A method for obtaining primordial germ cells by inducing haploid stem cells

Abstract

The present invention relates to the field of cell culture technology, and in particular to a method for obtaining primordial germ cells (PGCs) by inducing haploid stem cells from androecium. The method provided by the present invention is a highly efficient and stable in vitro PGC culture system. By pre-inducing haploid stem cells in a specific pre-treated culture medium and then transferring them to GK15 culture medium for induction culture, primordial germ cells (PGCs) can be obtained. After four days, the PGC ratio reaches 73%, a significant improvement over the existing technology (3%), providing new materials for beef cattle breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for obtaining primordial germ cells by inducing androgenic haploid stem cells. Background Art

[0002] Primordial germ cells (PGCs) are the embryonic precursors of germ cells. They develop into highly specialized germ cells, transmitting genetic material and epigenetic information to offspring, thereby ensuring the continuation of the species. PGC development is dynamic and complex, encompassing a series of processes including specialization, migration, proliferation, sex differentiation, and extensive epigenetic reprogramming. In mammals, PGC formation typically occurs during the gastrulation stage of embryonic development. After implantation in the uterus, proximal ectoderm cells differentiate into PGCs under the combined action of the BMP and Wnt signaling pathways. PGCs are a precursor cell population of gametes, derived from pluripotent stem cells. They provide an alternative source of gametes for reproduction, contributing to a deeper understanding of the fundamental mechanisms of reproductive biology, including sex determination, cell differentiation, and germ cell development, and providing a theoretical basis for optimizing breeding strategies.

[0003] This technology holds broad application prospects in beef cattle breeding. By gene editing or selectively breeding PGCs, individuals with specific traits can be rapidly obtained, significantly shortening the breeding cycle. These traits can also be stably passed on to offspring, achieving genetic improvement in beef cattle. Gene editing of PGCs can introduce superior genes into offspring, ensuring their retention and expression during the reproductive process. Furthermore, research on PGCs could lead to new methods for controlling the sex of offspring, enabling the selection of beef bulls or dairy cows in cattle breeding, thereby improving economic efficiency.

[0004] However, the acquisition, culture, and transplantation of PGCs remain challenging, particularly in large livestock, such as beef cattle. The isolation and purification of PGCs from embryos is complex, with low survival rates, and establishing an efficient and stable in vitro culture system for PGCs remains a challenge, limiting in-depth research and application. Summary of the Invention

[0005] To address the above issues, the present invention provides a method for obtaining primordial germ cells by inducing androgenetic haploid stem cells. The androgenetic haploid stem cells obtained by the present invention can be efficiently isolated and cultured into bovine PGCs, providing new materials for beef cattle breeding.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for obtaining primordial germ cells by inducing androgenic haploid stem cells, comprising the following steps:

[0008] Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and further includes the following components at concentrations: 5% KSR by volume, 80-120 ng / mL BMP4, 8-10 μM CHIR99021, 2-5 μM IWR1, and 8-12 μM Y-27632;

[0009] The pre-induced androgenic haploid stem cells are cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and also includes the following components at a volume concentration of 15% KSR, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / ml BMP4, 100 ng / mL SCF, 50 ng / ml epidermal growth factor, 10 ng / mlL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0010] Preferably, the pre-induction time is 24 hours, the pre-induction temperature is 37°C, the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO2 in the pre-induction environment is 5%; the culture time is 4 days, the culture temperature is 37°C, the culture environmental humidity is saturated humidity, and the volume concentration of CO2 in the culture environment is 5%.

[0011] Preferably, before the androecium haploid stem cells are pre-induced in the pretreatment culture medium, the pre-induction container is treated with recombinant human fibronectin.

[0012] Preferably, before culturing the pre-induced androgenic haploid stem cells in the GK15 culture medium, the method further comprises: performing a digestion treatment on the pre-induced androgenic haploid stem cells.

[0013] Preferably, the reagent used in the digestion treatment includes Accutase.

[0014] Preferably, the androecium haploid stem cells are bovine androecium haploid stem cells.

[0015] Preferably, the primordial germ cells express the SOX17 gene, the OCT4 gene and the NANOG gene, but do not express the SOX2 gene.

[0016] Preferably, the primordial germ cells have reduced expression levels of histone H3 lysine 9 dimethylation and 5-methylcytosine, and increased expression levels of histone H3 lysine 27 3 methylation.

