Method for differentiating bovine iPSCs (induced pluripotent stem cells) to form bovine primordial germ cell-like cells
By conducting 2D culture on Matrigel, differentiation is efficiently induced to form bovine proxygenic germ cell-like cells, solving the problem of difficult to efficiently obtain bovine proxygenic germ cell-like cells in the prior art, and achieving efficient cell differentiation and subsequent research in vitro.
Patent Information
- Application Number
- CN202510171279.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-05-13
AI Technical Summary
It is difficult to efficiently obtain bovine germ cell-like cells in the prior art, and the obtained cells differ from the isolated prairie germ cells in vivo.
2D culture of bovine iPSCs was carried out on Matrigel, and differentiation was efficiently induced to form bovine primitive germ cell-like cells through 2D culture during the pre-induction and differentiation stages.
The acquisition of bovine primitive germ cell-like cells efficiently induces in vitro provides convenience for subsequent related research and molecular research.
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Figure CN119979473A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell biology, and more particularly to a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells. Background Art
[0002] At present, the method of inducing bovine pluripotent stem cells to differentiate into bovine primordial germ cell-like cells and spermatogonial stem cell-like cells is still immature. For example, it is impossible to efficiently obtain bovine primordial germ cell-like cells for subsequent mechanism research, and the obtained bovine primordial germ cell-like cells are still different from the primordial germ cells isolated in vivo. Improving the efficiency of differentiation of bovine induced pluripotent stem cells into primordial germ cell-like cells and enabling them to exert a wider range of applicable capabilities has important biological significance.
[0003] In addition, Matrigel is a basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins, which contains about 60% laminin, 30% IV collagen and 8% nestin. Matrigel also contains basement membrane polysaccharide, TGF-β, epidermal growth factor, insulin-like growth factor, tissue plasminogen and other growth factors. Fibronectin is a type of macromolecular plasma protein located on the cell surface, which exhibits structural and adhesion properties in the fibrous matrix connected to the cells. Fibronectin is a major type of cell adhesion molecule and does not contain other growth factors. No research has been seen using it to solve the above technical problems.
[0004] Therefore, whether it is possible to provide a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells is an urgent problem to be solved by those skilled in the art. Summary of the invention
[0005] In view of this, the present invention provides a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells. During the primordial germ cell-like cell induction differentiation process, bovine iPSCs are cultured on Matrigel, and 2D culture is performed in the pre-induction and differentiation stages, so that bovine primordial germ cell-like cell samples can be efficiently obtained for subsequent related research.
[0006] Collection information:
[0007] iPSC-NC (bovine induced pluripotent stem cells) are deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC: NO.46317, and the deposit date is January 6, 2025.
[0008] In order to achieve the above object, the present invention adopts the following technical solution:
[0009] A bovine iPSCs, with the deposit number being: CGMCC No.46317.
[0010] The present invention also provides a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells, comprising the following steps:
[0011] (1) Differentiation of bovine iPSCs into bovine early mesoderm-like cells:
[0012] 11) culturing bovine iPSCs using culture medium at 38.5° C. and 5% CO2; digesting to obtain single cells; resuspending the cells using pre-induction culture medium to obtain a single cell suspension of bovine iPSCs;
[0013] 12) After adding Matrigel treatment to the culture plate in advance, add the single cell suspension of bovine iPSCs; after the pre-induction stage, wash the cells to obtain bovine early mesoderm-like cells;
[0014] (2) Inducing the differentiation of bovine early mesoderm-like cells into bovine primordial germ cell-like cells: adding the bovine early mesoderm-like cells into differentiation culture medium; culturing the cells at 38.5° C. and 5% CO 2 ; collecting the cells after differentiation to obtain bovine primordial germ cell-like cells.
