A bTSCM culture medium and a method for obtaining trophoblast stem cells

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a bTSCM culture medium and a method for obtaining trophoblast stem cells. The bTSCM culture medium comprises a basic culture medium and an additive component added in the basic culture medium; the additive component can synergistically regulate key signal pathways, epigenetic states and cell proliferation and survival, thereby inhibiting differentiation of non-trophoblast lineages, maintaining specific gene expression of trophoblast stem cells, keeping an undifferentiated state, and promoting establishment and stability of cell fate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bTSCM culture medium and a method for obtaining trophoblast stem cells. Background Technology

[0002] Trophoblastic stem cells (TSCs) originate from the trophoblast cells of the outer layer of the blastocyst and are capable of self-renewal in vitro and differentiating into placental-associated cell types. Existing research indicates that relatively mature culture systems for mouse and human TSCs have been established and are widely used in studies of early embryonic development and placental formation. However, research in large mammals such as cattle is still incomplete. TGF-β drives differentiation rather than maintaining self-renewal in bovine TSCs (unlike in mice), and the Wnt / EGF combination (key in human TSCs) is not central in cattle. Therefore, directly applying existing human or mouse culture media to cattle yields poor results. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a bTSCM culture medium and a method for obtaining trophoblast stem cells.

[0004] A bTSCM culture medium for trophoblast stem cells, the bTSCM culture medium comprising a basal culture medium and an additive, the composition of the additive and its concentration in the basal culture medium being as follows: The added ingredients include the following substances at concentrations: ITS-X supplement, NEAA, GlutaMAX, penicillin / streptomycin, sodium pyruvate, bovine serum albumin at 0.1-10% by weight / volume, serum substitute at 0.1-10% by weight / volume, CHIR99021 at 0.5-5 μM, IWP2 at 0.5-5 μM, A83-01 at 0.1-3 μM, SB431542 at 0.1-5 μM, Valproic acid at 0.1-1 mM, (S)-(+)-dimethindene malea at 0.5-5 μM, 1-Oleoyl-LPA sodium at 0.01-0.5 μM, human EGF at 10-100 ng / ml, Vitamin C at 0.5-5 μg / ml, BMP4 at 1-50 ng / ml, and Blebbistatin at 0.1-5 μM.

[0005] The bTSCM culture medium provided by this invention allows each component to synergistically regulate key signaling pathways, epigenetic states, and cell proliferation and survival, thereby inhibiting non-trophoblast lineage differentiation, maintaining trophoblast stem cell-specific gene expression, preserving the undifferentiated state, and promoting the establishment and stability of cell fate.

[0006] Preferably, the basal culture medium is a mixture of Neurobasal medium and DMEM / F12 medium in equal volumes.

[0007] A method for obtaining trophoblast stem cells using the bTSCM medium, wherein bovine extended pluripotent stem cells or bovine blastocysts are cultured in the bTSCM medium to obtain trophoblast stem cells.

[0008] Preferably, the steps for culturing bovine extended pluripotent stem cells are as follows: bovine extended pluripotent stem cells are seeded onto feeder cells and cultured in the bTSCM medium for 3-5 days to obtain trophoblast stem cells; the feeder cells are coated with matrix gel and bovine embryonic fibroblasts treated with mitomycin C. The cell density of bovine extended pluripotent stem cells was 1 × 10⁶ cells / cm² seeded on the matrix gel. 3 ~20×10 3 Each cell.

[0009] Preferably, the steps for culturing bovine blastocysts are as follows: the bovine blastocysts are placed on bovine embryonic fibroblast feeder cells treated with mitomycin C and cultured in the bTSCM medium until derivatives appear 7-10 days after blastocyst attachment. The derivatives are divided into small pieces and seeded onto bovine feeder cells coated with matrix gel and supplemented with 80% density. The cells are then cultured in the bTSCM medium for 6-8 days to obtain trophoblast stem cells.

[0010] Preferably, the amount of bTSCM culture medium added is 0.15~0.30 mL per square centimeter of culture area.

[0011] Preferably, the step of obtaining the bovine blastocyst is as follows: placing the fertilized egg obtained by in vitro fertilization in a Bio-IVC for incubation, removing the zona pellucida of the bovine blastocyst, and obtaining the bovine blastocyst.

