Method for preparing spermatogonial-like cells by xenoreconstructed testis and application thereof

Abstract

The application discloses a method for preparing spermatogonial-like cells from xenoreconstructed testis and application thereof, and belongs to the technical field of biology. The method comprises the following steps: mixing ex vivo mouse fetal testis somatic cells and pig primordial germ cell-like cells, and then culturing to obtain a xenopolymer; and the pig primordial germ cell-like cells are prepared by the following steps: inducing ex vivo pig gastrulated epiblast stem cells into mesendodermal precursor cells, and then into primordial germ cell-like cells. The xenopolymer is transplanted into a mouse kidney capsule, and spermatogonial-like cells are obtained in the formed xenoreconstructed testis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing spermatogonial-like cells from xenogeneic reconstructed testes and its application. Background Technology

[0002] Mammalian male germ cell (GC) development is a complex and ordered multi-step differentiation process, mainly comprising three distinct stages: primordial germ cell (PGC) specialization, sex determination and spermatogonial development, and subsequent spermatogenesis. Meiotic recombination events during haploid formation create genomic diversity, accelerating species evolution. Over the past two decades, pluripotent stem cells (PSCs) in mice and rats have been induced into primordial germ cell-like cells (PGCLCs), which subsequently differentiate into functional gametes. However, rodents have shorter lifespans, and their embryos are oval-cylindrical; their GC lineage specialization and developmental mechanisms differ significantly from those in humans and other species. Therefore, research findings in mice cannot be directly applied to clinical and livestock production and reproduction.

[0003] Recent research has shown that pluripotent stem cells from humans and non-human primates can be successfully induced into progenitor cell line cells (PGCLCs), which can then differentiate into anterior spermatogonia or early oogonia through epigenetic reprogramming in appropriate environments. However, due to ethical, moral, technical, and legal limitations, conducting in vivo functional studies on human PGCLCs is largely impractical. Compared to other laboratory animals, pigs are not only an important economic livestock but are also considered a reliable and representative biomedical model for studying human physiology, anatomy, and metabolism. Therefore, establishing a pig in vitro germ cell differentiation system not only has comparative biological significance but can also promote regenerative medicine, animal breeding, endangered species protection, and germplasm resource preservation. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to induce porcine gastrulated epiblast stem cells (pEpiSCs) to differentiate into porcine primordial germ cell-like cells (pPGCLCs) through mesoendothelial progenitor cells (pMEPCs), and then obtain spermatogonial-like cells by polymerizing them with mouse fetal testicular somatic cells and transplanting the xenogeneic polymer through the renal capsule. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: The present invention also provides a method for preparing xenografts, comprising culturing isolated mouse fetal testicular somatic cells and porcine primordial germ cell-like cells to obtain xenografts, wherein the porcine primordial germ cell-like cells are prepared by the following steps: inducing isolated porcine gastrulated epiblast stem cells into mesoderm precursor cells, and then inducing them into primordial germ cell-like cells.

[0006] In the above method, the ratio of mouse fetal testicular somatic cells to porcine primordial germ cell-like cells is 5:1-10:1.

[0007] In the above method, the mouse fetal testicular somatic cells are derived from the testicular tissue of mouse E12.5 fetuses.

[0008] E12.5 refers to the 12.5th day after conception in mice.

[0009] In the above method, the induction time for inducing isolated porcine gastrulated epiblast stem cells into mesoendothelial precursor cells is 30-42 hours. The induction is carried out using Composition 1, which includes an activator of the cell signaling pathway Activin / NODAL and an agonist of the cell signaling pathway Wnt / β-catenin.

[0010] The mesoendothelial precursor cells expressed OCT4, NANOG, SOX17, PRDM1, EOMES, and TBXT proteins.

[0011] SOX17 is SRY-box transcription factor 17, and its amino acid sequence is NCBI Reference Sequence:XP_001928411.1 (13-MAY-2017).

[0012] OCT4 (also called POU5F1) is a POU5 class homeobox 1, and its amino acid sequence is NCBI ReferenceSequence:NP_001106531.1 (MAM 02-APR-2025).

[0013] NANOG is a Nanog homeobox protein, and its amino acid sequence is NCBI Reference Sequence: NP_001123443.2 (03-NOV-2023).

[0014] PRDM1 is a zinc finger protein 1 with a PR domain. Its amino acid sequence is NCBI Reference Sequence: XP_005659397.1 (13-MAY-2017).

[0015] EOMES is the germin gene, and its amino acid sequence is NCBI Reference Sequence: XP_003132129.2 (13-MAY-2017).

[0016] TBXT is T-box transcription factor T, also known as brachyyury, and its amino acid sequence is NCBI Reference Sequence: XP_020930431.1 (13-MAY-2017).

[0017] GSC is the goosecoid homeobox gene, and its amino acid sequence is NCBI Reference Sequence: XP_003482361.2 (13-MAY-2017).

[0018] GATA4 is GATA-binding protein 4, and its amino acid sequence is NCBI Reference Sequence: NP_999458.1 (02-APR-2025).

[0019] The porcine gastrulation epiblast stem cells are derived from porcine embryos on day 10 of development. These porcine gastrulation epiblast stem cells express core pluripotent transcription factors, are positive for alkaline phosphatase staining, and have the ability to form embryoid bodies and teratomas, as well as to randomly differentiate into three germ layers. They exhibit multi-lineage differentiation characteristics and are in an intermediate pluripotent stage.

[0020] The mesoendothelial progenitor cells express SOX17, PRDM1, EOMES, and TBXT proteins, while the porcine gastrulation epiblast stem cells do not express SOX17, PRDM1, EOMES, and TBXT proteins.

[0021] The mesoendothelial precursor cells are cells with the potential to differentiate into primordial germ cells.

[0022] SOX17 was significantly overexpressed and SOX2 (NCBI ReferenceSequence: NP_001116669.1, 02-APR-2025) was significantly underexpressed 30-42 h after differentiation of porcine gastrulation epiblast stem cells. OCT4 was also co-expressed.

[0023] The Activin / NODAL cell signaling pathway plays an important role in early embryonic development, cell fate determination, and germ layer differentiation.

[0024] The Wnt / β-catenin pathway is a series of biochemical reactions triggered by Wnt protein ligands, which ultimately lead to the accumulation of the transcriptional coactivator β-catenin in the cell nucleus and the initiation of transcription of specific genes.

[0025] Agonists are substances that mimic endogenous signaling molecules and trigger receptor-mediated signal transduction. Activators are substances that enhance the inherent biochemical activity of target proteins and / or enzymes.

[0026] The activators of the Activin / NODAL cell signaling pathway include, but are not limited to, activin A.

[0027] Agonists of the Wnt / β-catenin cell signaling pathway include, but are not limited to, CHIR99021.

[0028] In some specific embodiments of this invention, the content of activator A is 50-100 ng / mL, and the content of WNT agonist is 0.3-1 μM.

[0029] In one specific embodiment of the present invention, CHIR99021 is a product of Selleckchem, named GSK-3α / β inhibitor, with catalog number S1263.

[0030] In one specific embodiment of the present invention, the activator A is a PeproTech product with product number 120-14E.

[0031] In some specific embodiments of the present invention, the culture medium used in the induction process contains, in addition to composition one, a ROCK inhibitor (Y-27632) and aRB27 basal medium.

[0032] In some specific embodiments of the present invention, the ROCK inhibitor (Y-27632) is a product of Selleckchem with catalog number S1049.

[0033] In some specific embodiments of the present invention, the ROCK inhibitor (Y-27632) is present in the aforementioned culture medium at a concentration of 5-10 μM.

[0034] The aRB27 basal medium consisted of: 1×B27 supplement, 1% penicillin-streptomycin, 1% GlutaMax, and 0.1 mM β-mercaptoethanol, with the balance being Advanced RPMI 1640 medium (Thermo Fisher, 12633012).

[0035] The B27 supplement is a product of Thermo Fisher Scientific, catalog number 12587010, with a concentration of 50×.

[0036] In the above method, the induction time for the mesoderm precursor cells to be induced into primordial germ cell-like cells is 12-24 hours. The induction is carried out using composition two, which includes bone morphogenetic protein 4, human leukemia inhibitory factor, stem cell factor and epidermal growth factor.

[0037] The primordial germ cell-like cells express marker proteins, including NANOS3, SOX17, OCT4, NANOG, PRDM1, and TFAP2C proteins.

[0038] NANOS3 for nanos C2HC Type zinc finger 3, its amino acid sequence is: MAPLCWCPLHPVSWTMHSFGRCIFGGAAASPPVTIRNLPQPAPPSSHPLGGIRRELTAQTPGLQREKGRGRGKGIEGRSLGWLGFFSLSALSPGTLCPAMGTFNLWTDYLGLARLVGALRGEEEPETRLDPQ PAPVPGPEGQRSPESSPAPERLCSFCKHNGESRAIYQSHVLKDEAGRVLCPILRDYVCPQCGATRERAHTRRFCPLTSQGYTSVYSYTTRNSAGKKLARPDKARTQDSGHRRGGGGGGASTGSKGAGKSSGTSPSPCCPSTSA.

[0039] TFAP2C is the transcription factor AP-2γ, and its amino acid sequence is NCBI Reference Sequence: NP_001116673.1(24-SEP-2024).

[0040] Primordial germ cell-like cells are cells that resemble primordial germ cells in the body in terms of gene expression, molecular characteristics, epigenetic characteristics, and developmental potential.

[0041] In some specific embodiments of the present invention, the content of bone morphogenetic protein 4 is 200-500 ng / mL, the content of human leukemia inhibitory factor is 10 ng / mL, the content of stem cell factor is 100 ng / mL, and the content of epidermal growth factor is 50 ng / mL.

[0042] In some specific embodiments of the present invention, the bone morphogenetic protein 4 (BMP4) is a product of PeproTech with catalog number 120-05.

[0043] In some specific embodiments of the present invention, the human leukemia inhibitory factor (hLIF) is a product of PeproTech with catalog number 300-05.

[0044] In some specific embodiments of the present invention, the stem cell factor (SCF) is a PeproTech product, catalog number 300-07.

[0045] In some specific embodiments of the present invention, the epidermal growth factor (EGF) is a PeproTech product with catalog number AF-100-15.

[0046] In some specific embodiments of the present invention, the culture medium used to induce the mesoderm precursor cells into primordial germ cell-like cells includes, in addition to Composition 2, a ROCK inhibitor (Y-27632) and aRB27 basal medium. The concentration of Y-27632 is 10 μM. The composition of the aRB27 basal medium is as described above.

[0047] In the above method, the culture medium used to culture isolated mouse fetal testicular somatic cells and porcine primordial germ cell-like cells to obtain xenografts contains composition three, which includes polyvinyl alcohol.

[0048] In the above method, the volume percentage of polyvinyl alcohol in the culture medium is 1%.

[0049] This invention also provides a method for preparing spermatogonial-like cells, including transplanting a xenograft prepared according to the aforementioned method into the renal capsule of a mouse to obtain spermatogonial-like cells.

[0050] Spermatogonia-like cells are cells that resemble spermatogonia in vivo in terms of morphology and molecular characteristics.

[0051] After the xenograft was transplanted into the renal capsule of a mouse, it developed into a xenogeneic reconstructed testis, which contained spermatogonial-like cells.

[0052] "Testis-like" refers to an organ with a seminiferous tubule-like structure that resembles a testis.

[0053] This invention also provides the following products: M1) is composed of the aforementioned composition one, the aforementioned composition two, and the aforementioned composition three; M2) Products used to prepare xenografts, including isolated mouse fetal testicular somatic cells, porcine primordial germ cell-like cells, the aforementioned composition one, the aforementioned composition two, and the aforementioned composition three.

[0054] The present invention also provides xenografts prepared by the aforementioned method or spermatogonial-like cells prepared by the aforementioned method.

[0055] The advantages of this invention lie in the use of porcine gastrulated epiblast stem cell lines (pEpiSCs) to construct a germ cell gene-targeted integration fluorescent reporter system. A two-dimensional (2D) adherent monolayer culture method is employed, and based on the transient mesendoderm precursor cell (MEPC) stage, a large number of pPGCLCs are rapidly induced. The obtained pPGCLCs are equivalent to early E14 PGCs in vivo. When male pPGCLCs aggregate with in vivo isolated mouse fetal testicular somatic cells, and the xenograft is transplanted through the mouse renal capsule, a xenogeneic reconstructed testis-like cell is generated. Within this testis-like cell, the pPGCLCs develop posteriorly into spermatogonial-like cells. Attached Figure Description

[0056] Figure 1 This study aims to determine the pedigree fate of porcine E10-E14 embryos. a) is a UMAP visualization of porcine embryo single-cell transcriptome sequencing (scRNA) data and published porcine embryo datasets obtained earlier in the inventors' laboratory. Colors and shapes represent different embryonic days and cell lineages, respectively. n=542 single cells. EPI: epiblast, PGC: primordial germ cells. b) is a UMAP plot showing the distribution and expression of pluripotency and lineage-specific genes. The gradient from white to red indicates expression from low to high. c) is a violin plot showing the average expression level of pluripotency and lineage-specific genes associated with each cell fate identified in extended data plot a. d) is a bubble chart showing the expression patterns of selected genes in different cell types. The size of the dots represents the percentage of significant expression (…). logP), and the color of the dots represents the average expression level of the corresponding genes. e is the gene ontology (GO) enrichment analysis of upregulated genes in E12 and E13_SOX17 positive cells compared with E10 and E11_EPI.