[0015] Preferably, step (1) is specifically:
[0016] 11) Cultivating bovine iPSCs using LCDM culture medium at 38.5° C. and 5% CO2; when the bovine iPSCs grow to a density of 80% to 90%, digesting them with TrypLE Select for 3 minutes to obtain single cells, and terminating the digestion with LCDM culture medium; resuspending the cells with pre-induction culture medium to obtain a single cell suspension of bovine iPSCs;
[0017] 12) Add Matrigel to the culture plate in advance, treat for 1 hour, discard Matrigel, add single cell suspension of bovine iPSCs, and inoculate 1 well cells, and the culture medium was replaced every day; after the 2-day pre-induction stage, the pre-induction culture medium was discarded, and the cells were washed with DPBS to obtain bovine early mesoderm-like cells.
[0018] The beneficial effects are: the number of starting cells in the pre-induction stage is half of the number of starting cells in the conventional 3D culture system;
[0019] After the pre-induction phase is completed, there is no need to digest the cells. The differentiation medium can be directly replaced after washing the cells with DPBS.
[0020] Preferably, step (2) is specifically as follows: adding the bovine early mesoderm-like cells to a differentiation culture medium; culturing the cells at 38.5° C. and 5% CO 2 ; collecting the cells after 6 to 8 days of differentiation to obtain bovine primordial germ cell-like cells;
[0021] Step (2) Collecting cells: Digest with TrypLE Select for 5 minutes, terminate the digestion with differentiation medium, and collect the cells after centrifugation at 1500 rpm for 5 minutes.
[0022] Preferably: LCDM culture medium has the following ingredients per 50 ml: DMEM / F12 24 ml, Neurobasal 24 ml, N2 0.5%, B27 1%, KSR 5%, NEAA 1%, L-glutamine 2 mM, β-mercaptoethanol 0.1 mM, Penicillin-streptomycin 1%, LIF 10 ng / mL, CHIR99021 1 μM, (S)-(+)-dimethindenemaleate 2 μM, minocycline hydrochloride 2 μM;
[0023] The components of the pre-induction culture medium per 50 ml: GMEM 41 ml, KSR 15%, NEAA 1%, L-glutamine 2 mM, sodium pyruvate 1 mM, β-mercaptoethanol 0.1 mM, CHIR9902 13 μM, Activin A 50 ng / mL, Y-27632 10 μM;
[0024] The components of differentiation culture medium per 50 ml: GMEM 41 ml, KSR 15%, NEAA 1%, L-glutamine 2 mM, sodium pyruvate 1 mM, β-mercaptoethanol 0.1 mM, BMP4 200 ng / mL, SCF 100 ng / mL, LIF 20 ng / mL, EGF 50 ng / mL, Y-27632 10 μM.
[0025] The present invention also provides bovine primordial germ cell-like cells prepared by any of the above methods.
[0026] The present invention also provides the use of the bovine primordial germ cell-like cells in inducing differentiation into bovine spermatogonial stem cell-like cells.
[0027] Preferably: comprising the following steps:
[0028] 81) Inducing differentiation of bovine primordial germ cell-like cells using differentiation medium at 38.5° C. and 5% CO2, and after 6 to 8 days of differentiation, digesting with TrypLE Select for 5 minutes to obtain single cells, and terminating the digestion with differentiation medium; resuspending the cells with spermatogonial stem cell-like cell differentiation medium to obtain a single cell suspension of bovine primordial germ cell-like cells;
[0029] 82) 0.2% gelatin was added to the culture plate in advance for 1 h, the gelatin was discarded, and a single cell suspension of bovine primordial germ cell-like cells was added; 2×10 5 cells, and the culture medium was replaced every day; the cells were collected after 8 to 12 days of differentiation to obtain bovine spermatogonial stem cell-like cells.
[0030] Preferred: The components of the differentiation culture medium for spermatogonial stem-like cells: DMEM 41 ml, ESC FBS 15%, NEAA 1%, L-glutamine 2 mM, sodium pyruvate 1 mM, β-mercaptoethanol 0.1 mM, RA 2 μM, GDNF 4 ng / mL, Testosterone 1 μM.
[0031] The present invention also provides the use of any of the above methods, the above bovine primordial germ cell-like cells, or the bovine spermatogonial stem cell-like cells prepared by any of the above applications in biopharmaceuticals.