[0012] Preferably, the conditions for culturing using the bTSCM medium are: 38.5°C, 5% CO2.

[0013] In bTSCM medium, ITS-X supplement, NEAA, GlutaMAX, sodium pyruvate, bovine serum albumin, and serum substitutes are basic cellular nutrition and metabolic support components. CHIR99021, IWP2, A83-01, SB431542, Valproic acid, (S)-(+)-dimethindene malea, 1-Oleoyl-LPA sodium, human EGF, Vitamin C, and BMP4 are key essential components determining cell fate transition and help induce and maintain the state of bovine trophoblast stem cells.

[0014] ITS-X supplements provide cells with key micronutrients such as insulin, transferrin, and selenium, maintaining cellular basal metabolic activity and survival, reducing serum dependence, and thus improving the stability and reproducibility of the culture system.

[0015] NEAAs (non-essential amino acids) are used to supplement the non-essential amino acids required for cell growth and proliferation, reduce the cell's own metabolic burden, promote cell growth, and maintain normal physiological functions.

[0016] GlutaMAX, as a stable form of glutamine source, provides cells with the substrates needed for energy metabolism, improves cell viability during culture, and reduces the adverse effects of ammonia accumulation on cells.

[0017] Sodium pyruvate serves as a supplementary carbon source for cellular energy metabolism, participates in the tricarboxylic acid cycle, and enhances cellular energy supply, thereby contributing to cell survival and functional maintenance.

[0018] Bovine serum albumin (BSA) is used as a carrier protein to bind and stabilize lipid-soluble molecules and small molecule regulatory factors in the culture system, while reducing the impact of external stress on cells and improving the stability of the culture system.

[0019] Serum substitutes are used to replace animal serum, providing cells with the nutritional factors and protective components needed for growth, reducing batch-to-batch variability while ensuring the controllability and consistency of culture conditions.

[0020] CHIR99021, as a GSK3 inhibitor, is used to activate the Wnt / β-catenin signaling pathway, thereby promoting the maintenance of cell self-renewal capacity and enhancing stem cell pluripotency-related status.

[0021] IWP2 is used to inhibit the activation of the endogenous Wnt signaling pathway. By blocking the production and secretion of Wnt ligands, it reduces the background activity of Wnt signaling in the culture system, thereby improving the stability and consistency of cell culture.

[0022] A83-01, as an inhibitor of the TGF-β / Activin / Nodal signaling pathway, is used to suppress cell differentiation signals and prevent cells from transforming into non-trophoblast lineages, thereby helping to maintain the undifferentiated state of bovine trophoblast stem cells and the stability of their state during culture.

[0023] SB431542, as a selective TGF-β receptor inhibitor, is used to further inhibit the activity of the TGF-β signaling pathway and work synergistically with other pathway regulators to reduce cell differentiation tendency and improve the consistency and reproducibility of bovine trophoblast stem cell culture.

[0024] Valproic acid, as a histone deacetylase (HDAC) inhibitor, can regulate the epigenetic state of cells, increase chromatin openness, and thus affect gene transcription activity. This is beneficial for maintaining the expression of trophoblast-related genes and improving cell stability and expansion capacity during in vitro culture.

[0025] (S)-(+)-dimethindene malea, as a bioactive signal-regulating small molecule, can be used to regulate the cellular response to external stimuli, thereby improving the survival status of cells under in vitro culture conditions.

[0026] 1-Oleoyl-LPA sodium, as a lysophosphatidic acid (LPA) signaling molecule, can activate LPA receptor-related signaling pathways, promote cell survival, and maintain cell structural stability.

[0027] Human EGF can be used to activate EGF receptor-mediated signaling pathways, promote cell proliferation and maintain cell viability, and is a commonly used growth factor in various cell culture systems.

[0028] Vitamin C, as an antioxidant, can scavenge reactive oxygen species and reduce oxidative stress, while also participating in various intracellular enzymatic reactions and epigenetic regulatory processes.

