Identification and application of porcine embryonic hair follicle placode progenitor cells

Abstract

The present invention relates to a method for identifying porcine embryonic hair follicle placode progenitor cells and its application, using BMP7 and TGFβ2 as marker genes for porcine embryonic hair follicle placode progenitor cells, the gene number of BMP7 in the NCBI database is 492315, and the gene number of TGFβ2 in the NCBI database is 397084, and the identification method is to immunofluorescently stain epidermal cells of a porcine embryo, and the cells in which BMP7 and TGFβ2 can be simultaneously detected are hair follicle placode progenitor cells. The porcine embryo is an embryo in which the porcine embryonic stage is 37 days or earlier. The present invention provides important marker genes BMP7 and TGFβ2 for identifying porcine embryonic hair follicle placode progenitor cells, and uses cell immunofluorescence detection technology to detect the expression of important marker genes in early skin cells of a porcine embryo, to accurately and efficiently select hair follicle placode progenitor cells, and provides a porcine hair follicle-related cell line that is valuable for research into hair follicle growth and regeneration.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for identifying porcine embryonic hair follicle placode progenitor cells and applications thereof. [Background technology]

[0002] Hereditary alopecia and thinning hair are of great interest to humans, and the morphological and functional stability of hair follicles (HFs) is key to maintaining a normal hair cycle. Hair follicles are composed of multiple cell types, including keratinocytes, hair follicle stem cells, melanocytes, basal cells, fibroblasts, and mesenchymal cells. Their structure is complex, and their genetic mechanisms have yet to be fully elucidated. Mammalian hair follicle development can be divided into prenatal (embryonic) hair follicle morphological growth and postnatal cyclical development. Embryonic hair follicle development occurs through interactions between epithelial and stromal cells. Analyzing embryonic hair follicle morphological growth is key to understanding hair follicle development and complex hair follicle-related diseases. During the early mammalian embryonic stage, signals from the dermis are thought to be the initial trigger for epidermal keratinocytes to rapidly expand in an upright position. Morphologically, this triggers the aggregation, proliferation, and growth of local epithelial cells into the dermis, forming a hair follicle placode (PC). The formation of the hair follicle placode is an important morphological marker of hair follicle growth initiation. It determines whether the hair follicle can be generated and developed normally, and the number of hair follicle placodes determines the density of postnatal hair. However, the cellular and molecular dynamics that occur before hair follicle placode formation have not been clearly defined. The origin of the hair follicle placode progenitor cells and the signals that trigger the migration and proliferation of hair follicle placode cells are still unknown and require further investigation.

[0003] The scarcity and ethical concerns of human hair follicle samples, especially those from embryonic stages, have limited in-depth research into the morphology and developmental mechanisms of embryonic hair follicle placodes. Pigs are increasingly attracting attention as a viable model for human hair follicle development. Pig and human genomes share a high degree of genetic homology, with genomes similar to those of humans, exhibiting similarities in complexity and chromosomal composition. Human and pig skin also share high similarities in anatomy and physiology. Our previous studies explored the morphological development of pig hair follicles and found that compared with mice, pigs share greater similarities with humans in terms of hair follicle type and morphogenesis. However, the signaling interactions between key cell subtypes in pig hair follicle placode formation remain unknown, and there are currently no reports on marker genes for pig embryonic hair follicle placode progenitors. Summary of the Invention [Means for solving the problem]

[0004] To solve the above problems, the present invention proposes a method for identifying porcine embryonic hair follicle placode progenitor cells, and aims to provide a reliable marker gene for porcine hair follicle placode progenitor cells, which can be applied to related research on hair follicle development.

[0005] The method for identifying porcine embryonic hair follicle placode progenitor cells provided by the present invention uses BMP7 and TGFβ2 as marker genes for porcine embryonic hair follicle placode progenitor cells. The gene number of BMP7 in the NCBI database is 492315, and the gene number of TGFβ2 in the NCBI database is 397084. The identification method involves immunofluorescent staining of porcine embryonic epidermal cells, and cells in which BMP7 and TGFβ2 can be simultaneously detected are hair follicle placode progenitor cells.