[0032] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells, and the technical effect achieved is: a 2D culture system can be used to efficiently induce bovine primordial germ cell-like cells in vitro, providing a technical means for improving primordial germ cell-like cells derived from bovine pluripotent stem cells, while laying a certain foundation for further related research on in vitro differentiation to form gametes, and also facilitating subsequent molecular research and sequencing research, such as fluorescent quantitative PCR, immunoblot analysis and transcriptome sequencing.
[0033] By identifying the differentiated cells, the results showed that under the stimulation of differentiation induction, the differentiated cells from bovine iPSCs were able to start the expression of early primordial germ cell marker genes TFAP2C, SOX17 and pluripotent gene NANOG on the 3rd day, and the expression of primordial germ cell marker gene PRDM1 on the 5th day. The primordial germ cell marker protein TFAP2C is synthesized in the pre-induction stage and then promotes the expression of PRDM1 protein. The bovine primordial germ cell-like cells (PGCLCs) obtained using this differentiation method can differentiate into spermatogonial stem cell-like cells (SSCLCs). BRIEF DESCRIPTION OF THE DRAWINGS
[0034] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0035] Figure 1 The accompanying drawing is a schematic diagram of the differentiation process provided by the present invention.
[0036] Figure 2 The accompanying drawing is a qPCR detection result diagram of the relative expression of bovine primordial germ cell-like cell marker genes provided by the present invention.
[0037] Figure 3 The attached figure is a result diagram of immunofluorescence staining of TFAP2C and PRDM1 in early mesoderm-like cells provided by the present invention, wherein the scale bar is 50 μm.
[0038] Figure 4 The attached figure is a result diagram of immunofluorescence staining of TFAP2C and PRDM1 of early mesoderm-like cells obtained on the 3rd day of differentiation provided by the present invention, wherein the scale bar is 50 μm.
[0039] Figure 5 The attached figure is a result diagram of immunofluorescence staining of TFAP2C and PRDM1 of early mesoderm-like cells obtained on the 5th day of differentiation provided by the present invention, wherein the scale bar is 50 μm.
[0040] Figure 6 The accompanying drawing is a differentiation flow chart of inducing the differentiation of bovine primordial germ cell-like cells into spermatogonial stem cell-like cells provided by the present invention.
[0041] Figure 7 The attached figure is a morphological diagram of bovine spermatogonial stem-like cells provided by the present invention, wherein the scale bar is 100 μm.
[0042] Figure 8 The accompanying drawing is a qPCR detection result diagram of the relative expression of bovine spermatogonial stem cell-like cell marker genes provided by the present invention.
[0043] Fig. 9 The attached drawing is a result diagram of immunofluorescence staining of DDX4, GFRA1 and PGP9.5 of bovine spermatogonial stem-like cells provided by the present invention, wherein the scale bar is 50 μm. DETAILED DESCRIPTION
[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0045] The embodiment of the present invention discloses a method for differentiating bovine iPSCs into bovine primordial germ cell-like cells.
[0046] Unless otherwise specified, the technical means not specifically described in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. The bovine iPSCs in the examples can be either preserved bovine iPSCs or other materials that meet the experimental requirements, which will not be described in detail.
[0047] Example 1
[0048] Preparation of bovine iPSCs
[0049] Bovine fetal fibroblasts (BFFs) were obtained from the ear tips and tail tips of bovine fetuses at slaughterhouses. BFFs were cultured in 6 cm culture dishes at 38.5°C in a 5% CO2 environment, and the bovine fetal fibroblast culture medium (DMEM+10% FBS+1% PS) was replaced every day. When the cell density reached about 90%, BFFs were passaged. For reprogramming, PiggyBac plasmids (including CAG-bovine OCT3 / 4, CAG-bovine SOX2, CAG-bovine KLF4 and CAG-bovine C-MYC) were transfected into BFFs by electroporation, and each electroporation was about About 5,000 transfected fibroblasts were seeded into a 12-well culture plate with a feeder layer and allowed to adhere using fibroblast culture medium. The next day, the culture medium was replaced with LCDM medium. From day 12 to day 23 after transfection, colonies were observed to gradually form. The resulting clones were selected and passaged using TrypLE Select.
[0050] The obtained bovine iPSCs were deposited under patent procedures.