[0017] Beneficial effects:

[0018] The method provided by the present invention is an efficient and stable PGC in vitro culture system. By pre-inducing haploid stem cells in a specific pretreated culture medium and then transferring them to GK15 culture medium for induction culture, primordial germ cells can be induced. The induction ratio reaches 73% after 4 days, which is a very significant improvement compared with the existing technology (3%). No haploid cells were found during the inspection of PGCs, providing new materials for beef cattle breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 Schematic diagram of the construction of PGC-specific dual-reporter gene vector and electrophoresis results;

[0021] Figure 2 Schematic diagram of the process of induction of haploid stem cells into primordial germ cells and the fluorescence results;

[0022] Figure 3 The results of flow cytometric analysis of haploid stem cells cultured for different days are shown;

[0023] Figure 4 These are the results of laser confocal microscopy observation of bovine haploid stem cells. DETAILED DESCRIPTION

[0024] The present invention provides a method for obtaining primordial germ cells by inducing androgenic haploid stem cells, comprising the following steps:

[0025] Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and further includes the following components at concentrations: 5% KSR by volume, 80-120 ng / mL BMP4, 8-10 μM CHIR99021, 2-5 μM IWR1, and 8-12 μM Y-27632;

[0026] The pre-induced androgenic haploid stem cells are cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and also includes the following components at a volume concentration of 15% KSR, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0027] The present invention pre-induces haploid stem cells in a pre-treated culture medium to obtain pre-induced haploid stem cells. In one embodiment, the haploid stem cells are bovine haploid stem cells. In one embodiment, the present invention utilizes the method described in steps 2 and 3 of Example 1 of Chinese patent CN119120578A to culture bovine haploid stem cells. The method provided by the present invention can efficiently isolate and obtain bovine haploid stem cells.

[0028] As an embodiment, before the pre-induction of the haploid stem cells in the pre-treatment culture medium, the pre-induction container is treated with recombinant human fibronectin. As an embodiment, the pre-induction container can be a culture dish.

[0029] As an embodiment, the pretreatment culture medium of the present invention uses N2B27 culture medium as the base culture medium and also includes the following components: KSR with a volume concentration of 5%, BMP4 of 100 ng / mL, CHIR99021 of 6 μM, IWR1 of 2.5 μM and Y-27632 of 10 μM.

[0030] As an embodiment, the pre-induction time is 24 hours, the pre-induction temperature is 37° C., the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO 2 in the pre-induction environment is 5%.

[0031] After obtaining pre-induced androgenic haploid stem cells, the present invention cultured the pre-induced androgenic haploid stem cells in GK15 culture medium to obtain primordial germ cells. In one embodiment, prior to culturing the pre-induced androgenic haploid stem cells in GK15 culture medium, the method further includes subjecting the pre-induced androgenic haploid stem cells to a digestion treatment. In one embodiment, the reagent used in the digestion treatment includes Accutase.

[0032] The GK15 culture medium described in the present invention is based on GMEM culture medium and further includes the following components at a volume concentration of 15% KSR, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0033] As an embodiment, the culture time is 4 days, the culture temperature is 37° C., the culture environment humidity is saturated humidity, and the volume concentration of CO 2 in the culture environment is 5%.

[0034] In one embodiment, the primordial germ cells express the SOX17 gene, the OCT4 gene, and the NANOG gene, but do not express the SOX2 gene.

[0035] As an embodiment, the primordial germ cells have reduced expression levels of histone H3 lysine 9 dimethylation and 5-methylcytosine, and increased expression levels of histone H3 lysine 27 3 methylation.

[0036] The method provided by the present invention is an efficient and stable PGC in vitro culture system. By pre-inducing haploid stem cells in a specific pretreated culture medium and then transferring them to GK15 culture medium for induction culture, primordial germ cells can be induced. The induction ratio reaches 73% after 4 days, which is a very significant improvement compared with the existing technology (3%). In addition, no haploid cells were found during the inspection of PGCs, providing new materials for beef cattle breeding.

[0037] To further illustrate the present invention, a method for obtaining primordial germ cells by inducing androgenic haploid stem cells provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] 1. The method described in steps 2 and 3 of Example 1 of Chinese patent CN119120578A was used to culture bovine haploid stem cells.

