Animals, human, and administration
The porcine fetuses were procured from Wanfu Group Co. Ltd (Qingdao, Shandong, China). The CD-1 mice utilized were procured from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). Additionally, the CAG/eGFP transgenic mice strain was graciously provided by Dr. Xiao Yang of the Institute of Biotechnology in Beijing. Human fetal genital ridge tissue samples were obtained from Anqiu Women and Children’s Hospital (Shandong, China). In accordance with national guidelines, written informed consent was obtained from all study participants, and the study was reviewed and approved by the Ethics Committee of Anqiu Women and Children’s Hospital as well as the Ethical Committee of Qingdao Agricultural University (QAU; Agreement No. 2020-018).
Epidermal growth factor (EGF, R&D Systems, 2028-EG, USA) and basic fibroblast growth factor (bFGF, PeproTech, 100-18B, USA) were dissolved in DMEM/F12 (Gibco, C11330, USA) containing 1% BSA and working solutions of EGF (20 ng/ml) and bFGF (40 ng/ml) were prepared in DMEM/F12 medium [25]. Melatonin (MLT, Sigma, M5250, Germany) was dissolved in anhydrous ethanol to 200 mM, and then diluted to 25 µM in differentiation medium as working solution [7]. Luteinizing hormone (LH, Sigma, L5269) was dissolved in DMEM medium to achieve a working concentration of 400 mIU for differentiation induction [26]. Retinoic acid (RA, Sigma, R4643) was dissolved with DMSO to 10mM storage solution, and then diluted to 5 µM in differentiation medium as working solution [27]. U0126 (Abcam, ab120241, UK) was dissolved in DMSO to 50 mM concentrated storage, and then diluted to 10 µM in differentiation medium for experimental use [27].
Dissociation of porcine skin and generation of SDSC spheres
The porcine SDSC culture method used in this experiment has been described above [7, 26, 27]. Dorsal skin was collected from female pig fetuses on gestation days 35–45 (E35-E45). Use a scalpel to cut into tissue blocks and wash with PBS (Solarbio, T8200, Beijing, China). Then, the tissue blocks were cultured with SDSC medium [DMEM/F12 (1:1), 20 ng/ml EGF, 40 ng/ml bFGF, 1% penicillin-streptomycin (Solarbio, P1400) and 1% B27 (Gibco, 17504044)] in 10 cm culture dishes (Sarstedt, Montreal, Canada). After 4 days, the tissue blocks were collected into a 15 ml centrifuge tube and the cells were mechanically dissociated using a 1 ml pipette. The single-cell suspension was continued in SDSC medium, and the medium was replaced every four days. The resulting SDSC spheres were harvested after 12 days of culture for further procedures. The diameters of SDSC colonies (four groups: control, EGF, bFGF, EGF and bFGF) were measured using ImageJ (V1.48) software (NIH, MD, USA).
Induction of porcine PGCLC
Porcine SDSC in vitro induction of porcine PGCLC was performed as previously [7, 26, 27]. In brief, SDSC clones are separated into single cells and inoculated into 60 mm cell dishes (Corning, 430166, USA) according to the density of 1×105 cells. The differentiation medium [DMEM (Gibco, C11995500BT), 5% FBS, 5% porcine follicular fluid, 1% GlutaMAX™ Supplement (Thermo Fisher Scientific, 35050061, USA), 0.1 mM 2-mercaptoethanol (2-ME, Sigma, M-7522), 1% non-essential amino acids solution (NEAA, Thermo Fisher Scientific, 11140-050), 1% sodium pyruvate] was changed every four days. The PGCLC was collected for follow-up experiments after about 16 days of differentiation.
Strategies for mouse SDSC differentiation of PGCLC in vitro
Previously published protocols detailed the isolation of mouse skin and the generation of SDSC clones [17]. The dorsal skin of newborn mice expressing the CAG/eGFP transgene was collected and subsequently dissected into smaller pieces using scissors. The single cell suspension was obtained using trypsin-EDTA solution (Sorlabio, T1300, China) in an incubator for 25 min and a 40 µm cell strainer (Biologix, 15-1040, USA). The individual cells obtained were cultured, with the medium being replaced every four days. After 12 days of cultivation, the generated SDSC spheres were harvested for subsequent experimentation.
The protocol and culture regimen for inducing PGCLC from mouse SDSC were executed in strict compliance with previously established protocol [17]. EpiLC was induced by P2 mouse SDSC, which was dissociated from single cell suspension and seeded into 24-well plates at 5 × 105 cells/well in EpiLC induction medium [17]. EpiLC colonies were dissociated into single cells after four days and were pipetting at a concentration of 5×105 cells/ml and plated on mitogenic inactivated MEF feeding cells for PGCLC induction in PGCLC differentiation medium [17, 28]. The half medium was replaced every two days and the generated suspended PGCLC were harvested for subsequent experimentation after about 12 days.