[0006] For the gene sequences of BMP7 (Gene ID: 492315) and TGFβ2 (Gene ID: 397084), please refer to the Sscrofa11.1 reference genome (ftp: / / ftp.ensembl.org / pub / release-95 / fasta / sus_scrofa / ).

[0007] Furthermore, the pig embryo is an early pig embryo in the critical period of hair follicle growth, and mainly refers to an embryo before day 37 of the pig embryonic stage.

[0008] The method for identifying porcine embryonic hair follicle placode progenitor cells of the present invention is applied to the study of hair follicle development. [Effects of the Invention]

[0009] First, the present invention uses bioinformatics and molecular testing to analyze hair follicle growth cells in the early skin of porcine embryos to clarify the origin of the primordial cells of porcine embryonic hair follicle growth, select key markers for expression detection, and accurately and efficiently select the key marker genes BMP7 and TGFβ2 of hair follicle placode progenitor cells, providing a method for selecting key markers of porcine hair follicle placode progenitor cells that are valuable for research into hair follicle growth and regeneration. Second, the present invention provides the key marker genes BMP7 and TGFβ2 for identifying porcine embryonic hair follicle placode progenitor cells. Using cell immunofluorescence detection technology, the expression of these key marker genes can be detected in early skin cells of porcine embryos, enabling accurate and efficient selection of hair follicle placode progenitor cells and providing a porcine hair follicle-related cell line that is valuable for research into hair follicle growth and regeneration. Third, in our preliminary study, we used single-cell and spatial transcriptome data to evaluate hair follicle morphogenesis in normal and hairless pigs at different stages (pre-initiation, induction, organogenesis, and cell differentiation). We identified various hair follicle-associated cell subtypes and analyzed the spatiotemporal spectrum of gene expression during hair follicle morphogenesis, revealing the origin of progenitor cells, cell signaling, and transcriptional regulatory networks involved in hair follicle placode generation. Based on these analyses, combined with cell experiments, we proposed that the optimal timing for identifying porcine embryonic hair follicle placode progenitors using BMP7 and TGFβ2 as marker genes is embryonic day 37 or earlier. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of phenotyping of porcine embryo samples and production of single cell samples. [Figure 2] FIG. 1 is a schematic diagram of cell clusters in the epidermis of a porcine embryo. [Figure 3] Cell ratio analysis of epidermal growth trajectories. [Figure 4] This is a diagram showing the cell type composition of each state in the cell trajectory. [Figure 5] 1 shows migratory cells in Example 2. [Figure 6] This is the differentiation trajectory of hair follicle placode progenitor cells. [Figure 7] Dynamic gene expression during hair follicle placode progenitor cell differentiation. [Figure 8] Key gene expression of hair follicle placode progenitor cell differentiation trajectory. [Figure 9] This is detection of the expression of hair follicle placode progenitor cell marker genes at E37. [Figure 10] 1 shows the expression detection of the TC2 key gene in E41 described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be further described below with reference to examples.

[0012] Example 1: Isolation and timing selection of porcine embryonic hair follicle placode progenitor cells Utilizing the hairless pig resources established in the previous phase of the research group, breeding of hairless male pigs and hairless female pigs (four breeding pairs) will be used to ensure that the genetic background is the same and that trait segregation occurs in the offspring of the same pair.

[0013] Studies have shown that pig embryos are in the pre-initiation stage of hair follicle growth at embryonic day 37 (E37), enter the induction phase at embryonic day 41 (E41), enter the organogenesis phase at embryonic day 52 (E52), and enter the cell differentiation phase at embryonic day 85 (E85). Therefore, at each of these four time points, one pregnant sow was aborted with a drug, and skin samples were collected from the pig embryos in the litter. Phenotyping was performed using a hematoxylin-eosin staining kit (HE) to identify hairless or normal fetuses. Embryos with fewer than one hair follicle per cm2 were classified as hairless (H), while embryos with more than four hair follicles per cm2 were classified as normal (N). Embryos with no initiation of hair follicle growth at E37 and lacking the characteristic structure of hair follicles were classified as indeterminate (U).

