A method for preparing hair follicle stem cells

Abstract

This invention belongs to the field of stem cell technology and regenerative medicine, specifically relating to a method for preparing hair follicle stem cells. The method includes preparing induced pluripotent stem cells (iPSCs) from urine cells, culturing the iPSCs in an epidermal induction medium to obtain epidermal progenitor cells, and then culturing the epidermal progenitor cells in a hair follicle stem cell induction medium to obtain hair follicle stem cells. This invention utilizes transcriptome sequencing of iPSCs, embryonic stem cells, and hair follicle stem cells to analyze differential gene expression, screening for several cytokines that are significantly differentially expressed in hair follicle stem cells. These cytokines are then combined in different ways to formulate hair follicle stem cell induction medium for the directed differentiation of iPSCs into hair follicle stem cells, ultimately resulting in a hair follicle stem cell induction medium formulation that can significantly improve the induction efficiency of hair follicle stem cells.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology and regenerative medicine, and specifically relates to a method for preparing hair follicle stem cells. Background Technology

[0002] Hair follicle stem cells are a type of epidermal stem cell found in the bulge region of hair follicles. They possess the ability to self-renew, differentiate into multiple lineages, and participate in hair regeneration. In recent years, hair follicle stem cells have shown promising applications in skin wound repair, hair regeneration, and re-epithelialization of biomaterials, becoming a hot topic in skin regeneration and tissue engineering research.

[0003] Currently, the main methods for obtaining hair follicle stem cells include: directly isolating primary HFSCs from animal or human tissues, which is cumbersome and resource-limited; inducing HFSCs from adult epidermal stem cells, but the expansion capacity and differentiation potential are limited; and differentiating hair follicle stem cells from induced pluripotent stem cells (iPSCs), which provides a new source.

[0004] However, existing methods for differentiating iPSCs into HFSCs still suffer from problems such as low induction efficiency, complex operation, unstable biomarker expression, and long differentiation time, which limit their clinical translation and industrialization. Summary of the Invention

[0005] Based on this, the present invention provides a method for preparing hair follicle stem cells with improved induction efficiency. By optimizing the induction system and simulating the microenvironment of hair follicle development, iPSCs are guided to differentiate into HFSCs in stages, thereby obtaining a cell population with typical HFSC phenotypes and functions, providing a reliable cell source and technical support for hair loss treatment, skin tissue engineering, and regenerative medicine.

[0006] The technical solution of the present invention:

[0007] This invention provides a method for preparing hair follicle stem cells, which includes the following steps:

[0008] (1) Collect urine cells;

[0009] (2) Urine cell reprogramming: After the reprogramming inducing factor is introduced into urine cells, they are cultured in a culture medium to obtain induced pluripotent stem cells;

[0010] (3) Differentiation of epidermal progenitor cells: Induced pluripotent stem cells were cultured in epidermal induction medium to obtain epidermal progenitor cells;

[0011] (4) Hair follicle stem cell differentiation: Epidermal progenitor cells were cultured in hair follicle stem cell induction medium to obtain hair follicle stem cells.

[0012] Furthermore, the method for collecting urine cells is as follows: Collect midstream urine, add 100x penicillin antibody P / S, centrifuge and discard the supernatant, then resuspend in PBS solution containing 50x penicillin antibody P / S, centrifuge again and discard the supernatant, add urine cell culture medium containing 1x penicillin antibody P / S and resuspend, and culture at 37℃ and 5% CO2 to obtain urine cells.

[0013] Furthermore, the urine cell culture medium is REGM medium.

[0014] Further, the reprogramming induction factor in step (2) is selected from one or more of OCT4, SOX2, NANOG, KLF4 and LIN28.

[0015] Further, the method for reprogramming urine cells in step (2) is as follows: after introducing the reprogramming inducing factor into the urine cells, they are cultured in a reprogramming culture medium. After ES-like clones appear, the culture medium is replaced with pluripotent stem cell culture medium.

[0016] Furthermore, the pluripotent stem cell culture medium is E8 medium or mTeSR1 medium.

[0017] Further, the epidermal induction culture medium in step (3) comprises: DMEM / F12, 10% KOSR, 50 ng / mL SHH, and 5 μM CHIR99021.

