Method for identifying porcine hair follicle bulge precursor cells and application thereof

Abstract

The present application relates to a kind of pig hair follicle matrix precursor cell identification method and its application.The present application uses OGN and UCHL1 as the marker gene of pig hair follicle matrix precursor cell, the gene number of the OGN in NCBI database is 106509723, the gene number of the UCHL1 in NCBI database is 396637, the identification method is: pig embryo epidermal cell is immunofluorescence dyeing, OGN and UCHL1 can be simultaneously detected cell is pig hair follicle matrix precursor cell, the pig embryo refers to the embryo of pig embryo period 37 days and before.This application provides the identification method of pig hair follicle matrix precursor cell, and is applied in hair follicle development research.The present application discloses that OGN and UCHL1 are more suitable as the marker gene of pig hair follicle matrix precursor cell than BMP7 and TGF beta 2.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for identifying porcine hair follicle substrate precursor cells and its application. Background Technology

[0002] Hair follicle development is a complex morphogenetic process, divided into embryonic morphological development and postnatal cyclical development. Embryonic hair follicle development involves the interaction of epithelium and mesenchyme, a complex process involving numerous molecules and signaling pathways. Induced by dermal signals, the epithelial layer thickens to form the hair follicle basal plate, marking the initial stage of hair follicle development and serving as a crucial morphological marker. The hair follicle basal plate can form almost all cell types of adult hair follicles, and its formation determines whether a hair follicle can form and develop normally. Furthermore, the number of basal plates determines the amount of hair. Much research has been conducted on events following the formation of the hair follicle basal plate due to its easy morphological observation and stable molecular markers. However, the lack of prospective molecular markers and the difficulty in distinguishing morphologies make it challenging to study the cellular origin and molecular mechanisms of the early formation of hair follicle basal plate cells. In-depth research on this process will contribute to understanding hair follicle development and provide insights into the study of human hereditary hair disorders.

[0003] In the field of hair follicle development research, mice are commonly used as a model due to their short generation intervals, synchronous hair development, ease of manipulation, and low cost. However, there are still some differences in embryonic development similarity between mice and humans. Furthermore, the difficulty in obtaining human embryos and the ethical issues they raise limit further research on embryonic hair follicle development. Pigs, as a species with very high homology to humans, share high similarities in anatomy and physiology with humans. Their embryonic development process, hair type, and morphology are highly consistent with humans, making them a suitable model animal for hair follicle development. However, research on early molecular events in hair follicle slab formation and markers of early hair follicle slab cells in pigs is scarce. Chinese Patent 202310153985.0 discloses a method for identifying porcine embryonic hair follicle slab precursor cells and its application, which is the research result of our team. It provides key gene marker genes BMP7 and TGFβ2 for identifying porcine embryonic hair follicle slab precursor cells. However, in our team's subsequent practice, we found that when screening hair follicle slab precursor cells using BMP7 and TGFβ2 as marker genes, some hair follicle slab precursor cells were missed because they did not express BMP7 and TGFβ2. Since early porcine embryos are used to screen hair follicle slab precursor cells, the sample source is relatively difficult. If there are more efficient marker genes for hair follicle slab precursor cells, the utilization efficiency of early porcine embryos will be greatly improved and more hair follicle slab precursor cells can be obtained. Summary of the Invention

[0004] To address the above issues, this invention proposes a method for identifying porcine hair follicle substrate precursor cells and its application. The aim is to provide an effective and stable marker for identifying porcine hair follicle substrate precursor cells, which can be used to track the early development process of hair follicles and explore the molecular mechanism of hair follicle substrate formation.

[0005] The method for identifying porcine hair follicle lamina precursor cells provided by this invention uses OGN and UCHL1 as marker genes for porcine hair follicle lamina precursor cells. The gene number of OGN in the NCBI database is 106509723, and the gene number of UCHL1 in the NCBI database is 396637. The identification method is as follows: porcine embryonic epidermal cells are stained with immunofluorescence. Cells that can detect both OGN and UCHL1 are porcine hair follicle lamina precursor cells. The porcine embryo refers to a porcine embryonic period of 37 days or earlier.