[0014] After phenotyping, one embryonic skin sample was collected on E37, and one hairless and one hairy pig embryo sample were collected on E41, E52, and E85, respectively, resulting in a total of seven embryonic skin samples at four time points, which were used for single-cell RNA sequencing and spatial RNA sequencing (Figure 1).

[0015] As can be seen from Figure 1, at day 41 of the pig embryos, haired (normal pig) samples and hairless pig samples can be distinguished by phenotypic identification. At day 37 of the pig embryos, hair follicle cell growth has not yet been observed and differentiation is not possible. Therefore, it was explained that the optimal timing for isolating pig embryonic hair follicle placode progenitor cells is at or before day 37 of the pig embryo.

[0016] (Example 2) Single-cell RNA sequencing and screening of candidate genes in hair follicle placode progenitor cells Single-cell suspensions were prepared from seven embryonic skin samples obtained at four time points in Example 1 and subjected to single-cell RNA sequencing. Analysis of the sequencing results for these samples yielded a total of approximately 18,000 genes. Cells from haired and hairless embryo samples were first clustered based on the top 2,000 highly variable genes, and cell type annotation was performed based on the expression of classical cell-type markers in the cells. Because hair follicle morphology occurs within the epidermal structure, the resulting epidermal-type cells were extracted and subclustered, and then further divided into nine cell subsets (see Figure 2). In Figure 2, numbers 0-8 represent the nine cell subsets, of which 0 is interfollicular basal cells, 1 is epidermal cells, 2 is mitotically active cells, 3 is hair follicle bulge cells, 4 is BMP7+ / TGFβ2+ cells, 5 is keratinocytes, 6 is progenitor cells (stromal / hair follicle stem cells), 7 is interfollicular granulosa cells, and 8 is keratinocytes (sebaceous gland cells).

[0017] Subgroup differential expression analysis was performed using Seurat software, and genes with subgroup-specific expression were identified using the "FindAllMarkers" function (AdjP-value < 0.05, detection method: Wilcoxon Rank Sum test). Cell type annotation was then completed by first calculating the genes with significant subgroup-specific expression and then screening within this range to determine which genes are involved in skin and hair follicle development. As can be seen in Figure 2, cell subgroup 1 did not express hair follicle-related markers and was labeled as epidermal cells. Cell subgroup 4 was identified as having specifically high expression of BMP7 and TGFβ2, and was designated as BMP7+ / TGFβ2+ cells or B / T cells.

[0018] Pseudo-time series analysis was used to construct epidermal growth trajectories and divide them into five growth states (states 1-5). Cell ratio analysis was used to statistically analyze the sample composition of each state, revealing that embryonic day 37 cells (E37U) are primarily located in state 1, embryonic day 85 cells (E85N and E85H) are primarily located in state 5, and embryonic day 41 and 52 cells (E41N, E41H, E52N, and E52H) are relatively located in the center of the trajectory (Figure 3). The results show that the differentiation direction of these cells can be categorized into two cell lineages: hair follicle growth and epidermal growth, from state 1 to state 5. Hair follicle growth includes states 2 and 4, and epidermal growth is state 5. At the same time, we found that both hair follicle placodes (PCs) and epidermal growth originate from state 1.

[0019] Subgroup differential expression analysis was performed using Seurat software, and subgroup-specific expression genes were identified using the "FindAllMarkers" function. Cell type annotation was then completed by first calculating the genes with significant subgroup-specific expression and then screening within this range, while also considering which genes are involved in the development of skin and hair follicles. Finally, cell subgroup 4 was determined to be cells with specifically high expression of BMP7 and TGFβ2 (BMP7+ / TGFβ2+ cells or B / T cells) (AdjP-value<0.05, detection method: Wilcoxon Rank Sum test).

