating the hair follicle regeneration cycle. Therefore, human hair follicle dermal stem cells may be effective in skin or hair follicle reconstruction, hair growth promotion, and skin disease treatment.

[0080] In some embodiments, products for reconstructing skin or hair follicles, promoting hair growth, or treating skin diseases also include drugs that promote hair growth, such as minoxidil and finasteride. In some embodiments, the multiple ingredients in the products for reconstructing skin or hair follicles, promoting hair growth, or treating skin diseases can be administered simultaneously or sequentially using one or more different methods. In some embodiments, the method of administration can be one or more of topical or oral administration.

[0081] In some embodiments, the skin disease includes vitiligo, scars, hair loss (such as androgenetic alopecia AGA and alopecia areata AA), etc.

[0082] In some embodiments, the product, when used, comprises applying human hair follicle dermal stem cells, or cell secretions, to a person's head.

[0083] The present invention is further described in detail below through specific examples.

[0084] It should be understood that these embodiments are only used to illustrate the present invention rather than to limit the scope of the present invention.

[0085] The following are some preferred examples of the present invention. Experimental methods, where specific conditions are not specified, generally followed conventional conditions or those recommended by the manufacturer. Human scalp tissue used in these examples was obtained from 22 healthy male volunteers (aged 3-77) undergoing scalp tumor resection surgery and observed using a Leica fluorescence microscope. Some conventional materials are detailed in Table 1. All other materials and reagents used were commercially available unless otherwise specified.

[0086] Table 1. Some experimental materials and sources

[0087]

[0088] Example 1: Identification of human hair follicle dermal stem cells

[0089] This embodiment provides a method for identifying human hair follicle dermal stem cells, the process is as follows:

[0090] (1) Tissue sample acquisition and pretreatment: Obtain scalp tissue from the back of the head of a healthy, non-hair-lossing individual, wash with PBS, and remove excess subcutaneous fat. Cut the washed scalp tissue into small pieces using sterile ophthalmic scissors to provide material for subsequent spatial transcriptome and single-cell sequencing.

[0091] (2) Spatial transcriptome sequencing experiment: Tissue samples were cut into small pieces according to the direction of hair growth, fixed with OCT and frozen at -80 °C. After the embedded tissue blocks were equilibrated to -20 °C, they were cut to a thickness of 12 μm using a freezing microtome, and the tissue sections were attached to the Stereo-seq chip and fixed with pre-cooled methanol (-20 °C) for 30 minutes. Subsequently, they were stained with ssDNA fluorescent dye for 5 minutes at room temperature in the dark, washed with 0.1 v / v% SSC buffer and air-dried, glycerol was applied to the tissue sections and placed on a coverslip. The staining was recorded using a fluorescence microscope, and the image quality was evaluated using ImageQC software. After imaging, the coverslip was removed from the chip, washed with 5 v / v% SSC buffer, and permeabilized with 0.1 v / v% pepsin at room temperature for 8 minutes. After permeabilization, in situ reverse transcription and cDNA amplification were completed at 42 °C on the chip, and finally sequenced using the DNBSEQ platform to obtain spatial transcriptome data. The read length of read 1 is 35 bp, and the read length of read 2 is 100 bp.

[0092] (3) scRNA-seq sequencing experiment: The tissues cut into small pieces were placed in 5 mL of preheated type I enzyme solution (4.0 mg / mL dispase II) and incubated at 37°C for 50 minutes. The tissue suspension was transferred to preheated type II enzyme solution (1.6 mg / mL type I and 2.0 mg / mL type IV collagenase) and incubated at 37°C for 30 minutes. Then, 20 mL of DMEM was added to stop the enzymatic reaction, and the cell suspension was collected through a 70 μm cell strainer. The remaining undigested tissue fragments were digested with 8 mL of 0.25% trypsin at 37°C for 15 minutes. The second digestion was stopped by adding 10 mL of DMEM containing 20 v / v% fetal bovine serum (FBS). The cell suspension was then filtered through a 70 μm cell strainer, and the filtrate was mixed with the previously collected filtrate. All subsequent operations were performed on ice or at 4°C. The mixed cell suspension was centrifuged, and the cell pellet was resuspended in PBS containing 2 v / v% FBS. This suspension was then passed through a 30 μm cell strainer to remove any residual debris. Finally, the filtered suspension was centrifuged, and the cell pellet was resuspended in PBS containing 0.04 v / v% bovine serum albumin (BSA) to generate the final single-cell suspension. Using the MGI DNBelab C Series high-throughput single-cell RNA library preparation process, single-cell transcriptome data were generated through cell barcoding, reverse transcription, cDNA amplification, fragmentation, and library circularization. Sequencing was performed using the DNBSEQ platform.