[0018] Further, the hair follicle stem cell induction culture medium in step (4) contains: DMEM / F12, 2% B27, 3 μM CHIR99021, 0.5 μM Purmorphamine (SHH activator), 150 ng / mL Wnt10b, 20 ng / mL FGF10, 20 ng / mL SHH, 100 ng / mL Noggin, and 50 ng / mL IGF-1.

[0019] Furthermore, the preparation method also includes culturing the obtained hair follicle stem cells in a maintenance culture medium.

[0020] Furthermore, the maintenance culture medium comprises: DMEM basal medium, FBS, 0.001–0.1% T-β, 1–100 ng / mL hEGF, 1–100 ng / mL VEGF, and 1–10 mmol / L L-glutamine; wherein the volume ratio of DMEM basal medium to FBS is 9:1.

[0021] Beneficial effects of the invention

[0022] This invention analyzes differential gene expression by performing transcriptome sequencing on induced pluripotent stem cells, embryonic stem cells, and hair follicle stem cells. Several cytokines that are significantly differentially expressed in hair follicle stem cells are screened and formulated into hair follicle stem cell induction culture media in different combinations to direct the differentiation of induced pluripotent stem cells into hair follicle stem cells. Finally, a hair follicle stem cell induction culture medium formula that can significantly improve the induction efficiency of hair follicle stem cells is obtained. Attached Figure Description

[0023] Figure 1 The image shows a volcano plot illustrating the differential analysis of RNA-seq sequencing for hiPSC and HFSC in Example 1 of this invention.

[0024] Figure 2 The image shows a volcano plot illustrating the differential analysis of RNA-seq sequencing for hESC and HFSC in Example 1 of this invention.

[0025] Figure 3 Showing Figure 1 and Figure 2 Venn diagram of the intersection of two groups of differentially expressed genes.

[0026] Figure 4 Showing Figure 3 The expression heatmap of the intersecting genes.

[0027] Figure 5 The flow cytometry data of hair follicle stem cells induced by formulation 4 in Example 2 are shown. A: CD200; B: ITGA6; C: p63; D: KRT15; E: SOX9. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0029] Example 1: Differential gene expression analysis of induced pluripotent stem cells, epidermal stem cells, and hair follicle stem cells

[0030] HiPSCs derived from resuscitated urine (Wuhan Saisios Biotechnology, SC-DRY0103) and human embryonic stem cells (hESCs) (Wuhan Saisios Biotechnology, CL-484h) were seeded into Matrigel-coated 6-well plates, mTeSR medium was added, and amplification culture was carried out in a 37°C, 5% CO2 incubator.

[0031] Resuscitated human hair follicle stem cells (HFSC) (Wuhan Shangen Biotechnology, SNP-H171) were seeded into collagen IV-coated 6-well plates, and keratinocyte system (Wuhan Shangen Biotechnology, SNPM-H171) was added. The cells were then expanded and cultured in a 37°C, 5% CO2 incubator.

[0032] When cells reached 40%–50% confluence, hiPSC, hESC, and HFSC cells were collected for transcriptome sequencing. Eukaryotic mRNA was enriched using Oligo magnetic beads, fragmented into short segments, reverse transcribed into cDNA strands, and end-repaired before ligation with sequencing adapters. Fragments of different sizes were selected by agarose gel electrophoresis, and cDNA was amplified and enriched by polymerase chain reaction (PCR) to complete library preparation. Paired-end sequencing was performed using an Illumina HiSeq2500. Sequencing was performed three times independently for each cell line.

[0033] Sequencing data was filtered to remove adapters, contaminants, and low-quality sequences. Hisat2 was used to match high-quality sequences to a reference genome. FeatureCounts software was used for gene-level quantitative analysis, calculating the read count for each gene in each sample. After standardization, hypothesis testing probabilities (P-values) were calculated based on the model, and multiple hypothesis testing corrections were applied to obtain adjusted P-values. DEGs were screened based on the criteria of |fold change| > 2.0 and P < 0.05.

[0034] The results are as follows Figure 1-4 As shown, hiPSC vs. HFSC yields 751 DEGs ( Figure 1 hESC vs. HFSC yielded 636 DEGs ( Figure 2 The intersection of the two gene groups yielded 107 DEGs. Figure 3 ), Figure 4 This is a heatmap of gene expression at intersection.

[0035] Since both hiPSCs and hESCs cultured in vitro can differentiate into HFSCs under specific induction conditions, these overlapping genes may play an important role in the induction of HFSC differentiation.