[0040] 2. To determine the ability of bovine haploid stem cells to differentiate into primordial germ cells in vitro, the primordial germ cell-specific genes PRDM1 (also known as BLIMP1) and TFAP2C were selected, and the reporter fluorescent gene was connected to the target gene to construct a PRDM1-tdTomato (PT) and TFAP2C-mNeonGreen (TG) dual reporter gene recombinant vector in vitro ( Figure 1 A and C in the middle); sgRNA in the CRISPR-Cas9 gene editing system is designed for the stop codon region of PRDM1 and TFAP2C, and is used to interact with Cas9 to bind to the target gene and specifically cut the stop codon region of the target gene, causing double-strand breaks in the target DNA near the stop codon, causing the cell to automatically start homologous recombination repair. The present invention transfects the synthesized dual-reporter gene linear fragment into the target cell, which can be used as a template for DNA repair, thereby guiding the target gene to synthesize DNA according to the sequence of the dual-reporter vector during DNA repair, and ultimately realizes that PRDM1 and TFAP2C carry fluorescent reporter genes respectively in bovine haploid stem cells. As the bovine haploid stem cells differentiate into primordial germ cells, PRDM1 and TFAP2C are expressed fluorescently while being expressed, and the differentiation ability of the bovine haploid stem cells into primordial germ cells is verified by fluorescence. The specific steps are as follows:

[0041] 1) The target fragments tdTomato and mNeonGreen were synthesized by Shanghai Bioengineering. The specific nucleotide sequences are as follows:

[0042] tdTomato (SEQ ID NO. 1):

[0043]

[0044] mNeonGreen (SEQ ID NO.2):

[0045] 5'--3'.

[0046] 2) PCR was used to splice tdTomato and mNeonGreen with the homology arm sequences selected from PRDM1 and TFAP2C, respectively, to form the target linear fragment. Multiple rounds of PCR reactions were required during the construction process, as follows:

[0047] An 1120-bp sequence upstream of the PRDM1 stop codon was selected as the left homology arm of the PRDM1 gene, a 1440-bp sequence upstream of the TFAP2C stop codon was selected as the left homology arm of the TFAP2C gene, a 987-bp sequence downstream of the PRDM1 stop codon was selected as the right homology arm of the PRDM1 gene, and a 1018-bp sequence downstream of the TFAP2C stop codon was selected as the right homology arm of the TFAP2C gene. Ultimately, the fluorescent gene tdTomato was inserted between the left and right homology arms of the target gene PRDM1, replacing the PRDM1 stop codon. The fluorescent gene mNeonGreen was inserted between the left and right homology arms of the target gene TFAP2C, replacing the TFAP2C stop codon. Consequently, the fluorescent genes tdTomato and mNeonGreen were co-expressed along with the expression of the PRDM1 and TFAP2C genes. The primers required for the PCR reaction of the homology arms are shown in Table 1.

[0048] Table 1 Primers required for PCR reaction of homology arms

[0049]

[0050] PCR reaction system for homology arms: 25 μL of 2× Phanta Flash Master Mix, 2 μL of upstream primer F (10 μM), 2 μL of downstream primer R (10 μM), 1 μL of template DNA, and 20 μL of ddH2O;

[0051] The left and right homology arms of PRDM1 and TFAP2C were amplified using the aforementioned system, yielding DNA for the left and right homology arms of PRDM1 and TFAP2C, respectively. The upstream primer for the left homology arm of PRDM1 was F1, and the downstream primer was R1. The same procedure was used for the other homology arms. Template DNA was extracted from bovine haploid stem cells using the FastPure Cell / Tissue DNA Isolation Mini Kit (Vazyme DC102).

[0052] The PCR reaction program for the homology arms was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 58°C for 5 s, and extension at 72°C for 10 s, for 35 cycles; and final extension at 72°C for 1 min.

[0053] PRDM1 left homology arm DNA was spliced ​​with the tdTomato sequence using PCR to generate PRDM1 left homology arm + tdTomato DNA. PCR was then used to splice the left homology arm + tdTomato DNA with the PRDM1 right homology arm into a complete sequence (PRDM1 left homology arm + tdTomato + PRDM1 right homology arm). Following the same method for TFAP2C, the TFAP2C left homology arm DNA was spliced ​​with the mNeonGreen sequence to generate TFAP2C left homology arm + mNeonGreen DNA. PCR was then used to splice the TFAP2C left homology arm + mNeonGreen DNA with the TFAP2C right homology arm into a complete sequence (TFAP2C left homology arm + mNeonGreen + TFAP2C right homology arm).