[0006] The gene sequences of OGN (Gene ID: 106509723) and UCHL1 (Gene ID: 396637) can be found in the Suscrofa11.1 reference genome (ftp: / / ftp.ensembl.org / pub / release-95 / fasta / sus_scrofa / ).

[0007] The method for identifying porcine hair follicle substrate precursor cells provided by this invention is applied in hair follicle development research.

[0008] The beneficial effects of this invention are as follows:

[0009] 1. This invention found that, compared with BMP7 and TGFβ2, OGN and UCHL1 were expressed at higher levels in a subset of porcine hair follicle lamina precursor cells and at lower levels in other subsets, indicating that OGN and UCHL1 are more suitable as marker genes for porcine hair follicle lamina precursor cells than BMP7 and TGFβ2. This invention combines bioinformatics and molecular biology to analyze hair follicle development-related cells at different time points during porcine embryonic development to explore the early development process of the hair follicle lamina. Specific markers were screened and detected, revealing that OGN and UCHL1 are more suitable markers, capable of confirming the presence of porcine hair follicle lamina precursor cells through co-expression, which is helpful for subsequent research into the molecular mechanisms of hair follicle lamina development.

[0010] 2. This invention provides two suitable biomarkers, OGN and UCHL1, which can be used to isolate and culture porcine hair follicle substrate precursor cells expressing the two biomarkers in the early stage of porcine embryos using flow cytometry. Based on this, a stable porcine hair follicle substrate precursor cell line can be established, providing materials for the study of the molecular mechanism of hair follicle substrate formation.

[0011] 3. In our preliminary experiments, we utilized single-cell transcriptome sequencing and spatial transcriptome sequencing to explore the process of hair follicle morphogenesis in pigs, identifying various cell subtypes related to hair follicle development and elucidating the spatiotemporal map of gene expression during hair follicle morphogenesis. We also elucidated the progenitor cell origin, cell signal transduction, and transcriptional regulatory network involved in hair follicle substrate formation. Based on the above analysis and combined with cell experiments, we propose that the optimal time to identify porcine hair follicle substrate precursor cells using OGN and UCHL1 as marker genes is 37 days or earlier in the embryonic period. Attached Figure Description

[0012] Figure 1 A schematic diagram illustrating the phenotypic identification of pig embryo samples and the preparation of single-cell samples;

[0013] Figure 2 This is a schematic diagram of cell clustering in the epidermis of a pig embryo.

[0014] Figure 3 The expression of the hair follicle substrate precursor cell marker gene in E37 was detected in Example 2.

[0015] Figure 4 This is for the detection of the expression of the TC2 key gene described in Example 2 in E41. Detailed Implementation

[0016] The present invention will be further described below with reference to the embodiments.

[0017] Example 1: Single-cell transcriptome sequencing and screening of candidate genes in early hair follicle basal cells.

[0018] The process of collecting cells from hairy and hairless pig embryo samples, clustering them, and annotating the cells based on the expression of classic cell type markers in the cells is consistent with the content disclosed in patent 202310153985.0.