[0020] Pseudo-time series analysis was used to construct epidermal growth trajectories and divide them into five growth states (states 1 to 5). Cell ratio analysis was used to statistically analyze the sample composition of each state, showing that E37 cells were primarily located in state 1, E85 cells were primarily located in state 5, and E41 and E52 cells were relatively located in the center of the trajectory (Figure 3). The results show that the differentiation direction of these cells can be categorized from state 1 to state 5 into two cell lineages: hair follicle growth and epidermal growth. Hair follicle growth includes states 2 and 4, and epidermal growth is state 5. At the same time, we found that both hair follicle placodes (PCs) and epidermal growth originate from state 1.

[0021] By analyzing the cell ratios within each state, we found that the most abundant cell type in state 1 was B / T cells. Because E37 cells were primarily located in state 1, the small number of B / T cells present in other states likely represent clustering errors and are not hair follicle placode progenitors (Figure 4). Further subdivision of state 1 cells revealed that B / T cells differentiated through migratory state 1 (TC1) to TC2 or TC3 (Figure 5). We constructed pseudo-temporal trajectories to further analyze the two distinct cell fate pathways (Figure 6). Next, gene expression analysis of this differentiation process revealed that TC1→TC2 primarily expressed genes related to hair follicle placode development, while TC1→TC3 primarily expressed genes related to epidermal development (Figure 7). This suggests that the molecular regulatory mechanisms for hair follicle placode formation occur between E37 and E41, leading to differentiation along the B / T→TC1→TC2 fate, while the alternative cell differentiation trajectory, B / T→TC1→TC3, is associated with the epidermal fate (Figure 7). Furthermore, we found that genes highly expressed in B / T cells are primarily related to the TGFβ and BMP signaling pathways, stem cell proliferation / differentiation, and cell migration. The BMP and TGFβ signaling pathway is the initial signaling pathway that triggers PC formation in B / T cells. During the B / T→TC1→TC2 process, genes are primarily involved in cell migration, hair cycle regulation, and hair follicle growth. During the B / T→TC1→TC3 process, gene functions are concentrated in epidermal growth (Figure 7). Analysis of the biological processes and ligands / receptors based on highly expressed genes in B / T cells, TC1, TC2, and TC3 revealed that B / T cells are primarily involved in mesenchymal cell growth, stem cell differentiation, and the regulation of BMP and TGFβ signaling pathways. Highly expressed genes in TC2 cells are primarily involved in classical hair follicle growth signaling pathways, such as the WNT and NFkB / EDA pathways. This confirms that BMP7- and TGFβ2-labeled cells (B / T cells) are hair follicle placode progenitors (Figure 8).

[0022] Between E37 and E41, B / T cell differentiation determines future cell fate, with some B / T cells differentiating into hair follicles and others into epidermal cells. We further demonstrated that porcine embryonic hair follicle placode progenitors must be isolated before embryonic day 37. We also demonstrated that two genes, BMP7 and TGFβ2, are key triggers for differentiation.

[0023] (Example 3) Verification of candidate gene functions In the above example, we screened for BMP7TGFβ2, an important marker gene for hair follicle placode progenitor cells, and then performed morphological analysis and verified the expression of the target gene by combining spatial transcriptome analysis technology and immunofluorescence technology.