[0057] Figure 2 This is the result of stem cell sex determination. Lane 1 is for D2000 molecular markers; lanes 2 and 4 are for pZFX gene detection (band size 506 bp); lanes 3 and 5 are for pSRY gene detection (band size 400 bp). Lanes 2 and 3 are 1-EPI cell lines, which express both pZFX and pSRY genes and are therefore male stem cell lines; lanes 4 and 5 are 2-EPI cell lines, which express only pZFX gene and are therefore female stem cell lines.

[0058] Figure 3This image shows the results of direct induction of porcine gastrulated epiblast stem cells (pEpiSCs) into primordial germ cells. a) Schematic diagram of three-dimensional (3D) induction of pEpiSCs into primordial germ cells (PGCs). b) Bright-field images of male pEpiSCs differentiating into PGCs via embryoid bodies within 0-4 days. Scale bar: 500 μm. c) Gene expression changes during PGC induction; error bars represent ±SD (n=3 biological replicates). P The value was calculated using Dunnett's multiple comparison test; ns, not significant. * P <0.05, *** P <0.001, where NANOS3 The differences between D1, D2, D3, and D4 and pEpiSCs were not significant, among which... SOX17 The differences between D1, D2, D3, and D4 and pEpiSCs were not significant, among which... TFAP2C The differences between D3 and D4 and pEpiSCs were not significant.

[0059] Figure 4 For pigs SOX17 and NANOS3 Verification of the cleavage efficiency of three target sgRNAs of the gene. 'a' represents the sgRNA target cleavage site on chromosome 4. SOX17 Sanger sequencing results. b shows the PCR amplification of the region spanning the sgRNA target site, which is close to the SOX17 stop codon and was used for T7 endonuclease I (T7E1) detection. c shows the Sanger sequencing results of the sgRNA target cleavage site NANOS3 on chromosome 2.

[0060] Figure 5 For reporter gene insertion identification. a) Screening of SOX17-targeting clones using genomic PCR of edited pEpiSCs. The integration of the reporter gene in the clones was screened using the internal primer pSOX17-3×Flag-P2A-NLS-tdTomato. #1-#7 are edited pEpiSCs, #8 is a wild-type pEpiSC. b) Screening of NANOS3-targeting clones using genomic PCR of edited pEpiSCs. The integration of the reporter gene in the clones was screened using the internal primer pNANOS3-3×Flag-P2A-NLS-tdTomato. #1-#17 are edited pEpiSCs, #18 is a wild-type pEpiSC.

[0061] Figure 6 for SOX17 - P2A - tdTomato Verification of homozygous knock-in pEpiSCs. a is... SOX17- P2A - tdTomato ( ST Representative images of clonal morphology (left) and wild-type (WT) pEpiSCs, and alkaline phosphatase (ALP) staining (right). Scale bar, 400 μm. b represents males. ST The karyotype of pEpiSCs is 37+XY. c represents the difference between the wild-type and the qRT-PCR detection result. ST Expression of core pluripotency markers in pEpiSCs. Data are presented as mean ± standard deviation (n = 3 biological replicates). P The values ​​were calculated using Dunnett's multiple comparison test. ns, not significant. d is... ST Representative immunostaining (IF) images of core pluripotency markers (NANOG, OCT4, and SOX2) in pEpiSCs. Scale bar, 50 μm. e represents... ST Representative images of tdTomato fluorescence and co-immunostained GATA4 and OCT4 in pEpiSCs-derived endoderm. Scale bar, 50 μm. f represents the contrast between the two images. ST Co-immunoimmunization analysis of pEpiSCs-derived endoderm showed co-localization of ST, SOX17, and 3×Flag fluorescence, indicating that the reporter gene faithfully mimics the pig's... SOX17 Endogenous gene expression patterns. Cell nuclei stained with DAPI. Scale bar, 50 μm.

[0062] Figure 7 pEpiSCs were induced to differentiate into porcine middle endoderm precursor cells (pMEPCs). SOX17 - P2A - tdTomato ( ST ) Schematic diagram of pEpiSCs inducing differentiation into porcine middle endoderm cells (ME). b is... ST Bright-field images (top) and real-time ST fluorescence images (bottom) of pEpiSCs during differentiation into MEs via monolayer culture. Scale bar: 200 μm. c represents the relative intensity of SOX17 protein during ME differentiation. d represents the ST fluorescence intensity during pEpiSCs differentiation as analyzed by flow cytometry. + Cell proportion. e represents the mRNA expression levels of SOX17 and core pluripotency markers during ME differentiation, analyzed temporally. SOX17 There was no significant difference between 12 h and pEpiSCs. f represents the expression levels of markers in the protostome, mesoderm, and early PGCs during ME differentiation, as detected by qRT-PCR. GSC There was no significant difference between 12 h and pEpiSCs. GATA4 There was no significant difference between 12 h and pEpiSCs. FOXA2There was no significant difference between 12 h and pEpiSCs. TET2 There was no significant difference between 12 h and pEpiSCs. g represents time-series IF analysis showing OCT4 co-expression during differentiation of ST reporter cell lines into ME. White arrows indicate individual ST cells. + Cells (red); clusters of yellow arrows point to individual OCT4 cells. + Cells (green). White dashed lines represent ST. + Strong expression but OCT4 + Cells with weak expression, and the purple dashed line represents ST. + Weak expression but OCT4 + Cells with strong expression. Scale bar, 50 μm. h is... Figure 7 The results of f were quantified, showing the co-expression of ST during the 0-48 hour process. + OCT4 + Cell percentage. Total cell number was calculated using nuclear counting (DAPI). Image i shows a representative co-immunostained image of ST-positive cells and SOX2 during ME differentiation. White arrows indicate individual ST cells. + Cells (red). Clusters of yellow arrows indicate individual SOX2 cells. + Cells (green). White dashed lines represent SOX17. + Strong expression and SOX2 + Cells with weak expression. Scale bar, 50 μm. j represents the relative intensity of OCT4 and SOX2 proteins during ME differentiation, where... OCT4 There were no significant differences between pEpiSCs and 12h, 24h, 36h, and 48h. k is a schematic diagram illustrating that during ME differentiation, the OCT4 heterodimer chaperone exchanges from SOX2 to SOX17 and enters the mesoendodermal lineage. l shows representative immunostaining images of NANOG and OCT4, ST and PRDM1, and TBXT during ME differentiation. White arrows indicate individual STs. + Cells. White dashed lines highlight cells co-expressing ST. + PRDM1 + Cells. Cell nuclei counterstained with DAPI. Scale bar, 50 μm. Scale bar, 50 μm. m represents the relative intensities of NANOG, PRDM1, and TBXT proteins during ME differentiation. n represents representative immunostaining images of mesoderm markers in 36-hour ST+ pMEPCs, where... NANOG There were no significant differences between pEpiSCs and pEpiSCs at 12h, 24h, and 36h. Scale bar, 50μm. The scatter plot compares the mean gene expression levels between pEpiSCs (left) and pMEPCs (right). Adjusted for... pDifferentially expressed genes (DEGs) were identified using threshold values ​​padj < 0.05 and absolute log2-fold change ≥ 1. Upregulated genes (log2-fold change ≥ 1) are shown in red, downregulated genes (log2-fold change ≤ -1) in blue, and insignificant genes in gray. p represents significant functional entries associated with differentially upregulated genes in pMEPCs compared to pEpiSCs, as shown by gene ontology (GO) enrichment analysis. Error bars for c, e, f, j, and m are shown as ± standard deviation (n = 3 biological replicates). P The values ​​were calculated using Dunnett's multiple comparison test.

[0063] Figure 8 Specialization of porcine primordial germ cell-like cells (pPGCLCs). a) Schematic diagram of the differentiation protocol for porcine primordial germ cell-like cells (pPGCLCs). b) Bright field (top) and real-time ST fluorescence (bottom) images of cells during ST pMEPCs monolayer culture 2. Scale bar, 200 μm. c) Time-series analysis of mRNA expression levels of pluripotency markers and early PGCs markers during pPGCLC differentiation, where... STELLA There was no significant difference between pMEPCs and pEpiSCs. NANOS3 There was no significant difference between pMEPCs and pEpiSCs. TFAP2C The differences between pMEPCs and pEpiSCs at 6h, 12h, and 18h ​​were not significant. PRDM14 There was no significant difference between pMEPCs and pEpiSCs. TFCP2L1 There were no significant differences between pMEPCs and pEpiSCs at 6h, 12h, and 18h. d shows bright-field (left) and real-time ST fluorescence (right) images of pPGCLCs cultured in ST pMEPCs monolayers for 24h. Scale bar, 200μm. e shows flow cytometry analysis of ST fluorescence in pPGCLCs at 24h of differentiation. +Cell proportions. f shows the expression of OCT4 during pPGCLC differentiation using ST fusion reporter cell lines and cytokine induction over 0-48 h, analyzed by time-series immunofluorescence staining. Representative images are shown at 0 h, 12 h, 24 h, and 36 h. Scale bar, 50 μm. g shows the relative intensity of OCT4 protein expression during pPGCLC differentiation. h shows the expression of PRDM1 and TFAP2C during pPGCLC differentiation using ST fusion reporter cell lines and cytokine induction over 0-48 h, analyzed by time-series immunofluorescence staining. Representative images are shown at 0 h, 12 h, 24 h, 36 h, and 48 h. Scale bar, 50 μm. i shows the relative intensity of SOX17, PRDM1, and TFAP2C protein expression during pPGCLC differentiation. j shows representative immunostaining images of TNAP, MKI67, and NANOS3 in ST+ pPGCLCs. Scale bar, 50 μm. k represents high levels of positive alkaline phosphatase (ALP) staining in the cytoplasm of NANOS3-tdTomato+ pPGCLCs. Scale bar, 50 μm. l represents representative co-immunostained images of SOX17, OCT4, NANOG, SOX2, and PRDM1 in NANOS3-tdTomato+(N3T) pPGCLCs. Scale bar, 50 μm. m represents the staining in pEpiSCs and pPGCLCs. H19 / I GF2 and SNRPN Methylation status of differentially methylated regions (DMRs). Each row represents a single allele. Black circles represent methylated CpG islands, and white circles represent unmethylated CpG islands. n is a representative image of OCT4 and TET2 co-immunostained in pPGCLCs compared to pEpiSCs. Scale bar, 50 μm. o is the relative intensity of TET2 protein in differentiated pPGCLCs compared to pEpiSCs. p is a representative image of OCT4 and 5-methylcytosine (5-mC) co-immunostained in differentiated pPGCLCs. Scale bar, 50 μm. q is the relative intensity of 5-mC protein expression in pPGCLCs compared to pEpiSCs. r is a representative image of OCT4 and H3K27me3 co-immunostained in differentiated pPGCLCs. Scale bar, 50 μm. s represents the relative intensity of H3K27me3 protein expression in pPGCLCs compared to pEpiSCs. t represents a representative image of OCT4 and H3K9me2 co-immunostained pPGCLCs compared to pEpiSCs. Scale bar, 50 μm. u represents the relative intensity of H3K9me2 protein in differentiated pPGCLCs compared to pEpiSCs. For c, i, and g, error bars represent ±SD (n=3 biological replicates), calculated using Dunnett's multiple comparison test. PValues. For o, q, s, and u, the error bars represent ±SD (n=3 biological replicates). P The values ​​were calculated using a two-tailed unpaired Student t-test.