[0019] Specifically, the samples used in this invention mainly come from the hairless pig genetic resource population previously constructed by our research group. Normal Large White pigs have a certain amount of hair on their backs. Our research group discovered a naturally hairless or sparsely haired population of Large White pigs. Through artificial mating experiments, we found that this hairless trait conforms to Mendelian inheritance laws and is an autosomal recessive trait. The dominant trait is normal hair, and the recessive trait is hairlessness. Heterozygotes exhibit normal hair. In the study of hair follicle basal cell precursor cells, our research group bred one heterozygous normal boar semen with four homozygous hairless sows. Then, on days 37, 41, 52, and 85, one of the sows was subjected to medical abortion, yielding more than 10 embryos from each sow (from the same litter). Our research group discovered that in normal Large White pigs, the epidermis is uniformly monolayered at 37 days of embryonic development. At 41 days, the epidermis grows into the dermis, invaging and forming the structure of a hair follicle substrate, which will develop into hair follicles and produce hair. In contrast, in hairless pigs, the epidermis does not form or has very few hair follicle substrate structures at 41 days of embryonic development. The epidermis of hairless pigs remains uniform from 41 days of embryonic development to adulthood, specifically exhibiting no hair follicle structure and no hair formation throughout their lives. Therefore, at 41 days of embryonic development and beyond, longitudinal sections of the skin can be prepared and stained with hematoxylin and eosin (HE) to identify whether the pig embryo is from a normal or hairless pig. The classification standard is based on the number of hair follicles per 1 cm. 2 Embryos are classified according to the number of hair follicles. Embryos with fewer than one hair follicle are hairless (H), and embryos with more than four hair follicles are normal (N). At day 37 of the embryonic period, hair follicle development has not yet begun in either normal or hairless pigs, and the phenotype of the pig cannot be determined; these are classified as unknown (U).

[0020] Based on this study, our research group collected one sample from littermates at 37 days of embryonic development, and one sample each from littermates of hairy and hairless pigs at 41, 52, and 85 days of embryonic development, obtaining a total of seven skin samples. We then prepared single-cell suspensions from these seven embryonic skin samples for single-cell transcriptome sequencing and spatial transcriptome sequencing (see [link to study]). Figure 1The sequencing results of the above samples were analyzed, yielding approximately 51,871 cells and about 18,000 genes. First, based on the top 2000 highly variable genes, cells from hairy and hairless embryonic samples were clustered. Cell type annotation was then performed based on the expression of classic cell type markers, resulting in six types: epidermal cells, dermal cells, peridermal cells, endothelial cells, vascular smooth muscle cells, and Schwann cells. Since the hair follicle substrate originates only from epidermal cells, after coarse classification and annotation of epidermal cell types, sub-clustering of epidermal cells was performed, further dividing them into nine cell subpopulations. Differentially identified genes and their functions were analyzed in epidermal cell subsets, and the subsets were annotated. Subset 0 consisted of interfollicular basal cells; subset 2 consisted of actively mitotic cells; subset 3 consisted of cells in the hair follicle ridge; subset 4 was considered to be precursor cells of the hair follicle basal plate; subset 5 consisted of keratinocytes; subset 6 consisted of progenitor cells (stromal / hair follicle stem cells); subset 7 consisted of interfollicular granular cells; subset 8 consisted of keratinocytes (sebaceous glands); and subset 1 did not identify any hair follicle-related markers and was labeled as epidermal 1 (see [link to relevant documentation]). Figure 2 ).

[0021] Our research group discovered that OGN and UCHL1 may be more suitable marker genes for hair follicle basal stem cell precursors than BMP7 and TGFβ2. Therefore, we used the "FindMarkers" function in Seurat software to compare the two groups of differentially expressed genes to find marker genes with stronger specificity. Here, pct.1 represents the proportion of the gene expressed in all cells of the corresponding cell subpopulation, and pct.2 represents the proportion of the gene expressed in all cells of other subpopulations. The larger the difference between pct.1 and pct.2, the more specific the gene is to a particular subpopulation. Compared to the previously discovered BMP7 and TGFβ2 genes disclosed in patent 202310153985.0, this embodiment found that the proportion of cells expressing OGN and UCHL1 (pct.1) in cell subpopulation 4 was significantly higher than that expressing BMP7 and TGFβ2. The proportion of cells expressing OGN and UCHL1 (pct.2) in all other subpopulations (excluding cell subpopulation 4) was much lower than that expressing BMP7 and TGFβ2. Furthermore, the difference between pct.1 and pct.2 for OGN and UCHL1 in cell subpopulation 4 was significantly greater than that for BMP7 and TGFβ2 (see Table 1). The results of this embodiment indicate that OGN and UCHL1 are expressed at higher levels in cell subpopulation 4 and at lower levels in other subpopulations compared to BMP7 and TGFβ2. This suggests that the OGN and UCHL1 genes are more specific and better represent the characteristics of cell subpopulation 4, indicating that OGN and UCHL1 are more suitable marker genes for porcine hair follicle lamina precursor cells.