[0093] (4) scRNA-seq data quality control:

[0094] 1. Raw Data Processing: After quality filtering of paired-end reads from the raw sequencing, the sequencing data were aligned to the human genome (hg38) using STAR (v2.7.1a). To identify single-cell droplets with valid cell barcodes, the EmptyDrops algorithm was employed to analyze the raw droplet-gene matrix by calculating the cosine similarity of oligonucleotide types and barcode counts between droplets. A cell-gene UMI count matrix was generated using PISA (v0.2).

[0095] 2. Removal of environmental free RNA: SoupX (v1.6.2) was used to remove environmental RNA contamination from single-cell RNA-seq data. First, a SoupChannel object was created using the original and filtered matrices. Clustering was then performed within the SoupChannel object, and the level of environmental RNA contamination was estimated using the autoEstCont function. Finally, the counts were adjusted to remove environmental RNA contamination using the adjustCounts function, and the results were rounded to integers.

[0096] 3. Doublet Detection: scDblFinder (v1.12.0) was used to detect doublets in scRNA-seq data. First, a SingleCellExperiment object was created for the raw count data of each library. Subsequently, the scDblFinder function was run on each library with the following parameters: clusters=TRUE, aggregateFeatures=TRUE, nfeatures=25, and processing="normFeatures". Doublet detection results were added to the Seurat object for further analysis.

[0097] 4. Data Quality Control: To ensure data quality, each sample was initially filtered to remove cells with UMI counts below 100 (UMI < 100). All samples were then merged into a single Seurat object, and the median and median absolute deviation (MAD) of all cells were calculated. Values that deviated from the median by more than four times the MAD were labeled as outliers. Finally, cells labeled as outliers and doublets were removed from all sample libraries.

[0098] (5) Identification of hHFDSC-specifically expressed genes:

[0099] 1. Use the Seurat software package to integrate batch effects, perform data dimensionality reduction and clustering, and use the FindMarkers function to capture highly expressed signature genes in cell populations. Select genes that meet p < 0.05 and log2 (Fold changes) > 0.5.

[0100] 2. Through gene signatures, we screened out dermal cell populations (cell populations with high expression of THY1, COL1A1, TPM1, and PDGFRA), and further identified different subtypes within the dermal cell populations. Finally, we identified (annotated) DP cells, DS cells, and hHFDSCs. The results are as follows: Figure 1 Characteristic genes of hHFDSC include multiple COL11A1, RBP4, MMP11, and SOX2, as well as highly expressed LEF1, HAPLN1, COL3A1, ASPN, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17, and TSPAN8.

[0101] (6) Spatial localization analysis of hHFDSCs:

[0102] 1. Process the raw data of spatial transcriptome sequencing and use STOmics Analysis Workflow (SAW v6.0) to align and denoise the sequencing data and generate a UMI matrix with spatial coordinates.

[0103] 2. Manually align the ssDNA staining image and the RNA grayscale image to precisely locate cells. Cell segmentation and cell labels are generated using a Gaussian weighted local threshold and Euclidean distance transform algorithm. UMIs in the DNBs are aggregated to generate a cellular gene expression matrix.

[0104] 3. Combined with the cell type annotation data of scRNA-seq in (5), the spatial position of hHFDSC was located. The technical noise introduced in the spatial transcriptome data acquisition process may affect the detection of tissue transcript expression levels, thereby affecting the identification of cell types. In addition, compared with scNRA-seq, the transcript signals of the spatial transcriptome are relatively sparse, which makes the accurate identification of cell types somewhat complex. Combined with the robust cell type decomposition (RCTD) method, the cell type annotation data of scRNA-seq in (5) was used to create a reference dataset, and the cells obtained by spatial transcriptome segmentation were labeled with cell types using a supervised learning method. The spatial expression matrix and coordinates were combined to determine the cell types of the spatial transcriptome. The results are shown in Figure 2. Figure 2 As shown, hHFDSCs are localized in the fusiform region of the hair bulb, adjacent to DP cells.

