Composition, cell culture medium and application of cell culture medium in establishment of trophoblast stem cells in bovine hatching blastocysts

Abstract

The invention provides a composition, a cell culture medium and application of the cell culture medium in establishment of trophoblast stem cells in bovine hatching blastocysts, and belongs to the technical field of cell engineering. The invention provides a composition. The composition comprises activin A, CHIR99021 and a recombinant human leukemia inhibition factor. After the composition is adopted to culture hatched blastocysts, bovine trophoblast stem cells are obtained in vitro, trophoblast cell specific markers are highly expressed in mRNA and protein levels, and the bovine trophoblast stem cells can be stably cultured in vitro for a long time. The composition not only can further enrich the culture system of the bovine trophoblast stem cells, but also can expand understanding of the difference of signal pathways causing the blastocysts in different stages of the cattle.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell engineering, and particularly relates to a composition, a cell culture medium and application thereof in establishing trophoblast stem cells in bovine hatched blastocysts. BACKGROUND

[0002] Trophoblast cells are specialized cells in the placenta responsible for mediating the interaction between mother and fetus, which originate from the trophoblast outer layer (TE) of the blastocyst. In 1998, Tanaka cultured mouse blastocysts or early post-implantation trophoblast cells in the presence of fibroblast growth factor 4 (FGF4), and a permanent mouse trophoblast stem cell line was isolated. In 2010, researchers first reported the successful isolation and culture of human trophoblast stem cells. Compared with mice, the isolation and culture conditions of bovine trophoblast stem cells (bTSC) are more complex, mainly affected by multiple factors such as embryo collection technology, culture medium formula and cell state.

[0003] Compared with humans and mice, the research on hTSCs is relatively less. In 2023, Jiang and Wu used a chemical mixture developed from human and mouse expanded pluripotent stem cells to derive bovine trophoblast stem cells from bovine 7.5-day blastocysts and stably culture them for a long time. The bovine TSCs retain the developmental ability to differentiate into mature trophoblast cells and exhibit the transcriptional and epigenetic (chromatin accessibility) characteristics of early bovine embryonic trophoblast cells. However, the existing technology uses bovine in vitro developed 7.5-day blastocysts to culture bovine trophoblast stem cells, which may be related to the signal pathways of different states of blastocyst stages. The hatched blastocysts do not derive related trophoblast stem cells, and the use of LCDM cannot derive trophoblast stem cells from hatched blastocysts. SUMMARY

[0004] In view of this, the present application provides a composition comprising Activin A, CHIR99021 and recombinant human leukemia inhibitory factor (hLif), which can induce hatched blastocysts to establish bovine trophoblast stem cells and stably culture them.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The present application provides a composition comprising Activin A, CHIR99021 and recombinant human leukemia inhibitory factor.

[0006] Preferably, the mass ratio of Activin A to CHIR99021 is (40-60 μg):(2x10 -5 ~4x10 -5 mol). The molar ratio of the CHIR99021 and the recombinant human leukemia inhibitory factor is 6: (0.5-1.5).

[0007] The application provides a bovine trophoblast stem cell culture medium, the cell culture medium is mTeSR 1 pluripotent stem cell culture medium; The concentration of activin A in the cell culture medium is 40-60 mu g / ml, the concentration of CHIR99021 is 20-40 mM, and the concentration of recombinant human leukemia inhibitory factor is 4-6 mM.

[0008] The application provides a culture medium composition, comprising the cell culture medium and bovine fetal fibroblasts.

[0009] Preferably, the culture medium composition further comprises a bovine trophoblast stem cell subculture medium.

[0010] The application provides application of the composition, the cell culture medium or the culture medium composition in establishment of bovine trophoblast stem cells.

[0011] The application provides a method for establishing trophoblast stem cells, comprising the following steps: The hatched blastocyst is inoculated into a feeder layer of bovine fetal fibroblasts treated with mitomycin, and is induced and cultured by using the cell culture medium, and after forming an expanded structure, subculture is carried out to obtain trophoblast stem cells.

[0012] Preferably, the concentration of the mitomycin is 0.3-0.7 mg / mL. The treatment time of the mitomycin is 2-3 h.

[0013] Preferably, the induction culture time is 3-10 d.

[0014] Preferably, the subculture time is 3-5 d.

[0015] Compared with the prior art, the application has the following advantages: The application provides a composition comprising Activin A, CHIR99021 and hLif. The application adopts three factors to culture the hatched blastocyst, and obtains bTSC in vitro, and can be long-term and stable in vitro, and expresses trophoblast cell specific markers at mRNA and protein levels. This can further enrich the culture system of bovine trophoblast stem cells, and can expand the understanding of the difference of signal pathways of bovine blastocysts at different stages.