[0022] Table 1. Comparison of differentially expressed genes in cell subset 4

[0023]

[0024] Meanwhile, although some literature mentions that OGN and UCHL1 are both related to the morphological development of hair follicles, this embodiment is the first to discover that OGN and UCHL1 are more suitable marker genes for porcine hair follicle substrate precursor cells than BMP7 and TGFβ2.

[0025] Example 2: Verification of candidate gene function

[0026] OGN and UCHL1 were identified as marker genes for hair follicle substrate precursor cells at both the spatial transcriptome and tissue protein levels.

[0027] In the spatial transcriptome analysis, the sample preparation, raw sequencing data processing, and analysis methods were consistent with those disclosed in patent 202310153985.0, the difference being that the marker genes for the mapped cells were OGN and UCHL1. Specifically:

[0028] S1. Visium spatial transcription was performed on the E37 and E41 embryos obtained in Example 1;

[0029] S2, Library preparation;

[0030] S3. Processing raw sequencing data to generate a UMI counting matrix;

[0031] S4. Spot Identification and Annotation: Following the aforementioned quality control, the spatial transcriptome data was processed and analyzed using Seurat. ST-captured spots were categorized into different cell types covering corresponding tissue regions. The spatial layout showed a thin epidermis with the dermis beneath and relatively thicker than the epidermis, consistent with actual anatomical location and morphology, thus depicting E37 (…). Figure 3 A) and E41N ( Figure 4 A) / E41( Figure 4 B) Spatial map of epidermal and dermal cell subtypes.

[0032] O / U cells were detected for the first time in E37 ST samples. Figure 3 B), and it overlaps with epidermal cells, indicating accurate localization. TC1 showed no significant difference in E41 normal embryos (E41N) and hairless embryos (E41H) samples. Figure 4 C). However, PC(TC2) cells were detected in the epidermis of normal pig sample E41N, while PC(TC2) cells were significantly reduced in the epidermis of hairless pig sample E41H. Figure 4C). This indicates that in normal pigs, O / U cells differentiate into hair follicle basal plate PC (TC2) cells, and the aggregation of a sufficient number of PC cells in the epidermis is the basis for hair follicle basal plate formation. The hair follicle basal plate is the observable initial structural feature in the hair follicle morphogenesis process, marking the beginning of hair follicle formation. However, in hairless pigs, O / U cells produce very few or no TC2 cells, indicating that the development of hair follicle basal plate precursor cells into normal hair follicles is inhibited in hairless pig samples. The overall results demonstrate the accuracy of the spatial localization of the two marker genes, OGN and UCHL1, and show high consistency with the single-cell transcriptome results, indicating that OGN and UCHL1 are suitable markers that can well reflect the characteristics of hair follicle basal plate precursor cells.

[0033] Regarding the validation of tissue protein levels, except for the selection of the primary antibody, the remaining steps are consistent with the content disclosed in patent 202310153985.0. Specifically:

[0034] (1) Paraffin sections were prepared from the E37 and E41 embryos obtained in Example 1, and the sections were placed in a constant temperature chamber at 60°C for 1 hour.

[0035] (2) Place them sequentially into xylene with a concentration of 100% (Ⅰ→Ⅱ→Ⅲ), 10 minutes each time, for a total of 30 minutes;

[0036] (3) Place them in ethanol of 100%, 95%, and 80% concentrations in sequence (Ⅰ→Ⅱ→Ⅲ), 10 min / time, for a total of 30 min;

[0037] (4) Rinse with running water for 5 minutes;

[0038] (5) Wash once with PBS (10-15 min);

[0039] (6) Pour the antigen retrieval solution into the slide box, cover it, and microwave it on high for 5 minutes, then on low for 20 minutes.