[0105] (6) Immunofluorescence verification of hHFDSC characteristic genes:

[0106] Human scalp tissue samples were fixed with 4 v / v% paraformaldehyde, embedded in paraffin, and sliced with a thickness of 7 μm. The paraffin sections were dewaxed, antigen retrieved, and blocked with 5 v / v% BSA at room temperature for 1 hour. Antibodies to hHFDSC marker genes (MMP11+RBP4, SOX2+RBP4, COL11A1+RBP4) were added to the sections and incubated overnight at 4°C. After washing three times with PBS, fluorescent secondary antibodies (see Table 1 for details) were added and incubated at room temperature for 1 hour. The cell nuclei were counterstained with 10 mg / mL Hoechst 33342 at room temperature for 15 minutes, and images were collected using an OLYMPUS FV4000 confocal microscope to analyze the characteristic gene-positive cells. The results are shown in Figure 2. Figure 3 As shown, the locations of the positive cells stained by the four antibodies are consistent with the hHFDSC positions locked in the spatial transcriptome sequencing, confirming the validity of the spatial transcriptome sequencing data and the accuracy of the hHFDSC spatial position.

[0107] (7) Analysis of differentiation potential of hHFDSCs:

[0108] 1. Based on Stereo-seq data, Dynamo was used to analyze the spatial dynamic rate of hHFDSC RNA. Dynamo calculated the ratio of uncut to cut RNA, established a gradient vector field for the transcriptome state of the cell as a continuous variable, estimated the direction and rate of dynamic changes in gene expression, and thus predicted the trend of gene expression changes in the future state of the cell and constructed a spatial lineage pseudo-time series trajectory. The results are as follows Figure 4 As shown in A, according to the Dynamo analysis results, hHFDSCs are located at the bottom of the hair follicle bulb, and the direction of change in their gene expression points to DP cells (red) and DS cells (light gray) as shown by the arrows. The hHFDSCs have a low pseudo-timing value and have the potential to differentiate into DP and DS cells.

[0109] 2. Use monocle3 to learn pseudo-sequential trajectory of scRNA-seq data. The results are as follows Figure 4 As shown in Figure B, the hHFDSC (green) population has a low pseudo-timing value, indicating it is at the starting point of the cell differentiation trajectory. As the pseudo-timing value of the cell population increases, hHFDSCs are able to differentiate into DP and DS cells. These results further verify that hHFDSCs have the potential to differentiate into DP and DS cells, confirming their stem cell characteristics.

[0110] Example 2: Isolation of human hair follicle dermal stem cells

[0111] This embodiment provides a method for isolating human hair follicle dermal stem cells, and the steps are as follows:

[0112] (1) Hair follicle acquisition

[0113] Hair follicles from the occipital region were removed from the heads of patients undergoing craniotomy or hair transplant surgery in the hospital. Figure 5 After ex vivo isolation, the hair follicles were immediately placed on ice for temporary storage to maintain cell viability. The hair follicles were then quickly transported (using cold chain transport equipment to maintain a low temperature during transportation) to the hospital laboratory for further processing. The hair follicles were washed with 1× PBS buffer and then blotted with clean tissue to prevent residual reagent from affecting the subsequent cell isolation process. Maintain aseptic technique during this step to avoid contamination of the hair follicles.

[0114] (2) Isolation of hHFDSC

[0115] 1. Hair follicle treatment: reference Figure 5 For sections B-D, the follicle bulb is first severed 1 mm from the distal end to create an entry point for subsequent cell isolation. Mechanical extrusion is then used to remove pigment and keratin from the follicle bulb, minimizing interference from non-target cells and impurities. The remaining dermal portion of the follicle after extrusion is digested with 1 mg / mL type I collagenase at 37°C for 55 minutes to achieve complete tissue dissociation while maintaining cellular integrity. During the digestion process, the mixture is gently pipetted periodically to ensure full contact between the collagenase and the dermal portion, improving digestion efficiency and ensuring complete tissue dissociation, laying the foundation for subsequent hHFDSC cell isolation and purification.