[0024] Spatial transcriptome analysis methods include: S1. Visium spatial transcriptomics was performed on E37 and E41 embryos obtained in Example 1: Skin samples were isolated using aseptic technique and placed in refrigerated sterile Hank's balanced salt solution. Fresh tissue was then immediately embedded in OCT blocks and frozen in liquid nitrogen-cooled isopentane. S2. Library Preparation: OCT was sectioned into 10 μm thick, 6.5 mm x 6.5 mm sections, three sections per sample, one per capture area, and placed on a single glass slide. These sections were stained with hematoxylin and eosin (H&E) according to the 10x Genomics fresh-frozen tissue processing procedure. After imaging the HE-stained skin tissue sections using a Zeiss Palm microbeam laser cap, tissue removal and library generation for the slides were performed according to the scheme established by 10x Genomics. The optimal membrane penetration time for 10 μm thick pig skin sections was 12 min. S3, Raw sequencing data processing: We process each sequencing spatial transcriptome library using 10x Genome's Space Ranger software (version 1.2.2), compare it with the Sscrofa11.1 reference genome, and compile the UMI counts for each spot to generate a UMI count matrix. S4. Spot Identification and Annotation: After the quality control described above, spatial transcriptome data were processed and further analyzed using Seurat. 2,000 highly variable genes were screened using the "VST" selection method. Subsequently, principal component analysis (PCA) was performed to reduce the dimensionality of the data to the top 20 principal components. Spots were clustered using the SNN algorithm and visualized in UMAP space. The resulting spot clusters represent anatomical regions in the tissue. The spots captured by ST were classified into different cell types covering the corresponding tissue regions, where the spatial layout of epidermal and dermal cell types matched their known anatomical locations. This resulted in the spatial spectrum of E37 (Figure 9A) and E41 (E41N / E41H) (Figure 10A, Figure 10B) epidermal and dermal cell subtypes. S5: Cell2location deconvolution was performed to determine the abundance of different cell states in ST samples and thereby determine the spatial localization of B / T cell differentiation. B / T cells were first detected in the E37 ST sample (Figure 9B). TC1 cells were not significantly different between E41 normal embryo (E41N) and hairless embryo (E41H) samples (Figure 10B). However, PC (TC2) cells were detected in the epidermis of the normal pig sample E41N, but were significantly reduced in the epidermis of the hairless pig sample E41H (Figure 10B). This suggests that in normal pigs, B / T cells differentiate into hair follicle placode PC (TC2) cells, and the aggregation of sufficient numbers of PC cells in the epidermis is the basis for hair follicle placode formation. The hair follicle placode is an early structural feature observable during hair follicle morphogenesis and indicates the initiation of hair follicle formation. We showed that hairless pig B / T cells produced few or no TC2 cells, indicating that the process by which hair follicle placode progenitor cells develop into normal hair follicles is inhibited in hairless pig samples. S6, tissue immunofluorescence detection of significant genes (1) The E37 and E41 embryos obtained in Example 1 were subjected to paraffin sectioning, and the sections were left in an incubator at 60°C for 1 hour. (2) Add 100% xylene (I → II → III) in order, 10 min each time, for a total of 30 min. (3) Ethanol concentrations of 100%, 95%, and 80% (I → II → III) were added in order, 10 min each time, for a total of 30 min. (4) Wash with running water for 5 minutes. (5) PBS wash once (10-15 min), (6) Fill the section box with antigen repair solution, cover it, and microwave on high heat for 5 minutes, then on low heat for 20 minutes. (7) The box containing the sections was removed and allowed to cool naturally for about 1 hour. (8) PBS was washed three times for 5 min each time. (9) Add PBS to the incubation box, dry the slides (be careful not to rub them against the tissue), place them flat in the humidity chamber, add 5% sheep serum (the specific amount depends on the number of slides), and use a pipette gun to draw up and drop the liquid onto the tissue. Each tissue requires about 30 μL of liquid, and the sheep serum must completely cover the tissue and keep it moist throughout the entire process. Then, place the slides in the humidity chamber and leave them at room temperature for 30 minutes. (10) Primary antibody: Two E37U samples were taken, the sheep serum on the slides was allowed to dry, and the liquid residue around the tissue was wiped off with clean paper. Then, BMP7 + KRT14 (epidermal marker) double-labeled and TGFβ2 + KRT14 double-labeled samples were used, respectively. (BMP7 antibody was purchased from Abmart, PA1101, a rabbit polyclonal antibody to BMP7, diluted 1:200; TGFβ2 antibody was purchased from Abmart, PA 2154 S, a rabbit polyclonal antibody to TGFβ2, diluted 1:200; KRT14 antibody was purchased from Santa, SC-53253, diluted 1:200). The primary antibodies were diluted in PBS and applied to the slides until they completely covered the tissue. The slides with the primary antibodies were placed in a humidity chamber and left overnight at 4°C. Similarly, E41N and E41H samples were selected and the expression of key TC2 genes (wnt5a and wnt10b) was verified (wnt5a was purchased from abmart, product number T 56869, and wnt10b was purchased from abmart, product number TD9038). (11) Secondary antibody: Goat anti-mouse IgG (H+L) labeled with Alexa Fluor 488 (green fluorescence) was mixed and diluted 1:500 with goat anti-rabbit IgG (H+L) labeled with Alexa Fluor 647 (red fluorescence), and then dropped onto the tissue. The sheet was placed in a humidity chamber and left at room temperature for 30 minutes. All operations after the start of secondary antibody application were performed in a light-shielded environment. (12) PBS wash 3 times, 5 min each time (13) The liquid around the tissue was wiped off, and ready-to-use DAPI was added and incubated for 2 min. (14) PBS wash 3 times, 5 min each time (15) Sealing the slide: Wipe off any liquid around the tissue, apply a drop of anti-fluorescence-attenuating sealant to the tissue, and then cover with a cover glass. Because the cover glass is slippery, you can apply nail polish around the cover glass to secure it in place. (16) Immediately after staining, the slides were photographed using a confocal microscope and analyzed. As shown in Figure 9C, BMP7+ / TGFβ2+ cells were detected in the E37 sample, consistent with the single-cell analysis and spatial transcriptome results. At the same time, the number of TC2 cells detected in the epidermis of the normal pig sample E41N was significantly higher than that in the epidermis of the hairless pig sample E41H (Figure 10C), consistent with the spatial transcriptome results.