[0064] Figure 9 Transcriptome expression profiling of pPGCLCs. a) Heatmap showing specific markers for a given cell cluster. b) Heatmap of correlation coefficients among pEpiSCs, pMEPCs, and pPGCLCs. c) Scatter plot comparing mean gene expression levels between pEpiSCs (left) and pPGCLCs (right). Differentially expressed genes (DEGs) were identified by an adjusted p-value threshold of padj < 0.05 and an absolute log2-fold change ≥ 1. Upregulated genes (log2-fold change ≥ 1) are shown in red, downregulated genes (log2-fold change ≤ -1) in blue, and insignificant genes in gray. d) Gene Ontology (GO) enrichment analysis showing significant functional entries associated with differentially upregulated genes in pPGCLCs compared to pMEPCs. e is a UMAP plot showing the integration of individual epiblast cells (EPI, E10-E14), primordial germ cells (PGCs, E14 and E31), pEpiSCs, pMEPCs, NANOS3-tdTomato+ PGCLCs, and D2.5 somatic cells. n=702 cells. f is a heatmap of selected marker genes in a specified cell cluster.

[0065] Figure 10 Spermatogonia-like cells differentiated from pig-mouse xenograft reconstructed testes (xrTestes) formed under the renal capsule. a. Schematic diagram of induced spermatogonia-like cells in xenograft reconstructed testes under the renal capsule. b. Graft samples collected at different time points. The left image shows the approximate appearance of the CD1 mouse xenograft under the renal capsule (white arrow). The right image shows a hematoxylin-eosin (HE) staining of a testis-like tissue section. White dashed lines delineate seminiferous tubule-like structures, and clusters of black arrows indicate a germ cell. Scale bar, left. Figure 1c. Representative immunofluorescence (IF) images show the expression of designated Sertoli cell (SC) markers (white arrow cluster, green: GATA4) and key spermatogonial markers (white arrows, red: DAZL, DDX4, PLZF) and DAPI (blue) in a 30-day xenografted testicular tissue section. The merged image is shown on the right. Scale bar, 50 μm, magnified 10 μm. d. Representative IF images of a 30-day xenografted testicular section show the expression of designated key germ cell (GC) markers (yellow arrow cluster: NANOG-positive cells, yellow arrows: cells co-localized with SOX17 and DAZL or NANOG and PLZF) and DAPI (blue). The merged image is shown on the right. Scale bar, 50 μm, magnified 10 μm. Representative IF images of testicular tissue sections from eE12.5 mouse fetuses, showing the expression of designated somatic genes (white arrow cluster, red: SOX17) and key GC markers (green, white arrows: DAZL, NANOG) and DAPI (blue). The merged image is shown on the right. Scale bar: 50 μm, magnification 10 μm. Representative IF images of testicular tissue sections from newborn pigs, showing the expression of designated somatic genes (white arrow cluster, red: SOX17) and key GC markers (yellow arrows: SOX17 co-localized with DAZL, yellow arrow cluster: SOX17 co-localized with NANOG) and DAPI (blue). The merged image is shown on the right. Scale bar: 50 μm, magnification 10 μm. g. Representative IF images of xenogeneic reconstructed testicular sections taken on day 100, showing designated key SC markers (green: GATA4) and GC markers (green: NANOOG; red: SOX17-P2A-tdTomato, γH2AX, RAD51), as well as DAPI (blue) and their combined plots. Scale bar, 50 μm, magnified 10 μm. All representative data were obtained from at least three independent experiments.

[0066] Figure 11Self-aggregating polymers of purified mouse fetal testicular somatic cells (mFTSCs) and pPGCLCs. a. Schematic diagram of the purification strategy for mouse fetal testicular somatic cells (mFTSCs). b. Successful purification of mFTSCs from mouse fetal male gonads. Scale bar: top 400 μm, middle 100 μm, bottom 200 μm. c. Representative IF images of E12.5 mouse fetal testicular tissue, showing specified Sertoli cell (SC) markers (green: GATA4) and key germ cell (GC) markers (green: OCT4, red: DAZL, DDX4, SSEA1), as well as DAPI (blue). Scale bar: 100 μm. d. Representative IF images of dissociated cultured mFTSCs, showing expression of specified SC markers (green: GATA4) and DAPI (blue), and absence of key GC markers (green: OCT4, red: DAZL, DDX4, NANOG). Scale bar, 200 μm. e. Representative IF image of purified mFTSCs self-polymers, showing expression of specified SC markers (green: GATA4) and DAPI (blue), and absence of key GC markers (green: OCT4, red: DAZL, DDX4, NANOG). Scale bar, 200 μm. f. Histological image of purified mFTSCs self-polymers. Scale bar, left 200 μm, right 50 μm. g. Representative IF image of mFTSCs self-polymer sections, showing expression of specified SC markers (green: GATA4) and DAPI (blue), and absence of key GC markers (green: OCT4, red: DAZL, DDX4, PLZF, STRA8, SSEA1). Scale bar, 50 μm. h. Morphological image of mFTSCs self-polymer development tissue 55 days post-transplantation. Scale bar, upper left 5 mm, upper right 1 mm, lower left 1 mm, lower right 100 μm. i. Representative IF image of a self-aggregating graft section of mFTSCs on day 55, showing expression of the indicated SC marker (green: GATA4) and DAPI (blue), but lacking key GC markers (red: DDX4, PLZF, STRA8). The merged image is shown at the top. Scale bar, 50 μm. j. Morphological image of self-aggregating tissue of pPGCLCs on day 30 post-transplantation. Scale bar, top left 5 mm, top right 1 mm, bottom left 500 μm, bottom right 100 μm. k. Representative IF image of a self-aggregating graft section of pPGCLCs on day 30, showing expression of the indicated SOX17 gene and DAPI (blue), but lacking key GC markers (red: DAZL, DDX4, STRA8) and SC markers (green: GATA4). The merged image is shown at the top. Scale bar, 100 μm, magnified 10 μm.Representative data must be obtained from at least three independent experiments.

[0067] Figure 12 Generation and characterization of xenograft testes (xrTestes) under the renal capsule in pigs and rats. a. HE staining of the xenograft polymer and renal capsule transplantation. Scale bar, left. Figure 5 mm, medium Figure 1 mm, right image 50μm. b Histological images of testicular tissue from pigs at different ages. Clusters of black arrows point to germ cells (GC) in the seminiferous tubules. Scale bar, 50μm. c Representative IF images of testicular tissue sections from 1-month-old pigs, showing designated Sertoli cell (SC) markers (white arrow cluster, green: GATA4) and key spermatogonial markers (white arrows, red: DAZL, DDX4, PLZF, and STRA8) as well as DAPI (blue). Merged image shown on the right. Scale bar, 50μm, magnified 10μm. d. Representative IF image of a pig-mouse xenograft reconstructed testicular tissue section on day 30, containing SOX17-tdTomato positive cells, showing key SC markers (white arrow cluster, green: GATA4) and GC markers (white arrows: SOX17-tdTomato, OCT4; yellow arrows: SOX17-tdTomato co-localization with DAZL; yellow arrow cluster: STRA8), and DAPI (blue), with a merged image. Scale bar: 50 μm, magnification 10 μm. e. Representative IF image of a pig-mouse xenograft reconstructed testicular tissue section on day 30, showing turbo-GFP (green), spermatogonial markers (red: DDX4, DAZL, STRA8), and DAPI (blue), with a merged image. Scale bar: 50 μm, magnification 10 μm. f. Representative IF images of pig-mouse xenogeneic reconstructed testicular tissue sections from day 100, showing the expression of designated SC markers (white arrow cluster, green: GATA4) and key GC markers (white arrows, red: DDX4, green: SYCP3), as well as DAPI (blue). The merged image is shown at the top. Scale bar, 50 μm, magnified 10 μm. g. Representative IF images of 3-month-old pig testicular tissue sections, showing the expression of designated meiotic markers (white arrows, red: DDX4, RAD51, and γH2AX; green: SYCP3) and DAPI (blue). The merged image is shown at the top. Scale bar, 50 μm, magnified 10 μm. h. qRT-PCR detection of GC gene (NANOS3) and meiosis-related markers (STRA8, SYCP3, PRM1, TNP1, and ACROSIN) in pig-mouse xenogeneic reconstructed testes from day 100. Data are presented as mean ± standard deviation (n = 3 biological replicates). The p-value was calculated using Dunnett's multiple comparison test; ns, not significant, **P<0.01, ***P<0.001, where TNP1 There was no significant difference between T103d and pEpiSCs. ACROSIN There was no significant difference between T103d and pEpiSCs. PRM1 There was no significant difference between T103d and pEpiSCs. For ag, representative data were obtained from at least three independent experiments.

[0068] In the above figures, all relative expression levels (unspecified values) showed significant or highly significant differences unless otherwise specified. Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] The terminology used in this invention: Differentiation of the mesendoderm (ME): During mammalian embryonic development, the mesendoderm, also known as the primitive streak, is the transitional period between the differentiation of the mesoderm and the endoderm. Here, it specifically refers to the in vitro differentiation of epiblast stem cells into the mesendodermal lineage.

[0072] E12-E13 EPIs refer to the epiblast tissues and cells of a pig fetus on days 12-13 of gestation.

[0073] In the gene expression analyses of the following examples, total RNA was extracted using the RNAprep Cell / Bacteria Kit (Tiangen, DP430) or TRIzol reagent (Thermo Fisher, 15596026CN) according to the manufacturer's instructions. cDNA was then reverse transcribed using the 5×All-In-One RTMaster Mix Kit (abm, G486).

[0074] In the quantitative experiments described below, all experiments were performed in triplicate, and GAPDH and EEF1A1 were used as housekeeping genes for standardization. The average value was taken, and the results were analyzed using the following methods: .

[0075] The following examples used GraphPad Prism v6.01 statistical software to process the data. Experimental results are expressed as mean ± standard deviation. One-way ANOVA and two-tailed unpaired t-tests were used. ns indicates no significance. P <0.05 (*) indicates a significant difference.

[0076] All experiments involving mice and pigs were conducted in accordance with the ethics guidelines of China Agricultural University and approved by the Animal Ethics Committee of China Agricultural University (License No.: AW02113202-3-1). CD1 and BALB / c nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) for use in feeding cell preparation and teratoma formation experiments. All animals were housed under specific pathogen-free conditions.

[0077] The histological analysis methods in the following examples were as follows: Tissues were dehydrated stepwise with a gradient of ethanol (70%, 80%, 90%, 95%, and 100%), then cleared in xylene and embedded in paraffin. All samples were serially sectioned to a thickness of 5 μm, attached to glass slides, dewaxed in xylene, and rehydrated with a gradient of ethanol (from high to low). They were then counterstained with hematoxylin (Sigma-Aldrich, MHS16) and eosin (Sigma-Aldrich, HT110116) and observed under a microscope (Leica, DM5500B).

[0078] The following examples illustrate the immunofluorescence assay method: Cells were fixed with 4% PFA at room temperature for 20 min or overnight at 4°C, then washed twice with DPBS for 3 min each time. Next, cells were permeabilized in 0.3% Triton X-100 / DPBS for 20 min, washed twice with DPBS for 3 min each time, and blocked with 1% BSA / 10% donkey serum for 40 min. Subsequently, cells were incubated overnight with a primary antibody at 4°C, washed three times with DPBS for 5 min each time. Immediately afterwards, cells were incubated with the appropriate secondary antibody at room temperature for 30 min, washed three times with DPBS for 5 min each time, counterstained with DAPI for 5 min, and finally washed with DPBS. Images were acquired using an Olympus IX71 fluorescence inverted microscope.

[0079] The karyotype analysis method in the following examples was as follows: When cells reached 70-80% confluence after passage, they were cultured in fresh medium containing 20 ng / mL KaryoMax Colcemid (Thermo Fisher, 15212012) for 2-3 h. The treated pEpiSCs were dissociated into single cells using Accutase and collected by centrifugation. After resuspending the cells, 0.075 M potassium chloride solution (pH 7.4) was added dropwise, and the cells were incubated at 37°C for 20 min. Then, the cells were fixed with freshly prepared methanol and glacial acetic acid (3:1) at 4°C for 3 min, followed by three fixation steps, each lasting 20 min. The fixed cells were dropped from a height onto pre-cooled, clean glass slides and then dried. The dried slides were stained with Giemsa stain, and chromosome diffusion images were taken under a microscope. The chromosome number was counted (at least 30 cells were analyzed).

[0080] The alkaline phosphatase staining method in the following examples was as follows: Cells in the logarithmic growth phase were washed with pre-warmed DPBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. After three washes with DPBS, cells were stained with BCIP / NBT ALP buffer in the dark for 10 min. Cells were washed again with DPBS and observed using an inverted microscope.