[0040] (7) Remove the box containing the slices and let it cool naturally for about an hour;

[0041] (8) Wash with PBS three times, 5 minutes each time;

[0042] (9) Place some PBS in the incubation box, wipe the slide dry (be careful not to wipe the tissue), place it flat in the humidified box, and add 5% sheep serum (the specific amount depends on the number of slides). Use a pipette to draw up the solution and drop it onto the tissue. Each tissue requires about 30 μL of liquid. Make sure the sheep serum completely covers the tissue, and keep the tissue moist throughout the process. Then place the slide in the humidified box and let it sit at room temperature for 30 minutes.

[0043] (10) Primary antibody: Take two E37U samples, shake off the sheep serum on the slides, wipe the residual liquid around the tissue with clean paper, and perform OGN+KRT14 (epidermal marker) double labeling and UCHL1+KRT14 double labeling respectively (OGN antibody purchased from Proteintech, 12755-1-AP, Rabbit polyclonal antibody to OGN, dilution ratio 1:100; UCHL1 antibody purchased from Booster, BM4990, Rabbit polyclonal antibody to UCHL1, 1:100 dilution; KRT14 antibody: purchased from Santa, sc-53253, 1:200 dilution). Dilute the primary antibody with PBS, add the diluted primary antibody and completely cover the tissue, place the slide with the added primary antibody in a humidifier box and incubate overnight at 4°C. Using the same method, samples from E41N and E41H were selected to validate the expression of important TC2 genes (wnt5a and wnt10b) (wnt5a was purchased from Abmart, catalog number T56869; wnt10b was purchased from Abmart, catalog number TD9038).

[0044] (11) Secondary antibody: Alexa Fluor 488 (green fluorescence) labeled goat anti-mouse IgG (H+L) and Alexa Fluor 647 (red fluorescence) labeled goat anti-rabbit IgG (H+L) were mixed and diluted at a ratio of 1:500 and then dropped onto the tissue. The slide was placed in a humidified chamber and left at room temperature for 30 minutes. All operations after the application of the secondary antibody were carried out in a dark environment.

[0045] (12) Wash 3 times with PBS, 5 min each time;

[0046] (13) Wipe the liquid around the tissue dry, add ready-to-use DAPI, and incubate for 2 min;

[0047] (14) Wash with PBS 3 times, 5 min each time;

[0048] (15) Mounting: Wipe the fluid around the tissue dry, place a drop of anti-fluorescence attenuation mounting medium on the tissue, and cover with a coverslip. Since the coverslip is easy to slide, you can fix it by applying nail polish around the edges after covering with the coverslip;

[0049] (16) Slides: Immediately after staining, take and analyze images using a confocal microscope. For example... Figure 3 As shown in C, OGN was detected in the E37 sample. + / UCHL1 +The results showed that the markers OGN and UCHL1 of cell subpopulation 4, obtained through data analysis in Example 1, were indeed expressed in early precursor cells of hair follicle substrate formation, consistent with the results of single-cell analysis and spatial transcriptomics. Both findings validated the accuracy of the hair follicle substrate precursor cell markers OGN and UCHL1 identified in this invention.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0051] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for identifying porcine hair follicle substrate precursor cells, characterized in that, OGN and UCHL1 were used as marker genes for porcine hair follicle lamina precursor cells. The gene number of OGN in the NCBI database is 106509723, and the gene number of UCHL1 in the NCBI database is 396637. The identification method was as follows: porcine embryonic epidermal cells were stained with immunofluorescence. Cells that could detect both OGN and UCHL1 were porcine hair follicle lamina precursor cells. The porcine embryos were porcine embryos at or before the embryonic stage of 37 days.

2. The application of the method for identifying porcine hair follicle substrate precursor cells as described in claim 1 in hair follicle development research.

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