[0116] 2. Cell separation: The cell suspension obtained after digestion was centrifuged at low speed (10 g, 2 minutes) to separate the supernatant containing hHFDSCs and the pellet containing DP cell mammospheres. This low-speed centrifugation parameter can effectively separate the target cells while reducing mechanical damage to the cells. The separated supernatant was collected and filtered through a 40 μm cell sieve to remove impurities and large cell aggregates in the cell suspension, thereby improving the purity of the final cell sample. The filtered supernatant was then centrifuged at 300 g for 5 minutes to obtain purified hHFDSCs (see Figure 5 The optimization of the centrifugation step ensures the high purity and activity of hHFDSCs, providing high-quality cell samples for subsequent cell culture.

[0117] Throughout the separation process, gentle manipulation is required to avoid damaging hHFDSCs. Through the above-mentioned optimization approach, this embodiment can provide an efficient and gentle cell separation method, significantly improving the separation efficiency and cell viability of hHFDSCs.

[0118] Example 3: Identification of isolated cells

[0119] (1) Morphology, proliferation ability and activity identification

[0120] The hHFDSCs isolated in Example 2 were cultured in high-glucose DMEM medium (supplemented with 20 v / v% FBS, 1 v / v% penicillin / streptomycin, 1 v / v% non-essential amino acids (NEAA), and 1 v / v% GlutaMAX) at 37°C and 5% CO2. The medium was changed every three days to ensure adequate nutrition and growth factors for the cells, promoting cell proliferation and maintaining their activity. Sterility was maintained during medium changes to avoid contamination. These culture conditions simulate the in vivo physiological environment, promoting normal cell growth and function.

[0121] When the cell density reaches 80%, aspirate the medium, wash with PBS, and digest with 0.05 v / v% trypsin at 37°C for 3 minutes. Terminate the digestion by adding high-glucose DMEM medium containing 10 v / v% FBS. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 300 g for 3 minutes. Once a cell pellet is visible, discard the supernatant and resuspend the cell pellet in high-glucose DMEM medium (same ingredients as above). Count the cells and seed them into new culture flasks at a density of 1 × 106 cells / mL under the same culture conditions. Repeat this method for passage.

[0122] The results are as follows Figure 6 As shown, the isolated primary hHFDSCs are basically consistent with the hHFDSCs after passage in morphology, proliferation ability and activity, and the experiment is highly reproducible. Therefore, the hHFDSCs isolated in the example can be normally expanded and the cell quality is good.

[0123] (2) Identification of characteristic genes

[0124] hHFDSCs isolated from Example 2 were blocked with 5 v / v% BSA in PBS at room temperature for 1 hour. Primary antibodies against marker genes were added, incubated at 4°C overnight, and then washed to remove unbound antibodies. Fluorescent secondary antibodies were added and incubated at 4°C for 1 hour, followed by washing to remove unbound secondary antibodies. DAPI was added to counterstain the cell nuclei for 15 minutes, and images were collected using an OLYMPUS FV4000 confocal microscope to analyze cells positive for signature genes. The results are shown in Figure 2. Figure 7 As shown, co-localization expression of MMP11+COL3A1, MMP11+RBP4, COL11A1+COL1A1, and SOX2+RBP4 was detected in hHFDSCs by co-staining, respectively. Therefore, the cells isolated in Example 2 are hHFDSCs and have the expected molecular characteristics.

[0125] (3) Identification of differentiation potential

[0126] 1. DP cell differentiation potential

[0127] After the isolated hHFDSCs were cultured to P3, they were replaced with DMEM / F12 medium (supplemented with 10 v / v% FBS) and cultured for another generation. Immunofluorescence staining of AQP1 and RBP4 was performed using the same method as in (2). Figure 8 As shown in Figure 2, microscopic observation revealed that the cells became more elongated, the expression of the DP cell marker AQP1 protein was upregulated, and the expression of RBP4 was downregulated, showing in vitro differentiation characteristics. Therefore, hHFDSCs successfully differentiated into DP cells.

[0128] 2. DS cell differentiation potential

[0129] When hHFDSCs were cultured in high-glucose DMEM medium (with the same ingredients as above) to P4, and continued to be cultured for one generation, immunofluorescence staining of SMA was performed using the same method as in (2). Figure 9 As shown in the figure, the cell morphology gradually changes from slender to flat, the number of antennae increases, the stem cell characteristics begin to decline, and SMA protein begins to be expressed. Therefore, hHFDSC cells have a tendency to differentiate into DS.

[0130] Example 4

[0131] This example provides a method for isolating human hair follicle dermal stem cells, which differs from Example 2 in that the centrifugal force used during low-speed centrifugation was 20 g. Results showed that while higher centrifugal forces facilitated separation, they also damaged the sedimentation of DP cell mammospheres, hindering the integrity and viability of the mammospheres.