[0081] The inventors revealed the dynamic process of germline fate emergence through experiments, integrating and analyzing single-cell resolution data of porcine embryos from day 10 (E10) to day 14 porcine epiblasts (pEPIs) (see...). Figure 1 (a) Around E10, core pluripotent transcription factors, such as..., were detected in pEPIs. OCT4 , NANOG , SOX2 and PRDM14 In E11's pEPIs, SOX2 and NODAL The expression of [something] was significantly downregulated. With further development to E12-E13, [something]... NANOG , OTX2 and PRDM14 Expression in EPIs is suppressed, while original markers, including SOX17 , PRDM1 , NODAL , Eomesodermin ( EOMES ), Goosecoid ( GSC ), GATA4 , GATA6 , PDGFRA , FOXA2 , GATA2 and GATA3 High expression of this gene indicates the onset of gastrulation (see...). Figure 1 (b and c). Given SOX17 It is the first factor regulating pPGC fate, and its expression can be detected in pEPIs of E12, increasing in E13. SOX17-positive cells in E12 and E13 EPIs co-express some lineage markers, such as... GATA6 , EOMES , NODAL , CHRD , FOXA2 , GATA4 , GSC and CER1 Similar to the original article in the report (see...) Figure 1 (d). These cells also showed significant expression of the Activin / NODAL and WNT signaling pathways. Gene ontology enrichment analysis revealed that, compared to E10 and E11 EPIs, the E12 and E13 SOX17-positive cell populations upregulated pathways including focal adhesion, tubular morphogenesis, gastrulation, endoderm development, and mesodermal formation. Therefore, the inventors defined these cells as pig mesendoderm precursor cells (pMEPCs) (see [reference needed]). Figure 1 (e) This cell refers to a cell state or type in pigs, which is different from pMEPCs mentioned later, which are cells induced to differentiate in vitro by pEpiSCs.

[0082] Primers used in this invention: pig EEF1A1-Fw:5'-AATGCGGTGGGATCGACAAA-3', Rv:5'-CACGCTCACGTTCAGCCTTT-3'; pig GAPDH-Fw:5'-TGGTGAAGGTCGGAGTGAAC-3', Rv:5'-CCTTGACTGTGCCGTGGAA-3'; pig NANOG-Fw:5'-CATCTGCTGAGACCCTCGAC-3', Rv:5'-GGGCTTGTGGAAGAATCAGG-3'; pig OCT4-Fw:5'-CAAACTGAGGTGCCTGCCCTTC-3', Rv:5'-ATTGAACTTCACCTTCCCTCCA-3'; pig SOX2-Fw:5'-CCAGAAGAACAGCCCAGACC-3', Rv:5'-GCTTCTCCGTCTCCGACAAA-3'; pig STELLA-Fw:5'-CCCGCCTTTCAATCTGTCTCC-3',Rv:5'-TCGCCGAACCGTGTATCGAA-3'; pig KLF4-Fw:5'-CATGAGTTGGGGGAGGGAAG-3',Rv:5'-ACTCACCAAGCACCATCGTT-3'; pig TFCP2L1-Fw:5'-TGCACGAAGAGACCTTGACC-3',Rv:5'-CGCGGATGGTACTCTTCCACA-3'; pig PRDM14-Fw:5'-GAGGTGCCACACTTCCTGAACA-3',Rv:5'-TCGGGTAGCAGAGAAGCATCCA-3'; pig TBXT-Fw:5'-GCTTCCCAGAGACCCAGTTC-3',Rv:5'-GCGTAAGATTGGGAGTACCCG-3'; pig EOMES-Fw:5'-ACTCCCATGGACCTCCAGAA-3', Rv:5'-TCGCTTACAAGCACTGGTGT-3'; pig GATA4-Fw:5'-CGACACCCTAATCTCGATATGTTT-3',Rv:5'-TCATCTTGTGGTAGAGGCCG-3'; pig GSC-Fw:5'-TGCTGCCCTACATGAACGTG-3',Rv:5'-TCATCGGTGAAGATGGTGCG-3'; pig FOXA2-Fw:5'-CCAACAAGATGCTGACCCTG-3',Rv:5'-AGGAAGCAGTCGTTGAAGGA-3'; pig PDGFRA-Fw:5'-GGTCACCTGTGCCGTCTTTA-3',Rv:5'-TTTGATGGACGGGACCTTGG-3'; pig NANOS3-Fw:5'-CTTCGAGACTACGTGTGCCC-3',Rv:5'-CGATGTCCAGAGTCCTGTGTC-3'; pig SOX17-Fw:5'-CGCACGGAGTTTGAACAATA-3',Rv:5'-CAGACGTCGGGGTAGTTACAG-3'; pig PRDM1-Fw:5'-CAGTGCCGTGAAGTTTCCA-3',Rv:5'-AAGGATGCCTCTGCCTGAAC-3'; pig TFAP2C-Fw:5'-AGGACTCCACATCCGTTTG-3',Rv:5'-TGGTCACCTTCAACCTCGT-3'; pig TET2-Fw:5'-GCCGAGTGATGAGAACAGACG-3',Rv:5'-GTCTGCTGCTGAATGTTGGC-3'; pig DNMT3B-Fw:5'-ATGACCGGCCTTTCTTCTGG-3',Rv:5'-AGCCGAAGATCCTGTTCATCC-3'; pig DAZL-Fw:5'-CTGCTGGGGAACAAAGGAGT-3',Rv:5'-CCACTGTCTGTATGCTTCGGT-3'; pig DDX4-Fw:5'-AGCTGATCGCATGCTGGATATGG-3',Rv:5'-TCCGCCCACTTGTCCAACAG-3'; pig STRA8-Fw:5'-CGTGTCTGCAGCCATCTCCC-3',Rv:5'-TCGGGGGTGGATACCAGTGA-3'; pig SYCP3-Fw:5'-GGAGAGTCAGTGGGTTCTGG-3',Rv:5'-GCCAGGCACCTTCCAGTTT-3'; pig PRM1-Fw:5'-AGATGTTGCCGCAGCCATAG-3',Rv:5'-AGTGCGGTGGTCTTGCTAC-3'; pig PRM2-Fw:5'-GACCAGGGCTGCAGACGGA-3',Rv:5'-TACTCAAGATCTCGTGGGCTCCT-3'; pig TNP1-Fw:5'-AAGCCCCTCATTTTGGCAGA-3',Rv:5'-GTCACAAGTGGGAGCGGTAA-3'; pig ACROSIN-Fw:5'-TCTTGCTGAACTCGCACTGG-3',Rv:5'-GTGGCTTCACCGGCTTATTG-3'; pig SRY-Fw:5'-GGGAAAGGCTCCTCACTATTT-3',Rv:5'-AGGGATACATCCTCTCCTCTAC-3'; pig ZFX-Fw:5'-GTGCTGCTTTGTCTTGGAATG-3',Rv:5'-GAGGGAGTTAGGTCTGGATACT-3'; pSOX17-sgRNA1-Fw:5'-caccGTAACTACCCCGACGTCTGA-3',Rv:5'-aaacTCAGACGTCGGGGTAGTTAC-3'; pSOX17-sgRNA2-Fw:5'-caccgTAACTACCCCGACGTCTGAC-3',Rv:5'-aaacGTCAGACGTCGGGGTAGTTAc-3'; pSOX17-sgRNA3-Fw:5'-caccGATGGGCACCCGTCAGACGT-3',Rv:5'-aaacACGTCTGACGGGTGCCCATC-3'; pSOX17-sgRNA test-Fw:5'-CGCACGGAGTTTGAACAATATC-3',pSOX17-sgRNA test-Rv:5'-TGACAGAGGCACTAGCAGAG-3'; pSOX17-5'arm-Fw:5'-cgaattgggcccggcgcgccACGCCTTAGCCTA-3',pSOX17-5'arm-Rv:5'-tggtccttatagtcggtaccGACGTCGGGGTAGTT-3'; pSOX17-3'arm-Fw:5'-ctatacgaagttatatcgatTGACGGGTGC-3',pSOX17-3'arm-Rv:caagctatgcatttaattaaTGATTGGCGAG-3'; pNANOS3-sgRNA1-Fw:5'-caccgAAAGGTGCCGGGAAGTCTTC-3',pNANOS3-sgRNA1-Rv:5'-aaacGAAGACTTCCCGGCACCTTTc-3'; pNANOS3-sgRNA2-Fw:5′-caccgCCTAAGAGGCTGGCGCGAGC-3′,pNANOS3-sgRNA2-Rv:5′-aaacGCTCGCGCCAGCCTCTTAGGc-3′ pNANOS3-sgRNA3-Fw:5′-caccgTCCACTTCTGCCTAAGAGGC-3′,pNANOS3-sgRNA3-Rv:5′-aaacGCCTCTTAGGCAGAAGTGGAc-3′ pNANOS3-sgRNA test-Fw:5′-AGAGTCCTATGGCCGCGAG-3′,pNANOS3-sgRNA test-Rv:5′-GATCCGCGAAGGGTAGGTCG-3′ pNANOS3-5′arm-Fw:5′-gcgaattgggcccggcgcgccCATTTGGGTGCCCCGAGT-3′,pNANOS3-5′arm-Rv:5′-tggtccttatagtcggtaccGGCAGAAGTGGAGGGACAGC-3′ pNANOS3-3' arm-Fw:5'-atgtatgctatacgaagttatatcgatTGGCGCGAGCAGGAC-3',pNANOS3-3' arm-Rv:5'-gaatactcaagctat gcatttaattaaGGCACTCTAATTGCCTCTCCAC-3′ pig NANOS3-KO test-Fw:5'-GGCAGGTGCATTTTTGGAGG-3',pig NANOS3-KO test-Rv:5'-AGCAACAGATCCAGAAGACTGT-3' EGFP-test-Fw:5′-CGAGGTAAACGGCCACAAGTTCAGC-3′,EGFP-test-Rv:5′-GATGGGGGTGTTCTGCTGGTAGTGG-3′ tdTomato-test-Fw:5'-GACAACAACATGGCCGTCATCAAAG-3',tdTomato-test-Rv:5'-GCTCGTCCATGCCGTACAGGAACAG-3' S0090-Fw:5′-CCAAGACTGCCTTGTAGGTGAATA-3′,Rv:5′-GCTATCAAGTATTGTACCATTAGG-3′ pig H19 / IGF2 DMR2 Inside-Fw:5′-AGGTGTTATTTTGTTTGTTGGT-3′,pig H19 / IGF2DMR2 Inside-Rv:5′-ATAAAATAACCTAAAAAAACTCAA-3′ pig H19 / IGF2 DMR3 Inside-Fw:5'-GATTTTTAGGTTTGTTATTATTT-3',pig H19 / IGF2 DMR3 Inside-Rv:5'-CAAATATTCAATAAAAAA ACCC-3' pig SNRPN DMR Inside-Fw:5'-TTATTGTGAAGGATTTTGTGTTTTG-3',pig SNRPN DMRInside-Rv:5'-AAAAAATAACCTCAAACATCT CCAA-3' IS PCR primer:5′-AAGCAGTGGTATCAACGCAGAGT-3′; 3′P2 primer:5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3′. QP2 primer:5′-CAAGCAGAAGAGGCATACGA-3′.