[0029] BMP4 belongs to the transforming growth factor β (TGF-β) superfamily and can participate in regulating cell fate and differentiation processes by activating SMAD-related signaling pathways. It can synergistically maintain or induce the target state of trophoblast cells.

[0030] Penicillin / streptomycin is used to inhibit bacterial contamination, and the addition of Blebbistatin improves cell survival and structural stability during cell culture. Both additions can further enhance the stability and reproducibility of the culture system.

[0031] CHIR99021, IWP2, A83-01, SB431542, Valproic acid, (S)-(+)-dimethindenemalea, 1-Oleoyl-LPA sodium, human EGF, and BMP4 work synergistically to establish an in vitro culture environment suitable for the growth and maintenance of bovine trophoblast stem cells. When used in combination, each functional component can synergistically regulate key signaling pathways, epigenetic status, and cell proliferation and survival, thereby inhibiting non-trophoblast lineage differentiation, maintaining trophoblast stem cell-specific gene expression, preserving the undifferentiated state, and promoting cell fate establishment and stability. Furthermore, CHIR99021, IWP2, A83-01, SB431542, 1-Oleoyl-LPA sodium, human EGF, and BMP4 regulate the activity of Wnt, TGF-β, Hippo, EGF, and BMP signaling pathways, maintaining the transcriptomic state of trophoblast stem cells and exhibiting high morphological and gene expression levels consistent with trophoblast stem cells. Valproic acid regulates epigenetic modifications, maintaining gene expression levels and undifferentiated state of trophoblast stem cells. (S)-(+)-dimethindene maleate improves cell stress response and survival rate by inhibiting histamine signaling, thus helping to maintain the stability of trophoblast stem cell status. This combination of functional components achieves long-term maintenance and stability of trophoblast stem cells in vitro through multi-pathway synergistic action.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The bTSCM culture medium provided by this invention allows each component to synergistically regulate key signaling pathways, epigenetic states, and cell proliferation and survival, thereby inhibiting non-trophoblast lineage differentiation, maintaining trophoblast stem cell-specific gene expression, preserving the undifferentiated state, and promoting the establishment and stability of cell fate.

[0033] This invention also provides a cell culture medium and induction method for directly converting bovine blastocysts and extended pluripotent stem cells into trophoblast stem cells without gene modification. The bovine trophoblast stem cells obtained from this cell culture medium have unlimited in vitro self-renewal capacity and can further differentiate into subtype trophoblast cells, which helps in the construction of bovine blastocysts or placental organoids, providing new tools for studying embryo-placental interactions and developmental regulation.

[0034] In existing technologies, bovine trophoblast stem cells (TSCs) can only be obtained from blastocysts or early-pregnancy placentas, which presents significant challenges in material sourcing. In this invention, bovine trophoblast stem cells can be induced from bovine EPSCs, eliminating the need to isolate primary cells from bovine placental tissues. They can be continuously derived from bovine EPSCs, improving the ease and efficiency of obtaining bovine TSCs. Furthermore, the bovine trophoblast stem cell culture medium of this invention can also be used to obtain bovine TSCs from bovine blastocysts, and these cells can be maintained through long-term in vitro passage. The bovine trophoblast stem cells prepared using the induction medium for bovine TSCs of this invention can serve as a model for studying bovine embryonic developmental lineage differentiation. Attached Figure Description

[0035] Figure 1 The images show the morphology of bovine EPSCs after being converted into TSCs and cultured for up to day 5, as well as after passage, in Example 1 of this invention.

[0036] Figure 2 This is an image showing the immunofluorescence staining results of GATA3 after converting bovine EPSCs into TSCs in Example 1 of the present invention.

[0037] Figure 3 The results show the gene expression of GATA3, TFAP2C, TEAD4, ELF5, etc. after converting bovine EPSCs into TSCs in Example 1 of this invention.

[0038] Figure 4 This is a morphological diagram of the process of inducing bovine blastocysts into TSCs in Embodiment 2 of the present invention.

[0039] Figure 5 This is an image showing the immunofluorescence staining results of GATA3 after bovine blastocysts were induced into TSCs in Example 2 of this invention.