[0054] The PCR reaction system for ligating the left homology arm of PRDM1 to tdTomato was as follows: 2×Phanta Flash Master Mix 25 μL, upstream primer F1 (10 μM) 2 μL, downstream primer R5 (10 μM) 2 μL, PRDM1 left homology arm DNA 1 μL, tdTomato DNA 1 μL, and ddH2O 19 μL;

[0055] The PCR reaction system for ligating the left homology arm of TFAP2C to mNeonGreen was as follows: 25 μL of 2× Phanta Flash MasterMix, 2 μL of upstream primer F3 (10 μM), 2 μL of downstream primer R6 (10 μM), 1 μL of TFAP2C left homology arm DNA, 1 μL of mNeonGreen DNA, and 19 μL of ddH2O.

[0056] The PCR reaction program for the left homology arm connected to tdTomato or mNeonGreen was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 58°C for 5 s, and extension at 72°C for 10 s, for 35 cycles; and final extension at 72°C for 1 min.

[0057] The PCR reaction system for PRDM1 left homology arm + tdTomato ligation to right homology arm was as follows: 2× Phanta FlashMaster Mix 25 μL, upstream primer F1 (10 μM) 2 μL, downstream primer R2 (10 μM) 2 μL, PRDM1 left homology arm + tdTomato DNA 1 μL, PRDM1 right homology arm DNA 1 μL, and ddH2O 19 μL;

[0058] The PCR reaction system for TFAP2C left homology arm + mNeonGreen linked to right homology arm was as follows: 2× Phanta FlashMaster Mix 25 μL, upstream primer F3 (10 μM) 2 μL, downstream primer R4 (10 μM) 2 μL, TFAP2C left homology arm + mNeonGreen DNA 1 μL, TFAP2C right homology arm DNA 1 μL, and ddH2O 19 μL;

[0059] The PCR reaction program for the left homology arm + tdTomato or mNeonGreen connected to the right homology arm was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 58°C for 5 s, and extension at 72°C for 10 s, for 35 cycles; and final extension at 72°C for 1 min.

[0060] 3) Construction of the plasmids required for CRISPR-Cas9 was performed as follows: The BbsI restriction site on the pSpCas9(BB)-2A-Puro(PX459) V2.0 (Addgene, 62988) plasmid was selected and digested with BbsI to obtain a linear plasmid fragment. This fragment was then ligated with synthetic sgRNAs targeting the stop codons of PRDM1 and TFAP2C to construct the recombinant plasmid. The sgRNA sequences are as follows:

[0061] PRDM1-sgRNA-F: 5'-caccctcgaggacttacgacttggcgagtca-3' (SEQ ID NO. 13);

[0062] PRDM1-sgRNA-R: 5'-aaactgactcgccaagtcgtaagtcctcgag-3' (SEQ ID NO. 14);

[0063] TFAP2C-sgRNA-F: 5'-caccctcgaggactcccagatcatctcgacc-3' (SEQ ID NO. 15);

[0064] TFAP2C-sgRNA-R: 5'-aaacggtcgagatgatctgggagtcctcgag-3' (SEQ ID NO. 16);

[0065] The ligation reaction system and conditions were as follows: 5 μL of Solution 1 (Solution Ⅰ) (Takara, 6022Q), 1 μL of the plasmid linear fragment, 1 μL of ddH2O, and 3 μL of sgRNA; ligation was performed at 16°C overnight.

[0066] 4) After obtaining target fragments containing target gene homology arms through multiple rounds of PCR in step 2), CRISPR-Cas9 technology can be used for homologous recombination to achieve site-specific knock-in of the exogenous gene. The steps are as follows:

[0067] The recombinant plasmid constructed in step 3) and the two dual-reporter gene recombinant linear vectors obtained in step 2) (PRDM1 left homology arm + tdTomato + PRDM1 right homology arm (3592 bp), TFAP2C left homology arm + mNeonGreen + TFAP2C right homology arm (3223 bp)) were co-transfected into the bovine haploid stem cells in step 1 via liposome transfection. After transfection, cell DNA was extracted and PCR reaction was used to determine whether the exogenous gene was integrated.