[0016] The present application provides a method for establishing trophoblast stem cells, wherein the hatched blastocyst is inoculated into a feeder layer of mytomycin-treated bovine fetal fibroblasts, and the hatched blastocyst is induced and cultured in the cell culture medium to form an expanded structure, and then subcultured to obtain trophoblast stem cells. In the technical route for generating trophoblast stem cells in vitro, the difficulty at present is to gradually transition the embryo in a specific culture medium to be able to be stably cultured in vitro for a long time. Many studies focus on how to change the culture state and try different combinations of small factors. The present application can directly establish the hatched blastocyst by combining the feeder layer cells and regulating the small factors, and improve the purity of bTSC. By using the culture of the cell culture medium, the bTSC can be easily observed at the cell level. This not only can simulate in vivo development, but also provides a new technical route for constructing bovine trophoblast stem cells in vitro: establishing trophoblast cells through blastocysts (hatched) in different states. The bTSC established in the present application with high efficiency in vitro can further verify the in vitro growth ability of the trophoblast cells, and can be stably cultured for a long time during the whole culture process. The method of the present application has low cost, simple operation and is suitable for large-scale in vitro culture. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Flow chart for establishing bovine hatched blastocyst-derived trophoblast stem cells; Figure 2 bTSC obtained by the ACL system (Activin A, CHIR99021 and hLif) electron microscope observation result graph; Figure 3 bTSC karyotype analysis result graph; Figure 4 Flow cytometry analysis results of bTSC (with BFF-feeder) for trophoblast cell markers CDX2 and GATA3; Figure 5 bTSC gene expression level detection result graph; Figure 6 bTSC immunofluorescence staining result graph. DETAILED DESCRIPTION

[0018] The present application provides a composition comprising Activin A, CHIR99021 and hLif.

[0019] In the present application, the composition can establish trophoblast stem cells in bovine hatched blastocysts and can be stably cultured in vitro for a long time.

[0020] In the present application, the mass ratio of Activin A to CHIR99021 is (40-60 μg):(2×10 -54 x 10 -5 mol); The molar ratio of CHIR99021 to hLif is 6: (0.5-1.5).

[0021] In the present application, the ratio of Activin A to CHIR99021 is preferably (45-55 μg): (2.5 x 10 -5 3.5 x 10 -5 mol), more preferably 50 μg: 3 x 10 -5 mol. The molar mass ratio of CHIR99021 to hLif is preferably 6: (0.8-1.2), more preferably 6:1. The present application defines the ratio of Activin A, CHIR99021 and hLif to promote the establishment of trophoblast stem cells in bovine hatched blastocysts.

[0022] The present application provides a bovine trophoblast stem cell culture medium, which is mTeSR 1 pluripotent stem cell culture medium; The concentration of Activin A in the cell culture medium is preferably 40-60 μg / ml; the concentration of CHIR99021 is preferably 20-40 mM; and the concentration of hLif is preferably 4-6 mM.

[0023] In the present application, the mTeSR 1 preferably purchased from Stem cell Technologies, item #85850. The mTeSR 1 preferably contains Basal Medium and 5 x Supplement. The volume fraction of the Basal Medium is preferably 80%, and the volume fraction of the 5 x Supplement is preferably 20%. In the present application, 505 ml of mTeSR 1 culture solution contains Basal Medium 400 ml, 5 x Supplement 100 ml and penicillin-streptomycin double antibody (PS) 5 ml. The Basal Medium is purchased from Stem cell Technologies, item #85851. The 5 x Supplement is purchased from Stem cell Technologies, item #85852.

[0024] In the present application, the concentration of Activin A is more preferably 45-55 μg / ml, and most preferably 50 μg / ml. The concentration of CHIR99021 is more preferably 25-35 mM, and most preferably 30 mM. The concentration of hLif is more preferably 4.5-5.5 mM, and most preferably 5 mM. The concentrations are conducive to establishing a new cell culture system and promoting the establishment of trophoblast stem cells in the hatched blastocyst of a bovine.

[0025] The present application provides a medium composition comprising the cell culture medium and bovine fetal fibroblasts.

[0026] In the present application, the medium composition preferably further comprises a bovine trophoblast stem cell subculture medium.