Single cell cDNA library preparation and sequencing
Dorsal skin tissue from at least three fetal pigs was separated into small pieces and subsequently digested in incubator with a trypsin-EDTA solution for 25 min to obtain single cell suspensions. The P2 SDSC spheres were dissociated by the above method to obtain single cell suspension. Samples were then filtered using a 40 µm cell filter and washed using with DMEM basic containing 0.1% BSA. We constructed a single cell cDNA library using Chromium single cell 3' Reagent Kit (10×Genomics, PN-1000075, USA). Cells with more than 90% viability were captured using the 10× Genomics Chromium controller (10× Genomics, DNBSEQ-T7, USA).
scRNA-seq data preprocessing and data visualization
The standard agreement to use CellRanger (v3.0.2) software for UMI does a comparison and quantitative. Seurat (v3) R package was used to conduct downstream quality control and visualization analysis of the processed expression matrix [29]. During quality control, low-quality cells are filtered using the default parameters in Seurat and the data is consolidated using the FindIntegrationAnchors function in the Seurat package. Then, the RunUMAP and FindClusters functions were used for reduction and cluster recognition. Single-cell trajectory inference was performed using the slingshot R package to infer dynamic gene expression along a pseudotime axis [30]. Everything else defaults to the official guidelines.
Hematoxylin-eosin (H&E) staining
The skin was fixed overnight at 4℃ with 4% PFA solution (Sorlabio, P1110). The following day, the fixed tissue was dehydrated in an ethanol solution and treated with xylene for 30 min. Then, the sample was embedded in paraffin. The slices were cut into 5 µm slices using Leica RM2255 microtome (Leica, RM2235, Germany), and the samples were transferred to adhesive slide. After the slides were dewaxed and rehydrated, they were stained with hematoxylin solution for 7 min and then rinsed with distilled water twice for 5 min. After rinsing with 1% HCl-ethanol solution for 3–5 sec, immediately rinse with 45°C water for 5 min. After dehydration, dye with 1% eosin ethanol solution and rinse with anhydrous ethanol for 10 min. Finally, the sheet was sealed with a neutral resin and photographed under microscope (Olympus, BX51, Japan).
Immunofluorescence (IF) staining, immunohistochemistry (IHC) staining, and immunocytochemistry (ICC) staining
The IF staining method used in this experiment has been described in recent studies [31]. In short, the slides were rehydrated and antigenic repair was performed. In brief, the slide was incubated with boiled 0.01 M sodium citrate buffer (pH = 6.0) for 10 min. After cooling to room temperature, the slide was closed with a blocking buffer (0.5 M Tris-HCI buffer containing 3% BSA and 10% goat serum (BOSTER, AR0009, China). Subsequently, then primary antibody and the secondary antibody were added and incubated. Details of the antibodies used in this experiment are shown in Table S6. The nucleus was then stained by DAPI. The image was taken under a confocal microscope (Leica Microsystems GmbH, TCS SP5 II). For IHC, in order to block endogenous peroxidase activity, 3% hydrogen peroxide was treated for 10 min, followed by color development with DAB kit (ZSGB-BIO, ZLI-9017, China).
ICC is used to detect protein localization and expression levels in cells. In short, cells are fixed with 4% PFA solution (Sorlabio, P1110), and then the cells are coated on a slide to dry. Then, PBST (PBS with 0.5% Triton X-100) was permeated for 10 min, and PBST containing 10% goat serum was sealed at room temperature for 30 min. After closure, the primary antibody was added. Then, the slides were rinsed with PBS containing 1% BSA (Solarbio, A8020) for three times, and an appropriate secondary antibody was added according to the species source of the primary antibody at 37℃ for 45 min. Details of primary and secondary antibodies are listed in Table S6. The nucleus was stained with DAPI or PI. Finally, confocal microscopy was used for image acquisition.
CCK-8 cell proliferation assay
The CCK-8 cell viability assay kit (Solarbio, CA1210) was used in this experiment. According to the standard requirements of the testing kit, the 10% CCK-8 solution was incubated in an incubator at 37℃ for 2 h. Subsequently, the absorption value at 450 nm wavelength was detected with a microplate reader (Bio-Rad, iMarkTM, USA).
Cell cycle analysis
Flow cytometry was used to measure DNA amount and cell cycle distribution. Briefly, the SDSC spheres for 4 groups (control, EGF, bFGF, EGF and bFGF) were digested into single cells by trypsin-EDTA solution, and fixed overnight at 4℃ in 70% ethanol. After the cells were washed with pre-cooled PBS, stained with the prepared propidium iodide staining solution (containing RNase A, Beyotime, C1052, China), and incubated at 37℃ for 30 min away from light. Finally, FACS Calibur flow cytometer (Becton Dickinson, FACS Calibur, USA) was utilized to measure 20 000 cells per sample, and then used FlowJo v10 software for analysis.