[0040] Figure 6 This is the result of gene expression detection of GATA3, TFAP2C, TEAD4, ELF5, etc. after bovine blastocysts were induced into TSCs in Example 2 of the present invention. Detailed Implementation

[0041] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0042] The companies and catalog numbers of the culture media and small molecules used in this invention are as follows: Neurobasal medium (Gibco, 21103049), DMEM / F12 medium (Gibco, 12634010), ITS-X supplement (Gibco, 51500056), NEAA (Gibco, 11140050), GlutaMAX (Gibco, 35050061), penicillin / streptomycin (Gibco, 15070) 063), Sodium pyruvate (Gibco, 11360070), Bovine serum albumin (Sigma, A7030), Serum substitute (Gibco, 10828028), CHIR99021 (Selleck, S1263), IWP2 (Selleck, S7085), A83-01 (MCE, HY-10432-1), SB431542 (MCE, HY-10431-5), Valproic acid (MCE, HY-10585), (S)-(+)-dimethindene malea (MCE, HY-107647), 1-Oleoyl-LPA sodium (MCE, HY-107614), human EGF (Peprotech, AF-100-15), Vitamin C (Sigma, V-047), BMP4 (Sino Biological, 10609-HNAE2), Blebbistatin (MCE, HY-13441).

[0043] Example 1 Methods for obtaining trophoblast stem cells from bovine extended pluripotent stem cells 1. Cultivation and maintenance of bovine EPSCs EPSCs is an abbreviation for Extended Pluripotent Stem Cells.

[0044] Bovine EPSCs were maintained on a feeder layer of bovine embryonic fibroblasts treated with mitomycin C, and passaged every 3-4 days using TryplE Express. The specific passage method was as follows: cells were dissociated into single cells using TryplE Express (38.5℃, 3 min), centrifuged (250g, 5 min), resuspended, and the resuspended cell suspension was divided into 6 equal portions according to the total volume. One portion was re-inoculated into bovine EPSC medium and cultured at 38.5℃ and 5% CO2.

[0045] Preparation of bovine extended pluripotent stem cell culture medium (bovine EPSC medium): Take mTeSR Plus basal culture medium and add 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin mixture, 1 μM CHIR99021, 0.3 μM WH-4-023, 5 μM MXAV939, 50 μg / ml Vitamin C, 10 ng / ml LIF, and 20.0 ng / ml Activin A. When passaged, use bovine EPSC medium supplemented with 5 μM Y27632. Freshly prepared medium can be stored at 4℃ for two weeks.

[0046] 2. Methods for converting bovine EPSCs into bovine TSCs: Bovine EPSCs cultured in bovine EPSC medium were distributed at a density of 1.6 × 10⁻⁶ cm². 4 Bovine TSCs were obtained by seeding cells at a density of 10% onto a feeder layer of bovine embryonic fibroblasts treated with mitomycin C, coated with Collagen IV matrix gel and at a density of 10%. After culturing in bTSCM medium for 3-5 days, bovine TSCs were obtained.

[0047] The amount of culture medium added is closely related to the size of the cell culture dish used, and is usually determined in the art based on the principle of culture area or liquid surface coverage. Generally, adjustments are made to ensure complete cell immersion and maintain adequate nutrient supply. Preferably, the amount of culture medium added is approximately 0.15-0.30 mL per square centimeter of culture area. In this example, approximately 0.20 mL of bTSCM medium is added per square centimeter of culture area.

[0048] The obtained bovine TSCs were passaged to expand and stabilize them. The specific passage method was as follows: the cells were dissociated into single cells using TryplE Select (38.5℃, 3 min), centrifuged (250g, 5 min), and resuspended. The resuspended cell suspension was divided into 5 equal parts according to the total volume. One part was seeded onto a feeder layer of bovine embryonic fibroblasts treated with mitomycin C and coated with Collagen IV at a density of 10%. The cells were cultured in bTSCM medium at 38.5℃ and 5% CO2.

[0049] Morphological images of bovine EPSCs converted to TSCs and cultured for up to day 5, and after passage, as shown. Figure 1 As shown.