[0132] Comparative Example 1

[0133] This comparative example provides a method for isolating human hair follicle dermal stem cells, differing from Example 2 in that the centrifugal force used during low-speed centrifugation was 4 g. The results showed that this step made it difficult to clearly distinguish between the hHFDSC supernatant and the precipitate containing DP cell mammospheres, resulting in unsatisfactory separation.

[0134] Comparative Example 2

[0135] This comparative example provides a method for isolating human hair follicle dermal stem cells, which differs from Example 2 in that type II collagenase, type III collagenase, type IV collagenase, trypsin, or neutral protease at the same enzyme concentrations are used instead of type I collagenase during the isolation process. Type II collagenase generally has a stronger digestive effect on the extracellular matrix, while type III and type IV collagenases may be more specific for binding to collagen on the cell surface. Trypsin primarily acts on proteins on the cell membrane, while neutral protease has a broader substrate specificity. The results showed that the isolation effect of hHFDSCs was affected when these alternative enzymes were used for digestion. Compared with type I collagenase, these enzymes may cause excessive digestion of the extracellular matrix and cell membrane, thereby damaging the integrity of hHFDSCs and affecting their vitality and subsequent function.

[0136] Comparative Example 3

[0137] This comparative example provides a method for isolating human hair follicle dermal stem cells. This method differs from Example 2 in that the concentration of type I collagenase was adjusted to 0.5 mg / mL (low concentration) and 2 mg / mL (high concentration). Results showed that low-concentration collagenase had insufficient digestion capacity, resulting in incomplete separation of hHFDSCs from surrounding tissue and affecting cell purity. While high-concentration collagenase enhanced digestion capacity, it may have disrupted the hHFDSC structure due to excessive digestion, reducing cell viability.

[0138] Comparative Example 4

[0139] This comparative example provides a method for isolating human hair follicle dermal stem cells. This method differs from Example 2 in that the digestion temperature is adjusted to 32°C (low temperature) and 42°C (high temperature). Results show that low temperatures reduce collagenase activity, resulting in slow digestion and poor isolation. High temperatures, while accelerating digestion, may cause thermal damage to hHFDSCs, affecting cell activity and function.

[0140] Comparative Example 5

[0141] This comparative example provides a method for isolating human hair follicle dermal stem cells. This method differs from Example 2 in that the digestion time is adjusted to 45 minutes (short) and 65 minutes (long). Results show that short digestion times are insufficient for fully isolating hHFDSCs, resulting in low purity; whereas long digestion times may lead to overdigestion, decreased cell viability, and even cell death.

[0142] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Identify markers of human hair follicle dermal stem cells, characterized by: The markers include COL11A1, RBP4, MMP11, SOX2, COL3A1, ASPN, LEF1, HAPLN1, POSTN, MME, TNMD, DPEP1, DKK3, TMEM119, CPXM2, SFRP1, LTBP2, SMOC2, COL8A2, FGFBP2, LAMC3, FAM180A, COL15A1, F2RL2, LRRC17 and TSPAN8.

2. The marker detection kit according to claim 1, characterized in that: The detection kit includes a molecule that specifically recognizes the marker.

3. The detection kit according to claim 2, characterized in that: The molecule includes at least one of an antibody, a primer, and a probe.

4. The detection kit according to claim 2, characterized in that: The molecule is coupled to a detectable label.

5. A method for identifying human hair follicle dermal stem cells, characterized in that: Detect whether the marker according to claim 1 is highly expressed in the cells to be identified.

6. A method for screening human hair follicle dermal stem cells, characterized by: The method comprises screening a cell population for cells that highly express the marker of claim 1.

7. The method according to claim 6, characterized in that: The cell population is derived from hair follicles.

8. Use of the detection kit according to any one of claims 2 to 4 in the preparation of products for identifying, screening and preparing human hair follicle dermal stem cells.

9. A method for producing human hair follicle dermal stem cells, characterized in that: The method comprises obtaining and culturing human hair follicle dermal stem cells according to any one of the methods of claims 6 to 7.

10. Use of the human hair follicle dermal stem cells obtained by the method according to any one of claims 6 to 7 in the preparation of products for reconstructing hair follicles and promoting hair growth.

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