Flow cytometry analysis
For single staining, the collected genital ridges and PGCLC (mouse) were digested into single cells and fixed with pre-cooled 80% methanol. They were then incubated with primary antibody (Table S6) for 60 min and secondary antibody (Table S6) for 45 min at 37℃. The samples were detected by flow cytometry and analyzed by WinMDI 2.9 software. For co-staining, the samples were incubated with primary antibodies including SOX17, PCNA (Table S6) and then incubated with secondary antibodies including fluorescein isothiocyanate (FITC) and Cyanine3 (CY3) conjugated antibodies (Table S6) for 45 min at 37°C. The cells suspension was detected by flow cytometer and the data was analyzed by FlowJo V10 software.
Teratoma formation and analysis
To investigate whether SDSC has properties similar to pluripotent stem cells, SDSC colonies were collected and transplanted to kidney capsules in SCID/Beige immune-deficient mice (Vital River Laboratory Animal Technology Co., Ltd, Beijing) for 10 weeks of ectopic development. The solid tumor was then observed, removed, fixed, sliced, and stained by H&E for histological analysis.
PCR and real time quantitative PCR (RT-qPCR)
Total RNA was extracted from tissues (solid tumor) and cells (SDSC, PGCLC) using RNAiso Plus (TaKaRa, 9109, Japan) and cDNA reverse transcription kit (SparkJade, AG0304, China) was used to synthesize the cDNA according to directions. PCR using Taq DNA Polymerase (TransGen, AP101, China) was performed in a 50 µl reaction volume according to instructions. Details of all primers are listed in Table S7. The amplified products were separated by electrophoresis using 2% agarose gel.
RT-qPCR used SYBR Premix Ex Taq™ II (Vazyme, Q711-02, China). The reaction was performed by Roche 480 Light Cycler RT-qPCR instrument (Roche, Germany) as previously described [32]. The RT-qPCR reactions were done in 20 µl reaction volume containing: 2 µl cDNA, 10 µl SYBR Premix Ex Taq™ II (2×), 7.2 µl RNase-free water, and 0.4 µl (10 µM) forward and reverse primers respectively. All primers are listed in Table S7. Real-time PCR and 2−(△△Ct) methods were used to measure relative gene expression [33]. Each sample was amplified by PCR three times, and porcine GAPDH as housekeeping gene.
Western blotting (WB) analysis
Firstly, the cells were collected by centrifugation in a 1.5 ml centrifuge tube, and the RIPA lysate (Beyotime, P0013C, China) was added for ice cracking. Protein was boiled with 5× SDS-PAGE Sample Loading Bufer (Beyotime, P0015L, China) for 5 min to denaturate protein. Then 12% SDS-PAGE was used for electrophoresis, 80 V for 30 min, then 120 V for 90 min. Bio-Rad Trans-Blot (Bio-Rad, Hercules, California, USA) was used to transfer proteins to polyvinadiene fluoride membranes (PVDF; Millipore, ISEQ00010, USA) for 150 min at 200 mA constant flow. Blocking was performed with TBST buffer (containing 5% skim milk powder) at room temperature for 2 h, the protein bands were incubated with primary antibody at 4℃ overnight and secondary antibody at room temperature for 75 min. All antibodies are listed in Table S6. Chemiluminescence was performed with BeyoECL Plus kit (Beyotime, P0018, China) and used AlphaView SA software for gray analysis of protein bands.
RNA sequencing (RNA-seq) analysis
Novogene Company (Beijing, China) was responsible for RNA extraction, library construction and sequencing of RNA-seq in this experiment. Three duplicate samples were detected in each group to ensure the reliability of the results. The data were quality-controlled, recounted and base-corrected with fastp in Linux, then compared with the reads in a pig reference genome using STAR, and finally tallied the number of reads using featureCounts. Differentially expressed genes (DEGs) with DESeq2 analysis, and |log2FoldChange| > 1 and P-value < 0.05 as the threshold for significant differences in gene expression.
Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
GO and KEGG enrichment of DEGs and representative gene sets were performed using ClusterProfiler software [34]. GO items and KEGG pathways with a P-value of less than 0.01 were regarded as significantly enriched.
Statistical analysis
The experimental data were expressed in the form of mean ± standard deviation, and each group of experiments was repeated at least 3 times. One-way analysis of variance was used to analyze data followed by Tukey's test for multiple comparisons using GraphPad Prism 8 software. *: 0.01 < P < 0.05 indicates a statistically significant difference and **: P < 0.01 indicates a highly significant difference.