[0083] Antibodies used in immunofluorescence staining in this invention: Rabbit anti-NANOG (1:1000, Peprotech, 500-P236), Mouse anti-SOX2 (1:500, Santa Cruz Biotechnology, sc-365823), Goat anti-OCT3 / 4 (1:1000, Santa Cruz Biotechnology, sc-8628), Rabbit anti-PRDM14 (1:300, Abcam, ab187881), Goat anti-OTX2 (1:300, R&D Systems, AF1979), Mouse anti-SSEA1(MC480) (1:200, Cell Signaling Technology, 4744p), Mouse anti-SSEA4(MC813) (1:300, Abcam, ab16287), Mouse anti-TRA-1-60 (1:500, Cell Signaling Technology, 4746), Mouse anti-TRA-1-81 (1:500, Abcam, ab16289), Rabbit anti-H3K27me3 (1:300, Cell Signaling Technology, 9733s), Rabbit anti-H3K27me3 (1:300, Millipore, 07-449), Rabbit anti-H3K9me2 (1:300, Abcam, ab1220), Rabbit anti-βIII-tubulin (1:300, Abcam, ab18207), Rabbit anti-α-SMA (1:300, Abcam, ab5694), Goat anti-GATA4 (1:10, Santa Cruz Biotechnology, sc-1237), Rabbit anti-GATA4 (1:100, Abcam, ab84593), Goat anti-BRACHYURY (N-19) (1:50, Santa Cruz Biotechnology, sc-17743), Goat anti-Brachyury (1:300, R&D Systems, AF2085), Rabbit anti-Eomes (1:500, Abcam, ab23345), Goat anti-GSC (1:50, Santa Cruz Biotechnology,sc-22234)、Rabbit anti-GATA6(1:300,Abcam,ab22600)、Goat anti-HAND1(1:300,R&D Systems,AF3168)、Rabbit anti-3×Flag(1:1000,Easybio,BE2005)、Rabbit anti-NANOS3(1:50,Invitrogen,PA5-115615)、Rabbitanti-MKi67(1:1000,Abcam,ab15580)、Mouse anti-TNAP(1:50,Santa CruzBiotechnology,sc-137213)、Goat anti-SOX17(1:500,R&D Systems,AF1924)、Rabbitanti-SOX17(1:1000,Cell Signaling Technology,81778s)、Rabbit anti-PRDM1(1:50,Cell Signaling Technology,9115S)、Rat anti-PRDM1(1:50,eBioscience,14-5963-82)、Goat anti-AP2γ(1:300,R&D Systems,AF5059)、Rabbit anti-TET2(1:600,GeneTex,GTX124205)、Mouse anti-5-mC(1:300,GeneTex,GTX60803)、Rabbit anti-DAZL(1:300,Abcam,ab34139)、Rabbit anti-DDX4(1:300,Abcam,ab13840)、Rabbit anti-PLZF(1:300,Abcam,ab39354)、Rabbit anti-TurboGFP(1:100,Invitrogen,PA5-22688)、Mouse anti-GFP (B-2)(1:100,Santa Cruz Biotechnology,sc-9996)、Rabbit anti-Stra8(1:200,Abcam,ab49602)、Rabbit anti-Rad51(1:100,Abcam,ab133534)、Rabbit anti-gammaH2A.X(1:100,Abcam,ab11174)、Mouse anti-SYCP3(1:100,Novus,NBP2 - 23487), Mouse anti - Acrosin (1:100, Abcam, ab1900), Mouse anti - Acrosin (1:100, Santa Cruz Biotechnology, sc - 51504), Rabbit anti - Protamine 1 (1:100, R&D Systems, NBP2 - 30949), Peanut Agglutinin (PNA) Rhodamine (1:300, Vector laboratories, RL - 1072 - 5), Rabbit anti - CDX2 (1:200, Proteintech, 82659 - 1 - RR), Alexa Fluor 488 conjugated donkey anti - rabbit IgG (1:1000, Life Technologies, A21206), Alexa Fluor 488 conjugated donkey anti - rat IgG (1:1000, Life Technologies, A21208), Alexa Fluor 488 conjugated donkey anti - goat IgG (1:1000, Life Technologies, A11055), Alexa Fluor 488 conjugated donkey anti - mouse IgG (1:1000, Life Technologies, A21202), Alexa Fluor 594 conjugated donkey anti - rabbit IgG (1:1000, Life Technologies, A21207), Alexa Fluor 594 conjugated donkey anti - goat IgG (1:1000, Life Technologies, A11058), Alexa Fluor 594 conjugated donkey anti - mouse IgG (1:1000, Life Technologies, A21203), Alexa Fluor 594 conjugated goat anti - chicken IgY (1:1000, Life Technologies, A11042).

[0084] Example 1. Amplification and culture of pEpiSCs 1. Preparation of MEF feeder layer cells Neutralization medium composition: 10% fetal bovine serum (FBS, Thermo Fisher product, catalog number 16000044), the remainder being DMEM medium (Thermo Fisher product, catalog number 11995073), the percentages mentioned above are volume percentages.

[0085] MEF medium composition: 10% FBS, 1% non-essential amino acids (NEAAs, Thermo Fisher, 11140050), 1% sodium pyruvate (Thermo Fisher, 11360070), 1% glutathione (GlutaMax, Thermo Fisher, 35050061), and 1% penicillin-streptomycin (Thermo Fisher, 15140122), with the balance being DMEM medium. All percentages mentioned above are volume percentages, and all contents mentioned above are final contents of each substance.

[0086] Pregnant female CD1 mice, 12.5 days post-mating, were euthanized by cervical dislocation, and embryos were isolated from the uterus, designated E12.5 embryos. These E12.5 embryos were placed in cold DPBS (Thermo Fisher product, catalog number 14190144) containing 1% penicillin-streptomycin. The embryos were dissected under a stereomicroscope, and the fetal torsos were collected in cold DMEM medium. The torsos were placed in 50 mL tubes and repeatedly minced with fine scissors for 10 min. The cells were washed once with DPBS, and digested with 0.25% trypsin-EDTA (Thermo Fisher, 25200072) for 10 min, gently agitated in a 37°C water bath. The cells were then quenched with neutralization medium to obtain mouse embryonic fibroblasts (MEFs). The mouse embryonic fibroblasts (MEFs) were resuspended in MEF medium and seeded in 10 cm culture dishes, approximately one dish per fetus, labeled P0 generation. Once the cells have reached 90% confluence, they can be frozen as primary cells or passaged into 15cm culture dishes. Cells are expanded to passage P3 at a ratio of 1:2 or 1:3, treated with 13 μg / mL mitomycin C (Selleck, S8146) for 2.5 hours, washed twice with pre-warmed DPBS, digested with 0.125% trypsin to form a single-cell suspension, neutralized, and counted. Cells are counted at a ratio of 2.4 × 10⁻⁶. 6 Cryopreserved at a density of 1 cell / mL for use as feeder cells in subsequent stem cell cultures.

[0087] 2. Culture and sex determination of porcine epiblast stem cells (pEpiSCs) Composition of 3i / LAF medium: 5% knockout serum substitute (KSR, Thermo Fisher, 10828028), 1×N2 supplement (Thermo Fisher, 17502048, 100×), 1×B27 supplement (Thermo Fisher, 12587010, 50×), 1% NEAAs, 1% penicillin-streptomycin, 0.5% glutathione, 0.1mM β-mercaptoethanol (Thermo Fisher, 21985023), 50μg / mL ascorbic acid (Sigma-Aldrich, A4544), 10ng / mL human leukemia inhibitory factor (hLIF, PeproTech, 300-05), 25ng / mL activin A. A, PeproTech, 120-14E), 10 ng / mL recombinant human basic fibroblast growth factor (bFGF, PeproTech, 100-18B), 2.5 μM IWR-1 (WNTi, Selleckchem, S7086), 1 μM CHIR99021 (GSK3α / β, Selleckchem, S1263), 1 μM WH-4-023 (LCK / SRCi, Selleckchem, S7565) and 5 μM Y-27632 (ROCK inhibitor, Selleckchem, S1049), with the balance being DMEM / F12 (Thermo Fisher, 10565018) and Neurobasal medium (Thermo Fisher, 21103049). The volume ratio of medium is 1:1, all percentages mentioned above are volume percentages, and the contents of each substance mentioned above are the final contents of the substance in 3i / LAF medium.

[0088] Porcine gastrulation epiblast stem cell lines (referred to as pEpiSCs in this patent): described in the non-patent literature “Zhi, ML et al. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res. 32, 383-400 (2022)”, and named pgEpiSCs in the aforementioned literature, are available to the public from China Agricultural University. This biomaterial is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0089] Undifferentiated porcine epiblast stem cell lines (pEpiSCs) were seeded onto MEF feeder cells obtained in step 1, MEF feeder cell preparation, for maintenance growth. The culture medium used was 3i / LAF, which was replaced daily with fresh 3i / LAF. Enzymatic passages were performed every 2-3 days with Accutase (Thermo Fisher, A1110501) for 3-5 minutes at a 1:3 ratio. Small clumps or single cells were reseeded onto new feeder cells. Sex of each cell line was determined by sex-specific PCR, using the following method: Using genomic DNA as a template, cell lines that amplified a 400bp fragment with pig SRY-F and pig SRY-R (primer information as described above) and amplified a 506bp fragment with pig ZFX-F and pig ZFX-R (primer information as described above) were male; cell lines that could not amplify a 400bp fragment with pig SRY-F and pig SRY-R but amplified a 506bp fragment with pig ZFX-F and pig ZFX-R were female. The identification results are shown below. Figure 2 Lane 1 was used for DL2000 marker detection, lanes 2 and 4 for pig ZFX gene detection, and lanes 3 and 5 for pig SRY gene detection. Male porcine epiblast stem cell lines (pEpiSCs) were selected for subsequent experiments, and will be referred to as pEpiSCs in subsequent experiments.

[0090] 3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in existing technologies aRB27 basal medium composition: 1×B27 supplement, 1% penicillin-streptomycin, 1% GlutaMax, and 0.1 mM β-mercaptoethanol, with the balance being Advanced RPMI 1640 medium (Thermo Fisher, 12633012). The percentages mentioned above are volume percentages.

[0091] The B27 supplement is a product of Thermo Fisher Scientific, catalog number 12587010, with a concentration of 50×.

[0092] The PGC induction medium consists of: 200-500 ng / mL BMP4, 10 ng / mL hLIF, 100 ng / mL stem cell factor (SCF, PeproTech product, catalog number 300-07), 50 ng / mL epidermal growth factor (EGF, PeproTech product, catalog number AF-100-15), and 10 μM Y-27632, with the remainder being aRB27 basal medium. All the contents of the aforementioned substances are final contents.

[0093] To clarify the germline differentiation capacity of pEpiSCs, they were first cultured in a medium containing cytokines such as BMP4 for 96 hours (h), and the differentiation of pPGCs was induced using a three-dimensional (3D) culture method. Figure 3 (a) and (b) were repeated three times, with each repetition as follows: pEpiSCs were digested into single cells, resuspended in PGC medium at a density of 3000 cells / 100 μL, and 100 μL was seeded per well. The cells were then cultured at 37°C with 5% CO2. During the culture process, the cells aggregated at the bottom of the U-shaped section of the 96-well plate, forming spherical structures. The results were observed at 24 hours (i.e.,...). Figure 3 D1 in c) 48h (i.e.) Figure 3 D2 in c) 72h (i.e.) Figure 3 D3 in c) and 96h (i.e. Figure 3 Cells are collected from D4 in c.

[0094] Total RNA was extracted from collected cells using the RNAprep Cell / Bacteria Kit (Tiangen, DP430) according to the manufacturer's instructions. cDNA was reverse transcribed using the 5×All-In-One RT Master Mix Kit (abm, G486). Quantitative PCR was performed using 2×RealStar Power SYBR Mixture (GeneStar, A311) on an Archimed X6 instrument (Kunpeng Gene). Changes in the expression levels of the genes pig NANOS3, pig SOX17, pig PRDM1, pig TFAP2C, and pig OCT4 in the collected cells were detected. Primer sequences were as described above. Each sample was tested three times, and normalization was performed using GAPDH and EEF1A1 as housekeeping genes.

[0095] The results showed that the cells obtained by the above three-dimensional (3D) culture method, SOX17 or NANOS3 The expression was not upregulated. Figure 3 c) indicates that pEpiSCs were not induced into porcine primordial germ cell-like cells by the aforementioned three-dimensional (3D) culture method.

[0096] Example 2: Method for inducing porcine primordial germ cell-like cells (pPGCLCs) provided by the present invention I. Targeted Integrated Fluorescence Reporting System 1. Construction of the cutting carrier Design three CRISPR-specific lines for pigs using the online design tool (http: / / crispr.mit.edu). SOX17 or NANOS3Single guide RNAs (sgRNAs) near the stop codon include sgRNA1, sgRNA2, and sgRNA3.

[0097] The primer pairs used for target vector construction mentioned above (Fw and Rv of pSOX17-sgRNA1, Fw and Rv of pSOX17-sgRNA2, Fw and Rv of pSOX17-sgRNA3, Fw and Rv of pNANOS3-sgRNA1, Fw and Rv of pNANOS3-sgRNA2, and Fw and Rv of pNANOS3-sgRNA3) were annealed to obtain six annealing products. The six annealing products were cloned into nucleotides 6853 to 6875 of pX330-puro (the sequence of which is described in "nucleotide sequence of pX330-puro plasmid" below) to obtain the cleavage plasmids pX330-sgRNA1, pX330-sgRNA2, pX330-sgRNA3, pX330-sgRNA4, pX330-sgRNA5 and pX330-sgRNA6.

[0098] The cleavage plasmid pX330-sgRNA1 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330-puro with the Fw and Rv sequences of pSOX17-sgRNA1 through annealing. The other sequences of pX330-puro remain unchanged. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA1 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-GUAACUACCCCGACGUCUGA-3', targeting nucleotides 76855151 to 76855170 of the porcine SOX17 gene (NCBI Reference Sequence: NC_010446.5, CON 12-JAN-2018).

[0099] The cleavage plasmid pX330-sgRNA2 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv sequences of pSOX17-sgRNA2 through annealing. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA2 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-UAACUACCCCGACGUCUGAC-3', targeting nucleotides 76855150 to 76855169 of the porcine SOX17 gene.

[0100] The cleavage plasmid pX330-sgRNA3 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv sequences of pSOX17-sgRNA3 and annealing them. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA3 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-GAUGGGCACCCGUCAGACGU-3', targeting nucleotides 76855139 to 76855158 of the porcine SOX17 gene.