[0050] Preparation of bovine trophoblast stem cell culture medium (bTSCM medium): Neurobasal medium and DMEM / F12 medium were mixed in a 1:1 (v / v) ratio and supplemented with 1% ITS-X supplement, 1% NEAA, 1% GlutaMAX, 1% penicillin / streptomycin, 1% sodium pyruvate, 0.3% bovine serum albumin, 0.5% serum substitute, 1.5μM CHIR99021, 2μM IWP2, 0.5μM A83-01, 1μM SB431542, 0.4mM Valproic acid, 1μM (S)-(+)-dimethindenemalea, 0.1μM 1-Oleoyl-LPA sodium, 50ng / ml human EGF, 2 μg / ml Vitamin C, 10ng / ml BMP4, and 0.5μM Blebbistatin.

[0051] Example 2 Methods for obtaining trophoblast stem cells from bovine blastocysts Bovine oocytes with multiple layers of cumulus cells were collected from follicles and matured in vitro for 24 hours. After in vitro fertilization for 8 hours, the cumulus cells were removed by treatment with hyaluronidase. The fertilized oocytes were then placed in Bio-IVC culture medium and incubated at 38.5℃, 5% O2, and 5% CO2 until the embryo developed into a blastocyst. The zona pellucida of the entire bovine blastocyst was removed with acidic thallium solution. Individual bovine blastocysts were then transferred to 12-well plates coated with mitomycin C-treated bovine embryonic fibroblast feeder cells, one blastocyst per well, and cultured in bTSCM medium, with approximately 1 ml of bTSCM medium added to each well. Outgrowth was observed 7-10 days after blastocyst attachment. The outgrowth cells were mechanically separated into small pieces of approximately 20 cells each and seeded into 12-well plates coated with Collagen IV and supplemented with 80% density bovine feeder cells, one piece per well. These pieces were cultured in bTSCM medium for 7 days to obtain bovine TSCs, with approximately 1 ml of bTSCM medium added to each well. bTSCM medium, culture conditions were 38.5℃ and 5% CO2.

[0052] Maintenance culture of bovine TSCs Bovine TSCs were maintained on a feeder layer of bovine embryonic fibroblasts treated with mitomycin C, coated with Collagen IV and supplemented with 80% density. Enzymatic digestion and passage were performed every 7 days. The enzymatic digestion and passage procedure was as follows: TSC clones were digested with 1 mg / ml Dispase II (38.5℃, 2 min), centrifuged (250 g, 2 min), resuspended in TryplE Select (38.5℃, 2 min), and further digested into small clumps of 10-20 cells. These clumps were then centrifuged (250 g, 2 min), resuspended in bTSCM, and the resuspended cell suspension was divided into two equal parts. One part was seeded into bTSCM medium, and 10 μM Y27632 was added before passage. The cells were removed after 24 h. Cells were cultured at 38.5℃ and 5% CO2.

[0053] The bTSCM culture medium used in Example 2 was the same as that used in Example 1.

[0054] Morphological images of bovine blastocysts induced into TSCs are shown below. Figure 4 As shown.

[0055] The maintenance culture step after obtaining TSCs in the example is used to expand and stabilize the obtained bovine TSCs to form a stable cell line that can be passaged for a long time.

[0056] The bovine trophoblast stem cells provided in the examples were identified.

[0057] Immunofluorescence staining identification: Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed twice with DPBS, permeabilized with 0.5% Triton X-100 in DPBS solution for 30 min, washed three times with DPBS, soaking for 5 min each time, and blocked with blocking buffer at room temperature for 1 hour. The primary antibody (anti-GATA3, Abcam, AB199428) was diluted in primary antibody dilution buffer and incubated at room temperature in the dark for 1 hour, followed by washing three times with DPBS, soaking for 5 min each time. Secondary antibody (goat anti-rabbit Alexa Flour488, Beyotime, A0423) was diluted with secondary antibody dilution buffer and incubated at room temperature in the dark for 1 hour. The sample was washed three times with DPBS, soaking for 5 min each time. Finally, the sample was stained with DAPI at room temperature in the dark for 15 min, the DAPI stain was discarded, and the sample was washed twice with DPBS, soaking for 5 min each time. The stained cells were then mounted on slides and photographed under a Zeiss inverted microscope.