[0025] These experiments further demonstrated that BMP7 and TGFβ2 specifically expressed in the epidermis of porcine embryos on day 37, before the hair follicle growth induction phase, are hair follicle placode progenitor cells. As cells grow, these progenitor cells differentiate toward two cell fates. In normal pigs, these progenitor cells are controlled by related factors and primarily differentiate into hair follicle placode, i.e., PC (TC2), cells. Therefore, BMP7 / TGFβ2 can be used as important screening markers for hair follicle placode progenitor cells.

[0026] This invention uses single-cell transcriptome data to evaluate hair follicle morphogenesis at different stages in normal and hairless pigs, identify the subtypes of various hair follicle-related cells, analyze the origin of the progenitor cells involved in hair follicle placode formation and related markers, and validate these markers in combination with morphological detection to discover important marker genes for porcine embryonic hair follicle placode progenitor cells, which can be applied to mammalian hair growth research.

[0027] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments set forth above, and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Accordingly, the embodiments are to be considered in all respects as illustrative and not limiting, and the scope of the present invention is limited not by the foregoing description but by the appended claims, and it is intended to cover within this invention all changes that come within the meaning and range of equivalent elements of the claims.

[0028] Furthermore, although this specification is described according to embodiments, each embodiment does not include only one independent technical solution, and such description form of this specification is merely for clarity, and those skilled in the art should understand that the specification must be considered as a whole, and the technical solutions of each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

[Claim 1] A method for identifying porcine embryonic hair follicle placode progenitor cells, comprising: A method for identifying porcine embryonic hair follicle placode progenitor cells, characterized in that BMP7 and TGFβ2 are used as marker genes for porcine embryonic hair follicle placode progenitor cells, the gene number of BMP7 in the NCBI database is 492315, and the gene number of TGFβ2 in the NCBI database is 397084, the identification method comprises immunofluorescently staining epidermal cells of a porcine embryo, and cells in which BMP7 and TGFβ2 can be simultaneously detected are hair follicle placode progenitor cells, and the porcine embryo is an embryo that is 37 days old at the porcine embryo stage.

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