[0101] The cleavage plasmid pX330-sgRNA4 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA1 through annealing. The DNA molecule formed by annealing the Fw and Rv of pX330 is kept unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA1 encodes an sgRNA targeting the porcine NANOS3 gene. The nucleotide sequence of this sgRNA is 5'-AAAGGUGCCGGGAAGUCUUC-3', targeting nucleotides 65275496 to 65275515 of the porcine NANOS3 gene (NCBI ReferenceSequence:NC_010444.4, REGION:65274249-65279289, CON 12-JAN-2018).

[0102] The cleavage plasmid pX330-sgRNA5 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA2 through annealing. The rest of the pX330 sequence remains unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA2 encodes an sgRNA targeting the porcine NANOS3 gene (NCBI ID: NC_010444, REGION: 65274249-65279289). The nucleotide sequence of this sgRNA is 5'-CCUAAGAGGCUGGCGCGAGC-3', targeting nucleotides 65275438 to 65275457 of the porcine NANOS3 gene.

[0103] The cleavage plasmid pX330-sgRNA6 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA3 through annealing. The rest of the pX330 sequence remains unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA3 encodes an sgRNA targeting the porcine NANOS3 gene (NCBI ID: NC_010444, REGION: 65274249-65279289). The nucleotide sequence of this sgRNA is 5'-UCCACUUCUGCCUAAGAGGC-3', targeting nucleotides 65275448 to 65275467 of the porcine NANOS3 gene.

[0104] 2. Cell transfection The aforementioned cleavage plasmids were transiently transfected into the male pEpiSCs cells prepared in Example 1 using Lipofectamine 3000 (Thermo Fisher, L3000150), with each plasmid replicated in two wells. 24 hours after transfection, the medium was replaced with fresh 3i / LAF medium, and puromycin (Puro, Thermo Fisher, A1113803) was added to the 3i / LAF medium at a final concentration of 0.5 μg / mL for selection. After culturing for 3 days, pEpiSCs cells transfected with the plasmids were obtained.

[0105] 3. Cutting efficiency verification According to the instructions, genomic DNA was extracted from pEpiSCs cells transfected with plasmids as described in "2. Cell Transfection" using the TIANamp Genomic DNA Extraction Kit (TIANGEN, DP316). PCR primers (pSOX17-sgRNA test, pNANOS3-sgRNA test, primers as described above) were designed to generate amplified fragments containing sgRNA target sites, and mutations were detected by Sanger sequencing. The PCR products were further digested with T7 endonuclease I (T7E1). The digestion system consisted of: 200-500 ng of gel-recovered DNA, 2 μL of 10×NEBuffer2, and 20 μL of deionized water. The annealing program was: 95℃ for 5 min, 95℃ for 5 sec, -2℃ / cycle, 85℃ for 5 sec, -0.1℃ / cycle, 25℃. After heterodimer formation, 0.5 μL of T7E1 was added, mixed thoroughly, and incubated at 37℃ for 1 h. Enzyme digestion products can be detected by 2% agarose gel electrophoresis or by the more sensitive 12% acrylamide gel electrophoresis. The gel electrophoresis bands are analyzed using ImageJ software to calculate the digestion efficiency.

[0106] Sequencing results showed that pSOX17-sgRNA1 and pNANOS3-sgRNA2 had higher sequencing peaks ( Figure 4 In the diagram, 'a' represents pSOX17-sgRNA1 to pSOX17-sgRNA3, and 'c' represents pNANOS3-sgRNA1 to pNANOS3-sgRNA3. Further enzyme digestion results confirmed that the cleavage efficiency of pSOX17-sgRNA1 was 51.6%, pSOX17-sgRNA2 was 28.2%, and pSOX17-sgRNA3 was 40.1%. Figure 4 (b); Therefore, pSOX17-sgRNA1 and pNANOS3-sgRNA2, which have high cleavage efficiency, were selected for further research.

[0107] 4. Donor plasmid construction Homologous arms of the SOX17 gene (5'arm: 1116bp, 3'arm: 842bp) and the NANOS3 gene (5'arm: 693bp, 3'arm: 699bp) were obtained by PCR amplification. Wild-type pEpiSCs genomic DNA was used as a template, and amplification was performed using PrimeSTAR GXL DNA polymerase (TaKaRa, R050A). The required primers included Fw and Rv of pSOX17-5'arm, Fw and Rv of pSOX17-3'arm, Fw and Rv of pNANOS3-5'arm, and Fw and Rv of pNANOS3-3'arm (as described above).

[0108] The nucleotide sequence of the amplified pSOX17-5'arm is nucleotides 76855154 to 76856269 of NCBI genbank number NC_010446 REGION (update: 12-JAN-2018).

[0109] The nucleotide sequence of the amplified pSOX17-3'arm is: nucleotides 76854312 to 76855153 from NCBI genbank number NC_010446.5 (update: 12-JAN-2018).

[0110] The nucleotide sequence of the amplified pNANOS3-5'arm is nucleotides 65275456 to 65276148 of NCBI GenBank number NC_010444.4 (update: 12-JAN-201).

[0111] The nucleotide sequence of the amplified pNANOS3-3'arm is nucleotides 65274749 to 65275447 in NCBI genbank number NC_010444.4 (update: CON 12-JAN-2018).

[0112] Subsequently, the 5'arm and 3'arm of the amplified pSOX17 from the genome were recombined into the plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' (the sequence of which is described in "nucleotide sequence of donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid" below) by enzyme digestion and ligation, thereby obtaining the donor plasmids (donor pSOX17-5'arm-3×Flag-PNP-tdtomato-3'arm and donor pNANOS3-5'arm-3×Flag-PNP-tdtomato-3'arm).

[0113] The donor plasmid donor pSOX17-5'arm-3×Flag-PNP-tdtomato-3'arm is a recombinant vector obtained by replacing nucleotides 17-1183 and 4192-4220 of the plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' with pSOX17-5'arm and pSOX17-3'arm respectively, while keeping the other nucleotides of the donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid unchanged.

[0114] Donor plasmid pNANOS3-5'arm-3×Flag-PNP-tdtomato-3'arm: This is a recombinant vector obtained by replacing nucleotides 17-1183 and 4192-4220 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' with pNANOS3-5'arm and pNANOS3-3'arm respectively, while keeping the other nucleotides of donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid unchanged.

[0115] 5. Construction of the pEpiSC system for SOX17-tdTomato and NANOS3-tdTomato key-in reports The aforementioned pEpiSCs identified as male were washed with DPBS and then dissociated using Accutase at 37°C for 5 min. Dispersed single cells were seeded onto DR4 MEF feeder cells (purchased from Thermo Fisher, catalog number A34966) for 6-8 hours (DR4 MEF feeder cells were pre-cultured in MEF medium for 12 hours). The medium was then replaced with antibiotic-free medium. Lipofectamine 3000 and 2 μg DNA (1 μg pX330-sgRNA plasmid (cutting plasmid pSOX17-sgRNA1 or cutting plasmid pNANOS3-sgRNA2) were gradually mixed with OptiMEM (Thermo Fisher, 31985070) and then added to pEpiSCs. 24 hours post-transfection, cells were screened with 0.5 μg / mL puromycin for 7 consecutive days. Viable single clones of pEpiSCs were manually picked using pipettes and amplified for 10-14 days. Genomic DNA was extracted from the gene-edited cells, and site-specific integration of the 5' and 3' ends of the targeting vector was examined. Using 1 μL of DNA as a template, 0.5 μL PrimeSTAR GXL DNA polymerase (TaKaRa, R050A), 5 μL 5×PrimeSTAR GXL buffer, 2 μL dNTP mixture, 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, and 12 μL nuclease-free water were added for PCR. The reaction program was as follows: 98℃ for 10 s, 55℃ or 60℃ for 15 s, and 68℃ for 1 min / kb for 35 cycles. Gel electrophoresis confirmed that the size of the PCR product matched the expected amplicon size. The primer pairs in the aforementioned system include the Fw and Rv of pSOX17-3×Flag-P2A-NLS-tdTomato-puro and the Fw and Rv of pNANOS3-3×Flag-P2A-NLS-tdTomato-puro (primer information as described above).

[0116] Two homozygous SOX17 knock-in pEpiSCs clones were obtained (numbered 5 and 6). Figure 5 a) and eight NANOS3 homozygous knock-in pEpiSCs clones (numbered 2, 4, 7, 8, 10, 15, 16 and 17, Figure 5 (b) (respectively named) SOX17 - P2A - tdTomato ( STpEpiSCs cells and NANOS3 - P2A - tdTomato ( N3T pEpiSCs cells.

[0117] Compared to male pEpiSCs, SOX17 - P2A - tdTomato ( ST The pEpiSCs cells have a 3×Flag-P2A-NLS-tdTomato-loxP-pgk-puro-loxP fragment inserted before the SOX17 gene stop codon in the male pEpiSCs genome (nucleotides 76855153 to 76855154 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3', nucleotides 1179-4196). This will be discussed later. SOX17 - P2A - tdTomato ( ST pEpiSCs cells are abbreviated as pEpiSCs. ST pEpiSCs cells.

[0118] Compared to male pEpiSCs, NANOS3 - P2A - tdTomato ( N3T The pEpiSCs cells have a 3×Flag-P2A-NLS-tdTomato-loxP-pgk-puro-loxP fragment inserted before the NANOS3 gene stop codon (nucleotides 65275455 to 65275456 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3') in the male pEpiSCs genome (NCBI number: NC_010444.4).

[0119] 6. SOX17 - P2A - tdTomato ( ST Functional identification of pEpiSCs (1) Alkaline phosphatase staining Logarithmic growth phase ST pEpiSCs cells were stained using the alkaline phosphatase staining method described above, and the results are as follows: Figure 6 As shown in Figure a, ST pEpiSCs cell clones maintain alkaline phosphatase (ALP) positivity.

[0120] (2) Karyotype analysis Analysis using the karyotype analysis method described above ST The karyotype of pEpiSCs cells showed that... ST pEpiSCs cells possess a normal male karyotype (37+XY). Figure 6 (b)

[0121] (3) Gene expression level analysis The detection was performed according to the method described in "3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in the prior art" of this embodiment. ST pEpiSCs cells NANOG Gene, OCT4 Gene, SOX2 The relative expression levels of genes were detected using primers including pig NANOG-Fw and pig NANOG-Rv, pig OCT4-Fw and pig OCT4-Rv, and pig SOX2-Fw and pig SOX2-Rv.

[0122] The results are as follows Figure 6 As shown in c, SOX17 - P2A - tdTomato ( ST pEpiSCs NANOG Gene, OCT4 Gene, SOX2 The genes remained unchanged compared to wild-type cells and can be used for subsequent analysis.

[0123] (4) Immunofluorescence The immunofluorescence method described above was used to... ST pEpiSCs cells were stained with primary antibodies NANOG, OCT4, and SOX2. Results are as follows: Figure 6 As shown in d, ST pEpiSCs cells express the core pluripotency transcription factors NANOG, OCT4, and SOX2, indicating that they maintain their pluripotency characteristics.

[0124] (5) Verification of endoderm differentiation The composition of the endoderm (DE) induction medium was: 10 ng / mL bone morphogenetic protein 4 (BMP4, PeproTech, 120-05), 5 μM SB431542 (Selleckchem, S1067) and 10 ng / mL bFGF, with the remainder being the aforementioned aRB27 basal medium.

[0125] Will STAfter culturing pEpiSCs cells in 3i / LAF medium for 48 hours, the medium was aspirated, the cells were washed with DPBS, and the medium was replaced with endoderm induction medium for 2-3 more days.

[0126] The results showed that, ST tdTomato fluorescence was detected in pEpiSC-derived cells that expressed GATA4 but not OCT4. Figure 6 (e). Immunofluorescence (IF) experiments further confirmed the co-localization expression of tdTomato and SOX17 or 3×Flag in the nuclei of DE cells, indicating that SOX17-P2A-tdTomato fluorescence accurately reflects the endogenous expression pattern of the SOX17 gene. Figure 6 (f)

[0127] two, ST pEpiSCs differentiate into mesoendothelial progenitor cells (pMEPCs) in vitro. The composition of the mesendoderm (ME) induction medium was as follows: 50 ng / mL Activin A (50-100 ng / mL is acceptable, 50 ng / mL in this example), 0.3 μM WNT agonist (CHIR99021) (0.3-1 μM is acceptable, 0.3 μM in this example), and 10 μM ROCK inhibitor (Y-27632) (5-10 μM is acceptable, 10 μM in this example), with the remainder being the aforementioned aRB27 basal medium.