[0027] The components of the medium composition in the present application are the best in vitro culture and establishment scheme after adjustment of experimental conditions and cell lines. The embodiments of the present application show that the hatched blastocyst is inoculated into a bovine fetal fibroblast feeder layer, and the trophoblast stem cells are obtained by inducing culture and subculture with the cell culture medium.

[0028] The present application provides the use of the composition, the cell culture medium or the medium composition in the establishment of bovine trophoblast stem cells.

[0029] In the present application, the establishment of bovine trophoblast stem cells preferably comprises the establishment of trophoblast stem cells in a hatched blastocyst of a bovine.

[0030] The present application provides a method for establishing trophoblast stem cells, comprising the following steps: The hatched blastocyst is inoculated into a feeder layer of bovine fetal fibroblasts treated with mitomycin, and the cell culture medium is used for inducing culture to form an expanded structure. After the expanded structure is formed, subculture is performed to obtain trophoblast stem cells.

[0031] The present application inoculates the hatched blastocyst into the mitomycin-treated bovine fetal fibroblast feeder layer, and uses the cell culture medium to induce culture, forming the expanded pattern structure. In the present application, the hatched blastocyst preferably includes the blastocyst hatched in vivo after in vivo fertilization, and more preferably is obtained by in vitro hatching of the blastocyst on the 7th to 8th day after in vivo fertilization for 3 to 5 days. The hatching culture medium preferably includes IVC culture solution rich in 10% FBS. In the embodiment of the present application, the hatched blastocyst is a 7.5-day blastocyst flushed from the oviduct of a cow in vivo, which is in vitro hatched for 4 days. The hatched blastocyst preferably includes the inner cell mass removal. The culture medium for culturing the bovine fetal fibroblast preferably includes M10 culture solution. The preparation method of the mitomycin-treated bovine fetal fibroblast feeder layer preferably comprises the following steps: treating bovine fetal fibroblasts with mitomycin, digesting the cells to obtain free mitomycin-treated bovine fetal fibroblasts, and inoculating the free mitomycin-treated bovine fetal fibroblasts into a gelatin-coated culture dish to obtain the mitomycin-treated bovine fetal fibroblast feeder layer. The concentration of the mitomycin is preferably 0.3 to 0.7 mg / mL, more preferably 0.4 to 0.6 mg / mL, and most preferably 0.5 mg / mL. The treatment time is preferably 2 to 3 hours, more preferably 2.2 to 2.8 hours, and most preferably 2.5 hours. The treatment temperature is preferably 38 to 39°C, and more preferably 38.5°C. The effect of the mitomycin treatment is to make the cells enter the division arrest, and promote the culture of stem cells. The reagent for digestion preferably includes Tryple enzyme. After obtaining the hatched blastocyst and the mitomycin-treated bovine fetal fibroblast feeder layer, the present application inoculates the hatched blastocyst into the mitomycin-treated bovine fetal fibroblast feeder layer, and adds the cell culture medium to induce culture, forming the expanded pattern structure. The addition amount is conducive to the formation of a clone of the hatched blastocyst. The induction culture time is preferably 3 to 10 days, more preferably 5 to 8 days, and most preferably 7 days. The induction culture temperature is preferably 37°C, and the CO2 volume concentration in the environment is preferably 5%. The effect of the induction culture is to culture the cells of the hatched blastocyst in vitro.

[0032] After forming the expanded pattern structure, the present application performs subculture to obtain the trophoblast stem cells. In order to obtain a stable cell line for stable and long-term culture in vitro, the embodiment of the present application performs subculture when the cell fusion degree in the expanded pattern structure reaches 80%.

[0033] In the present application, the method of subculture preferably comprises transferring the cells in the expanded blastocyst structure to a feeder layer of mytomycin-treated bovine fetal fibroblasts, and culturing with a subculture medium. The medium of subculture is preferably the same as the medium of the induced culture. The CO2 volume concentration of the subculture is preferably the same as the induced culture. The time of the subculture is preferably 3-5 days, which can be 4 days. The number of subculture is preferably 1-25 generations, more preferably 5-10 generations, and most preferably 6 generations. The bovine trophoblast stem cells are obtained after the subculture.

[0034] In the present application, the method of subculture preferably comprises transferring the cells in the expanded blastocyst structure to a feeder layer of mytomycin-treated bovine fetal fibroblasts, and culturing with a subculture medium. The medium of subculture is preferably the same as the medium of the induced culture. The CO2 volume concentration of the subculture is preferably the same as the induced culture. The time of the subculture is preferably 3-5 days, which can be 4 days. The number of subculture is preferably 1-25 generations, more preferably 5-10 generations, and most preferably 6 generations. The bovine trophoblast stem cells are obtained after the subculture.