[0058] Depend on Figure 2 and Figure 5 It can be seen that the bovine trophoblast stem cells obtained according to the embodiments of the present invention showed positive expression after GATA3 immunofluorescence staining (see...). Figure 2 , Figure 5 ).

[0059] RNA was extracted using the RNA extraction kit (Kangwei Century, CW0580S) according to the instructions, and cDNA was obtained by reverse transcription using the reverse transcription kit (abm, G592) according to the instructions. Following the instructions of the qPCR kit (Aikerui, AG11701), the cDNA was used as a template, and amplification was performed on a real-time quantitative PCR instrument using gene-specific primers and a SYBR Green fluorescence quantitative detection system. Normalization was performed using an internal reference gene (GAPDH), and the relative expression level of the target gene was calculated using the ΔΔCt method. Significance analysis was performed using the T-test (see [link to T-test]). Figure 3 and Figure 6 ).

[0060] Depend on Figure 3 and Figure 6 As can be seen from qPCR detection, the expression levels of trophoblast-specific marker genes in the bovine trophoblast stem cells obtained in this embodiment of the invention showed an upregulated trend compared to bovine EPSCs (see...). Figure 3 and Figure 6 ).

[0061] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0062] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0063] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A bTSCM culture medium for trophoblast stem cells, characterized in that, The bTSCM medium consists of a basal medium and an additive, and the composition and concentration of the additive in the basal medium are as follows: 1% (v / v) ITS-X supplement, 1% (v / v) NEAA, 1% (v / v) GlutaMAX, 1% (v / v) penicillin / streptomycin, 1% (v / v) sodium pyruvate, 0.1%–10% (w / v) bovine serum albumin, 0.1%–10% (w / v) serum substitute, 0.5–5 μM CHIR99021, 0.5–5 μM IWP2, 0.1–3 μM A83-01, 0.1–5 μM SB431542, 0.1–1 mM Valproicacid, 0.5–5 μM (S)-(+)-dimethindene malea, 0.01–0.5 μM 1-Oleoyl-LPA sodium, 10–100 ng / ml human EGF, 0.5–5 μg / ml Vitami n C, 1–50 ng / ml of BMP4 and 0.1~5μM of Blebbistatin.

2. The bTSCM culture medium according to claim 1, characterized in that, The basal culture medium is a mixture of Neurobasal medium and DMEM / F12 medium in equal volumes.

3. A method for obtaining trophoblast stem cells using the bTSCM culture medium of claim 1, characterized in that, Bovine extended pluripotent stem cells or bovine blastocysts were cultured using the bTSCM medium to obtain trophoblast stem cells.

4. The method according to claim 3, characterized in that, The steps for culturing bovine extended pluripotent stem cells are as follows: bovine extended pluripotent stem cells are seeded onto feeder cells and cultured in the bTSCM medium for 3 to 5 days to obtain trophoblast stem cells; the feeder cells are coated with matrix gel and bovine embryonic fibroblasts treated with mitomycin C. The cell density of bovine extended pluripotent stem cells was 1 × 10⁶ cells / cm² seeded on the matrix gel. 3 ~20×10 3 Each cell.

5. The method according to claim 3, characterized in that, The steps for culturing bovine blastocysts are as follows: the bovine blastocysts are placed on bovine embryonic fibroblast feeder cells treated with mitomycin C and cultured in the bTSCM medium until derivatives appear 7-10 days after the blastocysts adhere to the wall. The derivatives are divided into small pieces and seeded onto matrix gel with bovine feeder cells, and then cultured in the bTSCM medium for 6-8 days to obtain trophoblast stem cells.

6. The method according to claim 4 or claim 5, characterized in that, The amount of bTSCM culture medium added is 0.15~0.30 mL per square centimeter of culture area.

7. The method according to claim 5, characterized in that, The steps for obtaining the bovine blastocyst are as follows: fertilized eggs obtained by in vitro fertilization are placed in a Bio-IVC for incubation, and the zona pellucida of the bovine blastocyst is removed to obtain the bovine blastocyst.

8. The method according to claim 4 or claim 7, characterized in that, The conditions for culturing using the bTSCM medium are: 38.5℃, 5% CO2.

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