[0051] Example 2
[0052] Differentiation of bovine iPSCs into early mesoderm-like cells
[0053] Cultivating bovine iPSCs using LCDM culture medium at 38.5°C and 5% CO2; when the bovine iPSCs grow to an appropriate density (80% to 90%), digesting them with TrypLE Select for 3 minutes to obtain single cells, and terminating the digestion with LCDM culture medium; resuspending the cells with pre-induction culture medium to obtain a single cell suspension of bovine iPSCs;
[0054] Add Matrigel (CORNING, 354248) to the culture plate in advance and treat for 1 hour. Discard Matrigel and add single cell suspension of bovine iPSCs; inoculate one well of a 12-well plate Change the culture medium every day.
[0055] The composition of LCDM culture medium (50 ml) is shown in Table 1:
[0056] Table 1
[0057]
[0058]
[0059] The composition of the pre-induction culture medium (50 ml) is shown in Table 2:
[0060] Table 2
[0061]
[0062] Induction of bovine early mesoderm-like cells into bovine primordial germ cell-like cells
[0063] After the pre-induction phase (2 days), the pre-induction culture medium was discarded, and the cells were washed with DPBS to obtain bovine early mesoderm-like cells;
[0064] Add the differentiation medium to the bovine early mesoderm-like cells; culture the cells at 38.5°C and 5% CO2. Change the culture medium every day; collect the cells for subsequent research after 6 to 8 days of differentiation;
[0065] The method for collecting cell samples is as follows: digest with TrypLE Select for 5 minutes, terminate the digestion with differentiation medium, and collect the cells after centrifugation at 1500 rpm for 5 minutes.
[0066] The composition of differentiation culture medium (50 ml) is shown in Table 3:
[0067] Table 3
[0068]
[0069] The above differentiation process diagram is shown in Figure 1 .
[0070] Example 3
[0071] Identification of Primordial Germ Cell-like Cells Derived from Differentiation of Bovine iPSCs
[0072] 1. qPCR identification of marker genes of bovine primordial germ cell-like cells
[0073] RNA was extracted from bovine iPSCs and primordial germ cell-like cells obtained at different differentiation days (day 3, day 5, day 8) respectively, and then reverse transcribed into cDNA for (conventional) qPCR identification. The results showed that under the stimulation of differentiation induction, the differentiated cells from bovine iPSCs were able to activate the expression of early primordial germ cell marker genes TFAP2C, SOX17 and pluripotent gene NANOG on day 3, and the expression of primordial germ cell marker gene PRDM1 on day 5, as shown in Figure 2. Figure 2 .
[0074] The (conventional) primer sequences used are as follows:
[0075] GAPDH upstream primer: 5'-GGGTCATCATCTCTGCACCT-3'
[0076] Downstream primer: 5'-GGTCATAAGTCCCTCCACGA-3'
[0077] SOX17 upstream primer: 5'-AAGATGCTGGGCAAGTCG-3'
[0078] Downstream primer: 5'-CGGTACTTGTAGTTGGGATGG-3'
[0079] TFAP2C upstream primer: 5'-CGACATGGCACACCAGAT-3'
[0080] Downstream primer: 5'-GGAAATAGGACCTTTGCGAATAAC-3'
[0081] PRDM1 upstream primer: 5'-ATGGAGGACGCGGATATGAC-3'
[0082] Downstream primer: 5'-TGTTGGCGTTCTTGGGAACT-3'
[0083] NANOG upstream primer: 5'-TTCCCTCCTCCATGGATCTG-3'
[0084] Downstream primer: 5'-ATTTGCTGGAGACTGAGGTA-3'
[0085] 2 Immunofluorescence identification of bovine primordial germ cell-like cell marker proteins
[0086] See Figure 3 to Figure 5 The expression of marker proteins of the obtained mesoderm-like cells and bovine primordial germ cell-like cells (day3, day5) was identified.
[0087] The results showed that primordial germ cell marker protein TFAP2C was synthesized during the pre-induction stage and subsequently promoted the expression of PRDM1 protein.