[0036] This invention further analyzed a list of 107 DEGs, among which several cytokine expression genes were upregulated in both hiPSC vs. HFSC and hESC vs. HFSC, namely Wnt3a, Wnt10b, FGF2, FGF10, RA (retinoic acid), SHH (Sonic Hedgehog), Noggin (BMP antagonist), EGF, and IGF-1.

[0037] Example 2: Induction of hair follicle stem cell differentiation

[0038] Next, as shown in Table 1, the above cytokines were combined to prepare hair follicle stem cell induction culture medium, and the hair follicle stem cell induction culture medium with the highest induction efficiency was explored.

[0039] Table 1: Formula for Hair Follicle Stem Cell Induction Culture Medium

[0040] Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 DMEM / F12 + + + + + + + 2%B27 + + + + + + + 3 μM CHIR99021 + + + + + + + 0.5 μM Purmorphamine (SHH activator) + + + + + + + 150 ng / mL Wnt3a + - + - - - - 150 ng / mL Wnt10b + + - + + + + 10 ng / mL FGF2 + - - - - - - 20 ng / mL FGF10 + + + + + + + 200nM RA + + + - - - - 20 ng / mL SHH + + + - - + 100 ng / mL Noggin + + + + - + + 15 ng / mL EGF + + + - - - - 50 ng / mL IGF-1 + + + + + + -

[0041] The following experimental protocol was used to induce differentiation of hair follicle stem cells:

[0042] (1) Prepare a 250mL sterile collection bottle, add 100x of penicillin-peptide P / S (Gibco, catalog number 15140-122) in advance, collect about 200mL of midstream urine, aliquot the urine into 4 50mL sterile centrifuge tubes, centrifuge at 400g for 10 minutes at room temperature. Aspirate the supernatant and keep 1-2mL, then combine the solutions from the 4 tubes into 1 tube, add 40mL of PBS solution containing 50x penicillin-peptide P / S to resuspend, mix and wash, centrifuge at 400g for 10 minutes at room temperature, aspirate the supernatant and keep 0.5-1mL. Resuspend the pellet in 1mL of urine cell culture medium containing 1x P / S penicillin-peptide (REGM:10% FBS=1:1), add it to a 6-well plate pre-coated with gelatin, add 1mL of the above urine cell culture medium, and incubate at 37℃ in a 5% constant temperature incubator. After the urine cells adhere to the culture medium, aspirate the culture medium, wash with PBS, and then change the medium. When the urine cells reach 80% confluence, they can be passaged.

[0043] (2) Non-integrative attachment vectors expressing transcriptional regulatory factors OCT4, SOX2, NANOG, KLF4 and LIN28 (pEP4EO2SEN2K: 3.0 μg, pEP4EO2SET2K: 3.2 μg, pCEP4-M2L: 2.4 μg) were introduced into 1 million urocytes. The cells were then divided into three 10 cm plates pre-coated with Matrigel and cultured in reprogrammed medium (CIB, Cat#Rep-1102). The medium was changed every other day until ES-like clones appeared. The clones were then picked out, purified and expanded, and cultured in ECM (Sigma) coated culture plates in mTeSR1 (StemCell) medium. The medium was changed daily to obtain induced pluripotent stem cells.

[0044] (3) Set the iPSC to 5×10 4Epidermal progenitor cells were seeded at a density of 10 cells / cm² in collagen IV-coated 6-well plates and cultured in mTeSR1 + 10 μM Y-27632 medium. After 24 hours, the medium was replaced with epidermal induction medium (DMEM / F12 + 10% KOSR + 50 ng / mL SHH + 5 μM CHIR99021) and cultured for 7 days with daily medium changes. Successful differentiation of epidermal progenitor cells was verified by KRT14 immunostaining (>70% positive was considered acceptable).

[0045] (4) Epidermal progenitor cells were prepared at a concentration of 1×10⁻⁶. 5 10 cells / cm² were seeded in Matrigel (1:100 dilution) coated plates and cultured for 10 days, with the medium changed every 2 days.

[0046] (5) The obtained cells were cultured in maintenance medium (450 mL DMEM basal medium + 50 mL FBS + 0.1% T-β + 100 ng / mL hEGF + 1 ng / mL VEGF + 10 mmol / L L-glutamine).