[0128] The experiment was repeated 3 times, with each repetition as follows: Will ST After washing with DPBS, pEpiSCs were dissociated using Accutase at 37°C for 5 min. The dispersed single cells were seeded onto CD1 MEF feeder cells (CD1 MEF feeder cells were pre-cultured in MEF medium for 12 h), dissociated into small clumps using Accutase, centrifuged at 1000 rpm for 3 min, and then seeded into gelatin-coated culture dishes. Feeder cells were removed by differential adhesion. The collected cells were... ST pEpiSCs were re-inoculated into gelatin-coated culture dishes and cultured as a monolayer in mesendoderm (ME) induction medium. Mesendoderm (ME) differentiation was monitored every 6 hours. Figure 7 (a) The specific methods for monitoring the differentiation of the mesoderm are as follows: 1. ST Bright-field images and real-time ST fluorescence images of pEpiSCs differentiated through monolayer culture. Figure 7 b is ST Bright-field images and real-time ST fluorescence images of pEpiSCs during monolayer culture differentiation into ME were captured. Analysis was performed using software. Figure 7 The fluorescence intensity of SOX17 in b is shown in the results. Figure 7 As shown in Figure c, the relative strength of SOX17 increases significantly with increasing differentiation time.

[0129] 2. Flow cytometry analysis of pEpiSCs and STs during differentiation + Cell ratio After differentiation ST pEpiSCs cells were washed with DPBS and dissociated with 0.25% trypsin-EDTA. Single cells were resuspended in pre-chilled DPBS containing 1% BSA and passed through a 40 μm filter. The cell suspension was sorted and analyzed using a MoFlo® high-performance cell sorter (Beckman Coulter). FACS data were analyzed using Summit 5.2 software (Dako Cytomation, Copenhagen, Denmark) and Flowjo 10.1r5 (BD Biosciences). All experimental replicates used the same gating parameters.

[0130] The results are as follows Figure 7 As shown in d, with the increase of differentiation time, ST The proportion of SOX17-tdTomato-positive cells was significantly increased in pEpiSCs cells.

[0131] 3. Analyze the mRNA expression levels of SOX17, core pluripotency markers, and original streak-related genes during ME differentiation. The experiment was repeated three times, with the specific method for each instance as follows: Referring to the method shown in "3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in the prior art" in Example 1, the ME differentiation process was detected. SOX17 , SOX2 , OCT4 , NANOG , EOMES , GSC , TBXT (Brachyury) , GATA4 , FOXA2 , PDGFRA , PRDM1 , TET2 and DNMT3B The relative expression level changes.

[0132] The results are as follows Figure 7 As shown in Figure e, with the increase of differentiation time, SOX17 The relative expression level gradually increased. SOX2 , OCT4 and NANOG The relative expression level first increases and then gradually decreases. Figure 7 The results showed that the original marker gene EOMES and GSC The expression was moderately adjusted upwards, while GATA4 , FOXA2 , PDGFRA , PRDM1 and TET2 The expression of methyltransferases is induced in the later stages of ME differentiation. DNMT3B The expression is then downgraded. It is known that... TBXT (Brachyury) TBXT expression is a common characteristic that determines the fate of germ cells (GCs). The inventors observed that TBXT expression peaked at approximately 36 hours during ME differentiation, after which it gradually decreased. Figure 7 (e).

[0133] 4. Sequential co-immunofluorescence staining The experiment was repeated 3 times, with the following steps repeated each time: Refer to the cell immunofluorescence analysis method described above to detect ST-positive cells during ME differentiation ( SOX17 - P2A - tdTomato ( ST pEpiSCs gradually express SOX17 during differentiation, leading to tdTomato Cells that test positive at this stage are called ST-positive cells, or simply ST cells. + The expression of OCT4, SOX2, and PRDM1 in ST-positive cells was analyzed, and the expression of NANOG and TBXT was detected separately. Furthermore, the expression of EOMES, GSC, GATA4, HAND1, and NANOG in ST-positive cells at 36 hours was examined.

[0134] The results are as follows Figure 7 As shown in gk, relative to male pEpiSCs cells, starting from 24h, OCT4 + SOX17 expression in cells gradually increases ( Figure 7 (g and h), while SOX2 expression decreased sharply ( Figure 7 The expressions for i and j in the NANOOG expression remain largely unchanged. Figure 7 The "l" and "m" in the text indicate that differentiated cells, over time, exit pluripotency and enter a mesoendodermal fate rather than an ectoderm fate (e.g., ...). Figure 7 (As shown in Figure k). Notably, the proportion of cells co-localized with SOX17 and OCT4 reached its highest level at 36 h, at which time TBXT was also highly expressed ( Figure 7 (l and m in the text), and also, with ST +As the number of cells gradually increased, PRDM1 expression peaked at 36 hours. Figure 7 (l and m in the text).

[0135] Further staining at 36 hours revealed ST + The cells were positive for OCT4, NANOG, PRDM1, EOMES, TBXT, GSC, and GATA4; therefore, the cells cultured for 36 hours were... SOX17 - P2A - tdTomato ( ST pEpiSCs cells are called pMEPCs cells ( Figure 7 (n) in it has the ability to induce PGC.

[0136] 5. Conventional transcriptome sequencing collect ST pEpiSCs and their differentiated pMEPCs were sent to Beijing Novogene Technology Co., Ltd. for routine transcriptome sequencing analysis. Results are as follows: Figure 7 As shown in the middle (o), which is a scatter plot, the average gene expression levels of pEpiSCs (left) and pMEPCs (right) are compared. (Adjusted) p Differentially expressed genes (DEGs) were identified using thresholds padj < 0.05 and absolute log2-fold change ≥ 1. Red indicates upregulated genes (log2-fold change ≥ 1), blue indicates downregulated genes (log2-fold change ≤ -1), and gray indicates insignificant genes. Results showed that pMEPCs upregulated the expression of 1112 genes (119 transcription factors), including... SOX17 , EOMES , MIXL1 and BMP4 They play a central role in early mesodermal differentiation; meanwhile, the expression of 1086 genes (101 transcription factors) is specifically downregulated. Figure 7 (middle o). Figure 7 As shown in p, these genes are enriched with genes related to "epithelial-mesenchymal transition", "mesoderm formation", "anteroposterior axis pattern specialization", "tubular morphogenesis", "gastrulism", "endoderm development", and "focal adhesion". The results indicate that the genes cultured for 36 hours... SOX17 - P2A - tdTomato ( ST pEpiSCs cells are similar to SOX17-positive pMEPCs in E12-E13 EPIs in vivo.

[0137] III. pMEPCs are further induced into pPGCLCs Includes the following steps: Step 1, ST pEpiSCs cells follow the "two, SOX17 - P2A - tdTomato ( ST pEpiSCs were cultured for 36 hours using the method described above for in vitro differentiation into mesoendothelial precursor cells (pMEPCs). (During this stage, the cells were identified as pMEPCs according to the aforementioned description.) Step 2: Replace the culture medium of the cells from Step 1 with PGC induction medium (see Example 1), and culture for 48 hours. During this period, every 6 hours, identify whether the aforementioned pMEPCs cells have differentiated into pPGCLCs using the following method: 1. Temporal analysis of mRNA expression levels of pluripotency markers and early PGCs markers during pPGCLC differentiation. like Figure 8 As shown in Figures a and b, the expression of red fluorescence was observed every 6 hours, and cells were collected once. The expression of the genes pig OCT4, pig NANOG, pig STELLA, pig KLF4, pig NANOS3, pig SOX17, pig PRDM1, pig TFAP2C, pig PRDM14, pig DNMT3B, pig TET2, and pig TFCP2L1 was detected according to the method described in "3. Direct Induction of Porcine Primitive Germ Cell-like Cells (pPGCLCs) in the Prior Art" in Example 1. Primers were as described above.

[0138] The results showed that starting at 6 hours, STELLA and TET2 The expression of pluripotency markers gradually increased, while pluripotency markers such as NANOG , OCT4 , KLF4 , PRDM14 and DBNT3B The expression of these genes decreased significantly. However, the expression of primordial pluripotency genes... TFCP2L1 The expression of this substance can only be detected in cells 24 hours after differentiation. Figure 8 (c). Simultaneously, starting from 6 hours, cells express early germ cell-specific genes ( NANOS3 , SOX17 , PRDM1 and TFAP2C () Figure 8 (c)

[0139] 2. Flow cytometry analysis of ST in 24-hour pPGCLCs + Cell ratio Will STpMEPCs differentiated for 24 h were washed with DPBS and dissociated with 0.25% trypsin-EDTA. Single cells were resuspended in pre-chilled DPBS containing 1% BSA and passed through a 40 μm filter. The cell suspension was sorted and analyzed using a MoFlo® high-performance cell sorter (Beckman Coulter). FACS data analysis was performed using Summit 5.2 software (Dako Cytomation, Copenhagen, Denmark) and Flowjo 10.1r5 (BD Biosciences). All experimental replicates used the same gating parameters.

[0140] The results are as follows Figure 8 As shown in d and e, after 24 hours of treatment, the proportion of SOX17-tdTomato positive cells remained at a high level of 50.5%.

[0141] 3. Protein expression level detection First use ST Time-series analysis of pMEPCs during culture and differentiation process, ST + The co-expression of cells with OCT4, PRDM1, and TFAP2C proteins was used to identify true pPGCLCs.

[0142] like Figure 8 As shown in f and g, although the expression level of OCT4 decreased during differentiation, most OCT4-positive cells remained consistent with ST. + Cell colocalization, and simultaneously, dual fluorescence detection results based on ST / PRDM1 (fluorescence results of tmotdo and PRDM1) and ST / TFAP2C (fluorescence results of tmotdo and TFAP2C). Figure 8 (h and i in the text), the inventors believe that the ST differentiates 24h. + The cells should be assumed to be pPGCLCs.

[0143] Secondly, ST cultured for 24 hours after replacing the PGC induction medium... + The expression of TNAP, NANOS3, and SOX17 in the cells (i.e., ST pPGCLCs cultured for 60 h) was analyzed using the aforementioned immunofluorescence assay. Simultaneously, the previously constructed NANOS3-tdTomato cell line was used for replication and validation experiments.

[0144] Most ST + pPGCLCs were positive for NANOS3, the mitotic activity marker (MKI67), and TNAP. Figure 8 Using the same conversion method, the inventors obtained NANOS3-3×Flag-P2A-NLS-tdTomato+ (N3T)pPGCLCs, these cells exhibit ALP activity ( Figure 8 In addition, SOX17, OCT4, NANOOG, and PRDM1 are co-expressed, but SOX2 expression is low or absent. Figure 8 (l).

[0145] 4. Epigenetic level testing Experimental methods: Bisulfite sequencing: Genomic DNA was extracted from pEpiSCs and pPGCLCs using the TIANamp Genomic DNA Kit (Tiangen, DP304). DNA was bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research, D5005). Nested PCR amplification was performed using HotStart DNA polymerase (TAKARA, R007Q). Specific primers for the H19 / IGF2 and SNRPN DMR imprinted regions were as described previously. The PCR products were cloned into the pMD19T vector (TAKARA, 6013) and transformed into *E. coli*, with at least 20 insertion-positive clones sequenced. The bisulfite cytosine conversion rate was greater than 98%. The obtained data were analyzed using an online methylation analysis and quantification tool (QUMA, http: / / quma.cdb.riken.jp / ).

[0146] The results showed that the inventors compared pEpiSCs and pPGCLCs in terms of imprinted genes. H19 / IGF2 and SNRPN Methylation status of differentially methylated regions (DMRs). Bisulfite sequencing results showed that the methylation level of pEpiSCs was approximately 70% to 80%, while the level of pPGCLCs dropped sharply to half of that, indicating a significant DNA demethylation process. Figure 8 (m).

[0147] The fluorescence of 5-methylcytosine (5mC), methylcytosine dioxygenase 2 (TET2), H3K27me3, and H3K9me2 was detected using the aforementioned immunofluorescence assay. Immunofluorescence analysis of methylcytosine dioxygenase 2 (TET2) and its product 5-methylcytosine (5mC) showed that differentiated pPGCLCs exhibited low levels of overall DNA methylation. Figure 8 (n, o, p, and q). Furthermore, these cells also showed significant changes in histone modification levels, manifested as a decrease in H3K9me2 levels and an increase in H3K27me3 levels (n, o, p, and q). Figure 8(r, s, t, and u). In summary, the inventors' results demonstrate that pEpiSC-derived pPGCLCs undergo genome-wide epigenetic reprogramming.