[0068] It was identified that tdTomato and mNeonGreen have been knocked into the PRDM1 and TFAP2C genes ( Figure 1 (B and D) When haploid stem cells are induced into PGCs, PRDM1 and TFAP2C are specifically expressed, and the knocked-in fluorescent gene is also expressed, demonstrating that haploid stem cells can be induced into PGCs.

[0069] Example 2 Induction of PGCs from Haploid Stem Cells

[0070] The bovine haploid stem cells knocked into the fluorescent gene in Example 1 were pre-induced, and a 12-well plate was treated with recombinant human fibronectin (FC010, Millipore). 5 Individual bovine haploid stem cells were cultured in a preconditioned medium for 24 hours at 37°C, saturated humidity, and 5% CO2. The preconditioned medium was prepared by adding BMP4 (100 ng / mL), 6 μM CHIR99021, 2.5 μM IWR1, and 10 μM Y-27632 to N2B27 culture medium containing 5% (v / v) KSR (10828028, Thermo Fisher Scientific).

[0071] The pre-treated bovine haploid stem cells were digested with Accutase (A1110501, Thermo Fisher Scientific) and placed in GK15 culture medium. The culture was kept at 37°C, saturated humidity and 5% CO2 for 4 days to induce the haploid stem cells into PGC-like cells ( Figure 2(A) The GK15 culture medium is based on GMEM culture medium, to which the following reagents are added at final concentrations: 15% (v / v) KSR, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / ml penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1, and 10 μM Y27632.

[0072] The cells were observed under a fluorescence microscope on the second, third and fourth days of culture. Figure 2 Middle B. The results showed that red and green fluorescence could be observed after the second day of culture. Because the red and green fluorescence were linked to the primordial germ cell-specific expression genes PRDM1 and TFAP2C, respectively, the generation of fluorescence indicated that the primordial germ cell-specific expression genes could begin to be expressed, confirming that the induction culture system could quickly induce haploid stem cells into primordial germ cells.

[0073] The pre-treated bovine haploid stem cells cultured for different days were analyzed by flow cytometry. Figure 3 , where Hoechst represents DNA content, dG1 represents the diploid G1 phase of the cell cycle, dS represents the diploid S phase of the cell cycle, and dG2 represents the diploid G2 phase of the cell cycle. The results showed that the number of PT+::TG+ positive cells continued to increase during PGC induction. On days 2, 3, and 4 of induction, 31%, 65%, and 73% of the cells were PT+::TG+ positive, respectively. Compared with previous literature (Shirasawa A, Hayashi M, Shono M, Ideta A, Yoshino T, Hayashi K. Efficient derivation of embryonic stem cells and primordial germ cell-like cells incattle. J Reprod Dev. 2024 Apr 4;70(2):82-95. doi: 10.1262 / jrd.2023-087. Epub2024 Feb 15. PMID: 38355134; PMCID: PMC11017101.), the proportion increased significantly from 3% to 73%. Moreover, after the start of differentiation induction, haploid cells automatically restored diploid characteristics, and no haploid cells were found during the PGC examination. Therefore, the efficiency of inducing PGCs from haploid stem cells is higher.

[0074] Example 3 Expression of marker genes

[0075] Sox17 is a key transcriptional regulator for the development of definitive endoderm, Oct4, Sox2, and Nanog are specific genes for maintaining stem cell pluripotency, and PRDM1 and TFAP2C are primordial germ cell-specific genes.

[0076] The culture medium of the PGC cells induced and differentiated in Example 2 was discarded, paraformaldehyde was added to fix for 30 minutes, and the cells were permeabilized and blocked with 3% (v / v) bovine serum albumin. Subsequently, antibodies against the PGC endogenous PRDM1 and TFAP2C genes, the PGC-specific expression genes SOX17, OCT4 and NANOG, and the stem cell-specific expression gene SOX2 were incubated overnight at 4°C, washed three times with phosphate-buffered saline (PBS), and incubated with secondary antibodies at 37°C. The cell nuclei were then stained with 4',6-diamidino-2-phenylindole (DAPI), and finally, anti-fluorescence quencher was added and sliced. The sources of the primary antibodies used and the dilution ratios are as follows:

[0077] SRY-box transcription factor 17, SOX17 (Santa Cruz), dilution ratio 1:200;