[0047] Example 3: Flow cytometry detection of surface markers of hair follicle stem cells

[0048] (1) 0.25% trypsin digestion. Example 2: Cells were prepared using different induction culture medium formulations and the cells were collected to make the cell number 2×10. 5 ;

[0049] (2) The cells were resuspended in 1 mL of DPBS and washed twice, then centrifuged at 200 g for 5 min.

[0050] (3) Add 1 mL of 70% pre-cooled alcohol to each sample tube for resuspending and fixation for 2 h;

[0051] (4) Centrifuge and discard the fixative, add 200 μL of mouse CD200, ITGA6, p63, KRT15, ​​and SOX9 primary antibody (dilution of 1:100) and incubate at room temperature for 30 min;

[0052] (5) After washing with 1 mL of PBS, incubate with FITC-labeled secondary antibody (dilution of 1:200) at room temperature in the dark for 1 h.

[0053] (6) After washing twice with PBS, discard the PBS, add an appropriate amount of PBS to resuspend the cells according to the cell volume, and detect them using a flow cytometer.

[0054] CD200+ITGA6+p63+KRT15+SOX9+ were used as indicators for identifying positive hair follicle stem cells. The positive rate of hair follicle stem cells in cells prepared using different induction culture medium formulations in Example 2 was detected, and the results are shown in Table 2.

[0055] Table 2: Positive rate of hair follicle stem cell induction

[0056] Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 Positive rate 89% 87% 74% 84% 42% 62% 68%

[0057] As shown in Table 2, Formula 4 is a superior formula for hair follicle stem cell induction culture medium, which can achieve a high positive induction rate with the addition of fewer cytokines. Figure 5 AE shows the positive rates of various markers detected by flow cytometry in hair follicle stem cells induced by Formula 4.

[0058] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing hair follicle stem cells, characterized in that, Includes the following steps: (1) Collect urine cells; (2) Urine cell reprogramming: After the reprogramming inducing factor is introduced into urine cells, they are cultured in a culture medium to obtain induced pluripotent stem cells; (3) Differentiation of epidermal progenitor cells: Induced pluripotent stem cells were cultured in epidermal induction medium to obtain epidermal progenitor cells; (4) Hair follicle stem cell differentiation: Epidermal progenitor cells were cultured in hair follicle stem cell induction medium to obtain hair follicle stem cells; The hair follicle stem cell induction culture medium in step (4) contains: DMEM / F12, 2% B27, 3 μM CHIR99021, 0.5 μM Urmorphamine, 150 ng / mL Wnt10b, 20 ng / mL FGF10, 20 ng / mL SHH, 100 ng / mL Noggin, and 50 ng / mL IGF-1; The epidermal induction culture medium in step (3) contains: DMEM / F12, 10% KOSR, 50 ng / mL SHH and 5 μM CHIR99021.

2. The preparation method according to claim 1, characterized in that, The method for collecting urine cells is as follows: Collect midstream urine, add 100x penicillin P / S, centrifuge and discard the supernatant, then resuspend in PBS solution containing 50x penicillin P / S, centrifuge again and discard the supernatant, add urine cell culture medium containing 1x penicillin P / S and resuspend, and culture at 37℃ and 5% CO2 to obtain urine cells.

3. The preparation method according to claim 2, characterized in that, The urine cell culture medium is REGM medium.

4. The preparation method according to claim 1, characterized in that, The reprogramming induction factor mentioned in step (2) is selected from one or more of OCT4, SOX2, NANOG, KLF4 and LIN28.

5. The preparation method according to claim 1, characterized in that, The method for reprogramming urine cells in step (2) is as follows: after introducing the reprogramming inducing factor into urine cells, they are cultured in a reprogramming culture medium. After ES-like clones appear, the culture medium is replaced with pluripotent stem cell culture medium.

6. The preparation method according to claim 5, characterized in that, The pluripotent stem cell culture medium is E8 medium or mTeSR1 medium.

7. The preparation method according to claim 1, characterized in that, This also includes culturing the obtained hair follicle stem cells in a maintenance culture medium; The maintenance culture medium includes: DMEM basal medium, FBS, 0.001-0.1% TGF-β, 1-100 ng / mL hEGF, 1-100 ng / mL VEGF and 1-10 mmol / L L-glutamine; The volume ratio of DMEM basal medium to FBS was 9:1.

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