[0148] 5. Conventional transcriptome and single-cell transcriptome sequencing Experimental Methods: pEpiSCs and their differentiated pMEPCs and pPGCLCs were collected and sent to Beijing Novogene Technology Co., Ltd. for routine transcriptome sequencing analysis. Single-cell RNA sequencing libraries were prepared using a modified Smart-seq2 method. Briefly, single cells were transferred via pipette to lysis buffer containing 8-base barcodes. Subsequently, first-strand cDNA was synthesized and amplified in reverse transcription (RT) using a mixture containing 4U RNase inhibitor, 100U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1M mdNTPs (TAKARA, 4019), 60mM magnesium chloride, 3μM RT primers, and 10μM TSO primers. After PCR amplification, the products were purified using 0.8×AMPure XP magnetic beads (Beckman, A63882). Biotin PCR was then performed for enrichment. Finally, a single-cell RNA sequencing library was constructed according to the instructions for the KAPA Hyper PrepKit series (KAPA, KK8054). The high-quality library was sequenced at 150 bp paired ends using Illumina NovaSeq X Plus (Novogene).

[0149] Plain transcriptome analysis showed that differential gene heatmaps and correlation coefficient heatmaps indicated that pPGCLCs and pMEPCs were more similar than pEpiSCs. Figure 9 (a and b in the text). Figure 9 The c-analysis results showed that differentially expressed genes that were significantly upregulated during pPGC specialization included SOX17 , PRDM1 , CXCR4 , ITGB3 and DAB2 These genes are significantly enriched in the extracellular space, calcium ion binding, extracellular matrix, and cell migration involved in gastrulation. Figure 9 (d). To further elucidate the differentiation trajectory of pPGCLCs, the cell types involved in the differentiation process of pPGCLCs (pEpiSCs, ST) were analyzed. + pMEPCs, D2.5 somatic cells and N3T +Single-cell RNA sequencing analysis was performed on pPGCLCs. The results showed that in the UMAP diagram, pEpiSCs roughly corresponded to porcine epiblast cells (pEPIs) at E10-E11 stages, pMEPCs clustered with pEPIs at E12-E13 stages, somatic cells at D2.5 stage were similar to somatic cells at E14 stage, while pPGCLCs were equivalent to early PGCs at E14 stage in vivo. Figure 9 (e). Furthermore, the expression of key marker genes was consistent with the inventors' cell annotation results ( Figure 9 (f)

[0150] In conclusion, SOX17 - P2A - tdTomato pEpiSCs cells ( ST pEpiSCs cells differentiated into pMEPCs after being induced and cultured in mesoendothelial induction medium for 36 hours. pMEPCs then differentiated into pPGCLCs after being induced and cultured in PGC induction medium for 24 hours.

[0151] It should be understood that constructing male pEpiSCs cells as SOX17 - P2A - tdTomato ( ST pEpiSCs or NANOS3 - P2A - tdTomato ( N3T The pEpiSCs cells are used to determine or assist in determining the cell differentiation stage by using the expression or expression level of the marker proteins SOX17 and NANOS3. However, this should not be used to limit the methods provided in this invention for inducing pMEPCs into pPGCLCs, pEpiSCs into pPGCLCs, or pEpiSCs into pMEPCs.

[0152] pMEPCs have at least the following characteristics: high expression of SOX17 protein and ability to differentiate into primordial germ cells.

[0153] pPGCLCs possess at least the following characteristics: morphology, gene expression, epigenetic features, and developmental potential similar to primordial germ cells in an in vivo day 14 embryo.

[0154] Example 3: pPGCLCs develop into spermatogonial-like cells in pig-mouse xenograft testes Experimental methods: 1. Isolation of fetal testicular somatic cells Pregnant CD1 female mice (obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., catalog number 201) were euthanized by cervical dislocation, and E12.5 embryos were isolated from the uterus. Gonads containing the mesonephric fossa were collected in pre-cooled DPBS containing 1% penicillin-streptomycin. Fetal testes were carefully identified based on the appearance of the gonads (testicular cord structure) and dissected under a dissecting microscope using a tungsten needle. The homologous gonadal tissue was repeatedly minced with fine scissors for 5 min, washed with DPBS, and then dispersed by gentle shaking with 0.25% trypsin-EDTA in a 37°C water bath for 5 min. Once the tissue samples dissociated into single cells, the reaction was terminated with MEF medium. The cell suspension was filtered through a 40 μm filter and counted. The difference in adhesion ability between germ cells and somatic cells, combined with culture medium selection, was used to remove endogenous mouse germ cells. In short, single-cell suspensions were inoculated into pre-coated culture dishes at 37°C for 2 hours, then the suspension cells were removed. This step was repeated twice, and the culture medium was changed every 12 hours for two days to obtain high-purity mouse fetal testicular somatic cells (mFTSCs).

[0155] 2. Generation of pig-mouse xenogeneic reconstructed testes The polymer medium consisted of 10% KSR (volume percentage), 0.1 mM β-mercaptoethanol, 100 U / mL penicillin / streptomycin, 150 μM ascorbic acid, 10 μM Y-27632, with the remainder being the minimum essential medium (α-MEM, Invitrogen, 12561056).

[0156] Polymerization medium containing 1% polyvinyl alcohol (PVA): Add 1% PVA by volume to the aforementioned polymerization medium.

[0157] To generate a suspension polymer, mFTSCs and pPGCLCs (derived from...) ST pEpiSCs cell group or pEpiSCs cells) were mixed at a cell number ratio of 10:1 (a ratio of 5:1 to 10:1 is also acceptable) and seeded into U-shaped 96-well plates (Greiner, 650185) at a seeding density of 2 × 10⁶ cells / well. 4Cells / well were cultured in polymeric medium containing 1% PVA. After two days of incubation, the resulting aggregates were collected and transplanted under a stereomicroscope (Olympus, SZX16) using a pipette into the subcapsular region of the kidneys of 8-week-old male CD1 mice. 10-20 cells were transplanted from each kidney. Following transplantation, the host mice were euthanized at different time points, and the xenogeneic reconstituted testes (xrTestes) were collected and fixed for subsequent histological and immunofluorescence analysis.

[0158] Experimental results: To assess the developmental capacity of pPGCLCs, the inventors employed a xenogeneic reconstruction strategy, reassembling pPGCLCs with mouse fetal testicular somatic cells (mFTSCs), followed by mouse kidney capsule transplantation. Figure 10 (a) Using differential adhesion, sufficient somatic cells were isolated from the testes of mouse fetuses. As a first step in applying this method to livestock, the inventors first tested whether the dissociated somatic cells could re-aggregate and grow under the renal capsule after the removal of endogenous mouse primordial germ cells (mPGCs). Figure 11 (ad). The inventors inoculated E12.5 mFTSCs, from which mPGCs were completely removed, into U-shaped dishes and cultured them for 2 days, after which they formed dense aggregates (ad). Figure 11 (e.g.) 55 days after renal capsule transplantation, the graft exhibited numerous tubular structures composed of GATA4-positive Sertoli cells (SCs). Figure 11 In the h and i samples, neither DDX4 (a marker of spermatogonium to round spermatogonium stage) nor STRA8 (a marker of spermatogonium differentiation) positive GCs were present, indicating that mPGCs had been successfully removed. Figure 11 Furthermore, 30 days after transplantation of the self-aggregated pPGCLCs, no tumor material or tubular structure development was observed in the grafts. Figure 11 (j). It is worth noting that, SOX17 - P2A - tdTomato ( ST Positive cells were maintained in the graft, but did not express germ cell and stem cell-specific markers. Figure 11 (k), which further confirms the reliability of the strategy.

[0159] Subsequent histological examination by the inventors revealed that 30 days after transplantation, the xenograft developed into testicular-like tissue, termed xenograft reconstructed testes (xrTestes). Figure 10 b and Figure 12 (a) Approximately 100 days after xenograft transplantation, the tubular structures in the testes enlarged and exhibited a vacuolated appearance, remarkably similar to the seminiferous tubules in the testes of mature boars. Figure 10 b and Figure 12 (b) Notably, no teratoma formation was observed in the host animals at any of the analyzed time points. Co-immunostained seminiferous tubules from day 30 xenografts exhibited normal cellular tissue structure, with GATA4+ Sertoli cells encapsulating (pre)spermatogonia markers (DAZL, DDX4, PLZF)-positive germ cells, faithfully reproducing the testicular microenvironment in age-matched (one-month-old) porcine testes. Figure 10 c and Figure 12 (c). By detecting co-localization of SOX17 with DAZL, NANOG, and PLZF, as well as OCT4 and STRA8, the identity of GCs in xrTestes on day 30 was further clarified. Figure 10 d and Figure 12 (d). The inventors discovered that SOX17 is only a marker of endoderm lineage in mouse fetal testes, but in pigs, it is also expressed in GCs, as observed by immunofluorescence. Figure 10 (e, f). Correspondingly, on day 30 xrTestes, key markers of ST+ cells and spermatogonia (NANOG and DAZL) ( Figure 10 d and Figure 12 The presence of both GFP and d indicates that they originate from pPGCLCs rather than endogenous mouse PGCs. Furthermore, immunofluorescence analysis showed that in xrTestes 30 days after transplantation, GFP-positive pPGCLCs were able to efficiently differentiate into spermatogonial-like cells (…). Figure 12 (d, e). The inventors performed immunofluorescence analysis on xrTestes on day 100 to determine whether pPGCLCs could develop into more advanced male germ cell types. The results showed that ST+ pPGCLCs-derived germ cells still strongly expressed NANOG, and spermatocytes undergoing first meiosis (γH2AX+ / RAD51+ / DDX4+ / SYCP3+) appeared in tubular structures composed of GATA4+ SCs. Figure 10 Zhongg and Figure 12 (f), indicating that pPGCLCs develop further posteriorly. As a positive control, immunostaining showed that 3-month-old pig testicular tissue expressed DDX4, RAD51, γH2AX and SYCP3 (f), indicating that pPGCLCs develop further posteriorly. Figure 12 (g). Subsequently, the inventors detected the expression of NANOS3, STRA8, and SYCP3 in these germ cells at the mRNA level, but not the haploid marker genes PRM1, TNP1, and ACROSIN (g). Figure 12 (h), which is sufficient to prove that they have initiated the first meiotic recombination, but have not yet completed it. The above results confirm that mFTSCs in xrTestes support the survival of pPGCLC and promote its further development into differentiated spermatogonial-like cells.

[0160] The nucleotide sequence of the pX330-puro plasmid is as follows:

[0161] The nucleotide sequence of the donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid is as follows:

[0162] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing xenografts, characterized in that, This includes culturing isolated mouse fetal testicular somatic cells and porcine primordial germ cell-like cells to obtain xenografts. The porcine primordial germ cell-like cells are prepared by the following steps: inducing isolated porcine gastrulated epiblast stem cells into mesoderm precursor cells, and then inducing them into primordial germ cell-like cells.

2. The method according to claim 1, characterized in that, The ratio of mouse fetal testicular somatic cells to porcine primordial germ cell-like cells was 5:1-10:

1.

3. The method according to claim 2, characterized in that, The mouse fetal testicular somatic cells were derived from the testicular tissue of mouse E12.5 fetuses.

4. The method according to claim 3, characterized in that, The induction time for inducing isolated porcine gastrulated epiblast stem cells into mesoendothelial precursor cells is 30-42 hours. The induction is carried out using Composition 1, which includes an activator of the cell signaling pathway Activin / NODAL and an agonist of the cell signaling pathway Wnt / β-catenin.

5. The method according to claim 4, characterized in that, The induction time for the mesoderm precursor cells to be induced into primordial germ cell-like cells is 12-24 hours. The induction is carried out using composition two, which includes bone morphogenetic protein 4, human leukemia inhibitory factor, stem cell factor and epidermal growth factor.

6. The method according to claim 5, characterized in that, The culture medium used to obtain xenografts by mixing isolated mouse fetal testicular somatic cells and porcine primordial germ cell-like cells contains composition three, which includes polyvinyl alcohol.

7. The method according to claim 6, characterized in that, The volume percentage of polyvinyl alcohol in the culture medium is 1%.

8. A method for preparing spermatogonial-like cells, comprising transplanting a xenograft prepared according to any one of claims 1-7 into the renal capsule of a mouse to obtain spermatogonial-like cells.

9. The product, characterized in that, The product is any one of the following: M1) is composed of composition one as described in claim 4, composition two as described in claim 5, and composition three as described in claim 6 or 7; M2) Products for preparing xenografts, including isolated mouse fetal testicular somatic cells, porcine primordial germ cell-like cells, composition one of claim 4, composition two of claim 5, and composition three of claim 6 or 7.

10. The xenograft prepared by the method of any one of claims 1-7 or the spermatogonial-like cells prepared by the method of claim 8.

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