[0088] Example 4
[0089] Induction of bovine primordial germ cell-like cells into bovine spermatogonial stem cell-like cells
[0090] Differentiation process instructions Figure 6 , using the differentiation medium of primordial germ cell-like cells (Table 3) to induce differentiation of bovine primordial germ cell-like cells at 38.5°C, 5% CO2. After 6 to 8 days of differentiation, digestion was performed with TrypLE Select for 5 minutes to obtain single cells, and digestion was terminated with the differentiation medium of primordial germ cell-like cells (Table 3); the cells were resuspended with the differentiation medium of spermatogonial stem cell-like cells (Table 4) to obtain a single cell suspension of bovine primordial germ cell-like cells;
[0091] Add 0.2% gelatin to the culture plate in advance and treat for 1 hour. Discard the gelatin and add a single cell suspension of bovine primordial germ cell-like cells; inoculate one well of a 12-well plate. The culture medium was changed every day; cells were collected after 8 to 12 days of differentiation for subsequent studies. Figure 7 .
[0092] The components of the differentiation culture medium for spermatogonial stem cell-like cells are shown in Table 4:
[0093] Table 4
[0094]
[0095] Example 5
[0096] Identification of spermatogonial stem cell-like cells derived from induced differentiation of bovine primordial germ cell-like cells
[0097] 1. qPCR identification of bovine spermatogonial stem-like cell marker genes
[0098] RNA from bovine iPSCs, obtained primordial germ cell-like cells, and obtained spermatogonial stem cell-like cells was extracted (conventionally), reverse transcribed into cDNA, and then qPCR identification was performed.
[0099] The results showed that under the stimulation of differentiation induction, spermatogonial stem cell-like cells differentiated from bovine primordial germ cell-like cells expressed spermatogonial stem cell marker genes THY1, CDH1 and PAX7. Figure 8 .
[0100] The (conventional) primer sequences used are as follows:
[0101] GAPDH upstream primer: 5'-GGGTCATCATCTCTGCACCT-3'
[0102] Downstream primer: 5'-GGTCATAAGTCCCTCCACGA-3'
[0103] THY1 upstream primer: 5'-TCTACACGTGTGAACTCCGC-3'
[0104] Downstream primer: 5'-CTTATGCCCCCACACCTGAC-3'
[0105] DDX4 upstream primer: 5'-CCGGCTTCATCGTCAGATCCT-3'
[0106] Downstream primer: 5'-TTCCAACTCCAAAACCACCCAC-3'
[0107] CDH1 upstream primer: 5'-CGTGACCGACCAGAATGACA-3'
[0108] Downstream primer: 5'-GGCTGGGATTGTGTAACCGA-3'
[0109] PAX7 upstream primer: 5'-CCACCTACCCCACCACTACT-3'
[0110] Downstream primer: 5'-AAGAGGACGGTTAGGCTCCA-3'
[0111] 2 Immunofluorescence identification of bovine spermatogonial stem cell-like cell marker proteins
[0112] The expression of the obtained bovine spermatogonial stem cell-like cell marker proteins was identified. The results showed that the obtained bovine spermatogonial stem cell-like cells expressed spermatogonial stem cell marker proteins DDX4, GFRA1 and PGP9.5. Fig. 9 .
[0113] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0114] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A bovine iPSCs, characterized in that: The deposit number is: CGMCC No.46317.
2. A method for differentiating bovine iPSCs into bovine primordial germ cell-like cells, characterized in that: The following steps are involved: (1) Differentiation of bovine iPSCs into bovine early mesoderm-like cells: 11) culturing bovine iPSCs using culture medium at 38.5° C. and 5% CO2; digesting to obtain single cells; resuspending the cells using pre-induction culture medium to obtain a single cell suspension of bovine iPSCs; 12) After adding Matrigel treatment to the culture plate in advance, add the single cell suspension of bovine iPSCs; After the pre-induction stage, the cells were washed to obtain bovine early mesoderm-like cells; (2) Inducing the differentiation of bovine early mesoderm-like cells into bovine primordial germ cell-like cells: adding the bovine early mesoderm-like cells into differentiation culture medium; culturing the cells at 38.5° C. and 5% CO 2 ; collecting the cells after differentiation to obtain bovine primordial germ cell-like cells.