[0078] Octamer-binding transcription factor, OCT4 (Santa Cruz), dilution ratio 1:200;

[0079] Nanog Homeobox, NANOG (Santa Cruz), dilution ratio 1:100;

[0080] SRY-box transcription factor 2, SOX2 (Santa Cruz), dilution ratio 1:250;

[0081] The sources and dilution ratios of the secondary antibodies used are as follows:

[0082] AlexaFluor 594 goat anti-rabbit IgG, Thermo, dilution ratio 1:1000;

[0083] AlexaFluor 488 goat anti-rabbit IgG, Thermo, dilution ratio 1:1000;

[0084] AlexaFluor 594 goat anti-mouse IgG, Thermo, dilution ratio 1:1000.

[0085] The images were observed by NIKON A1R laser confocal microscope. Figure 4 Middle A. The results showed that on day 4 of culture, the induced primordial germ cells expressed PRDM1 and TFAP2C, but also the stemness-related genes Oct4 and Nanog, and the endoderm-specific gene Sox17. This is highly consistent with existing research results. Human primordial germ cells in the early stages of development express a series of genes related to stem cell pluripotency, such as OCT4 and NANOG, but do not express the key transcription factor gene SOX2. Instead, they express two other SOX family genes (SOX15 and SOX17).

[0086] At the same time, the expression of histone H3 lysine 9 dimethylation (H3K9me2, Abcam, dilution ratio 1:50), histone H3 lysine 27 trimethylation (H3K27me3, Abcam, dilution ratio 1:50) and 5-methylcytosine (5mC, Abcam, dilution ratio 1:50) was detected by immunofluorescence. The results are shown in Figure 4 Middle B. The results showed that compared with negative cells, the levels of DNA methylation (5mC) and H3K9me2 in induced primordial germ cells were reduced, while the levels of H3K27me3 were increased. Studies have confirmed that in primordial germ cells, H3K9me2 and 5mC undergo extensive demethylation to clear somatic epigenetic memory and establish germ cell-specific regulation; while H3K27me3 maintains the silence of key genes through dynamic regulation and maintains a high methylation state in the early stage of PGC. Figure 4 The results of medium B are also consistent with this conclusion, confirming that the primordial germ cells induced by the solitary haploid stem cells meet the characteristics of primordial germ cells, proving that GK15 culture medium can accurately induce the solitary haploid stem cells to produce primordial germ cells

[0087] In summary, in addition to the intra-species / inter-species chimera ability, haploid stem cells are also suitable for direct induction of PGCs in vitro.

[0088] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for obtaining primordial germ cells by inducing haploid stem cells, characterized in that: The following steps are involved: Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and supplemented with the following components at concentrations: 5% KSR by volume, 80-120 ng / mL BMP4, 8-10 μM CHIR99021, 2-5 μM IWR1, and 8-12 μM Y-27632; The pre-induced androgenic haploid stem cells were cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and supplemented with the following components: 15% KSR by volume, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / ml penicillin / streptomycin, 200 ng / ml BMP4, 100 ng / ml SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1, and 10 μM Y27632; The androecium haploid stem cells are bovine androecium haploid stem cells.

2. The method according to claim 1, characterized in that The pre-induction time is 24 hours, the pre-induction temperature is 37°C, the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO2 in the pre-induction environment is 5%; the culture time is 4 days, the culture temperature is 37°C, the culture environmental humidity is saturated humidity, and the volume concentration of CO2 in the culture environment is 5%.

3. The method according to claim 1 or 2, characterized in that Before the androecium haploid stem cells are pre-induced in the pre-treatment culture medium, the pre-induced container is treated with recombinant human fibronectin.

4. The method according to claim 1 or 2, characterized in that Before the pre-induced androgenic haploid stem cells are cultured in the GK15 culture medium, the method further comprises: performing a digestion treatment on the pre-induced androgenic haploid stem cells.

5. The method according to claim 4, characterized in that The reagent used in the digestion treatment includes Accutase.

6. The method according to claim 1, characterized in that The primordial germ cells express the SOX17 gene, the OCT4 gene and the NANOG gene, but do not express the SOX2 gene.

7. The method according to claim 1, characterized in that The primordial germ cells have reduced expression levels of lysine dimethylation at position 9 of histone H3 and 5-methylcytosine, and increased expression levels of lysine 3-methylation at position 27 of histone H3.

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