3. The method according to claim 2, characterized in that Step (1) is specifically as follows: 11) Cultivating bovine iPSCs using LCDM culture medium at 38.5° C. and 5% CO2; when the bovine iPSCs grow to a density of 80% to 90%, digesting them with TrypLE Select for 3 minutes to obtain single cells, and terminating the digestion with LCDM culture medium; resuspending the cells with pre-induction culture medium to obtain a single cell suspension of bovine iPSCs; 12) Add Matrigel to the culture plate in advance, treat for 1 hour, discard Matrigel, add single cell suspension of bovine iPSCs, and inoculate 1 well cells, and the culture medium was replaced every day; after the 2-day pre-induction stage, the pre-induction culture medium was discarded, and the cells were washed with DPBS to obtain bovine early mesoderm-like cells.
4. The method according to claim 3, characterized in that Step (2) is specifically as follows: adding differentiation culture medium to bovine early mesoderm-like cells; culturing the cells at 38.5° C. and 5% CO2; collecting the cells after differentiation for 6 to 8 days to obtain bovine primordial germ cell-like cells; In step (2), the cells were collected by digesting with TrypLE Select for 5 minutes, terminating the digestion with differentiation medium, and centrifuging at 1500 rpm for 5 minutes before collecting the cells.
5. The method according to claim 4, characterized in that The components of the LCDM culture medium per 50 ml are as follows: DMEM / F12 24 ml, Neurobasal 24 ml, N2 0.5%, B27 1%, KSR 5%, NEAA 1%, L-glutamine 2 mM, β-mercaptoethanol 0.1 mM, Penicillin-streptomycin 1%, LIF 10 ng / mL, CHIR99021 1 μM, (S)-(+)-dimethindene maleate 2 μM, minocycline hydrochloride 2 μM; The components of the pre-induction culture medium per 50 ml: GMEM 41 ml, KSR 15%, NEAA 1%, L-glutamine 2 mM, sodium pyruvate 1 mM, β-mercaptoethanol 0.1 mM, CHIR9902 13 μM, Activin A 50 ng / mL, Y-27632 10 μM; The components of differentiation culture medium per 50 ml: GMEM4 1 ml, KSR 15%, NEAA 1%, L-glutamine 2mM, sodium pyruvate 1mM, β-mercaptoethanol 0.1mM, BMP4 200 ng / mL, SCF 100ng / mL, LIF 20ng / mL, EGF 50ng / mL, Y-27632 10μM.
6. Bovine primordial germ cell-like cells prepared by the method according to any one of claims 2 to 5.
7. Use of the bovine primordial germ cell-like cells according to claim 6 in inducing differentiation into bovine spermatogonial stem cell-like cells.
8. The use according to claim 7, characterized in that The following steps are involved: 81) Inducing differentiation of bovine primordial germ cell-like cells using differentiation medium at 38.5° C. and 5% CO2, and after 6 to 8 days of differentiation, digesting with TrypLE Select for 5 minutes to obtain single cells, and terminating the digestion with differentiation medium; resuspending the cells with spermatogonial stem cell-like cell differentiation medium to obtain a single cell suspension of bovine primordial germ cell-like cells; 82) 0.2% gelatin was added to the culture plate in advance for 1 h, the gelatin was discarded, and a single cell suspension of bovine primordial germ cell-like cells was added; 2×10 5 cells, and the culture medium was replaced every day; the cells were collected after 8 to 12 days of differentiation to obtain bovine spermatogonial stem cell-like cells.
9. The use according to claim 8, characterized in that The differentiation culture medium of the spermatogonial stem-like cells contains the following components: DMEM 41 ml, ESC FBS 15%, NEAA 1%, L-glutamine 2 mM, sodium pyruvate 1 mM, β-mercaptoethanol 0.1 mM, RA 2 μM, GDNF 4 ng / mL, and Testosterone 1 μM.
10. Use of the method according to any one of claims 2 to 5, the bovine primordial germ cell-like cells according to claim 6, or the bovine spermatogonial stem cell-like cells prepared by the use according to any one of claims 7 to 9 in biopharmaceuticals.