[0035] All the results of the present application are biologically repeated and various related detections are performed. It is proved by multiple experiments that the method route of the present application can successfully obtain bTSC with high purity, and compared with other cell line establishment methods, the bTSC obtained by the present application is more conducive to observing cell morphology and conducting subsequent experiments.

[0036] In order to further illustrate the present application, the scheme provided by the present application is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0037] 1. Medium and solution components.

[0038] The components of the bovine trophoblast stem cell culture solution are shown in Table 1. The components of 505 ml mTeSR 1The components of the culture solution are shown in Table 2. The components of 50 ml M10 culture solution are shown in Table 3. The components of 50 ml R termination solution are shown in Table 4.

[0039] Table 1 Components of bovine trophoblast stem cell culture solution

[0040] Table 2 Components of 50 ml M10 culture solution Culture solution components In the present application, the method of subculture preferably comprises transferring the cells in the expanded blastocyst structure to a feeder layer of mytomycin-treated bovine fetal fibroblasts, and culturing with a subculture medium. The medium of subculture is preferably the same as the medium of the induced culture. The CO2 volume concentration of the subculture is preferably the same as the induced culture. The time of the subculture is preferably 3-5 days, which can be 4 days. The number of subculture is preferably 1-25 generations, more preferably 5-10 generations, and most preferably 6 generations. The bovine trophoblast stem cells are obtained after the subculture.

[0041] Table 3 Ingredients per 50 ml M10 medium

[0042] Table 4 Ingredients per 50 ml R stop solution

[0043] Ingredients per 10 ml 10% DMSO stock solution: DMSO 1 ml, FBS 9 ml.

[0044] IVC medium was purchased from BIO, Cat# 71005.

[0045] 0.5 mg / mL mitomycin: 2 mg / vial of mitomycin was added with 4 mL DPBS, inverted to mix well, and stand for 5-10 min. 4 mg of mitomycin was transferred to 50 mL centrifuge tube, and adjusted to 8 mL with DPBS.

[0046] 0.075 mol / L preheated KCL solution: 0.0559 g KCL powder was weighed into a 15 mL centrifuge tube, and 10 mL ultrapure water was added to prepare 0.075 mol / L KCL solution, which was preheated in a 37°C water bath for standby.

[0047] Pre-cooled fixative: 30 mL of methanol and 10 mL of glacial acetic acid were mixed and pre-cooled in a -20°C refrigerator for standby.

[0048] Preheated Giemsa staining solution (freshly prepared): 2.5 mL of Giemsa stock solution was added to 47.5 mL of ultrapure water, mixed well, and filtered twice to remove impurities in the staining solution, then poured into a staining jar, and preheated in a 37°C water bath for 30 min.

[0049] The method for preparing bovine fetal fibroblasts (BFF) is disclosed in the prior art (Zhao, L. X., Gao, X. F., Zheng, Y. X., Wang, Z. X., Zhao, G. P., Ren, J., … Li, X. H. (2021). Establishment of bovine expanded potential stem cells. Proceedings of the National Academy of Sciences of the United States of America, 118(15), e2018505118. doi:10.1073 / pnas.2018505118) and is preserved in the 432 laboratory of the School of Life Sciences, Inner Mongolia University.

[0050] Example 1 The method for establishing bovine feeder layer stem cells in vitro (see schematic diagram Figure 1 ), comprising the following steps: 1. Preparation of bovine fetal fibroblasts: 37 ℃ water bath thawing bovine fetal fibroblasts (BFF) cells (5.0×10 5 cells) into 3 ml R stop solution in advance, centrifuged at 1300 rpm for 3 min, discarded the supernatant, resuspended the cell pellet with 20 mL M10 culture medium and inoculated into 2 15 cm culture dishes (the culture dishes were coated with gelatin in advance 20 min in advance), and incubated in a 38.5 ℃ CO2 incubator for 3-4 days. On the fifth day, the BFF cells in the two culture dishes were subcultured at a ratio of 1:10 into 20 dishes (the culture dishes were coated with gelatin in advance 20 min in advance) and cultured for 5 days.

[0051] After 5 days, 400 μL of 0.5 mg / mL mitomycin was added to each of the 20 15 cm dishes and incubated in the incubator for 2.5 h. The culture medium was discarded and the cells were washed with 5 mL of DPBS. 5 mL of preheated Tryple enzyme (Gibco Company) was added to each dish and incubated for 5-10 min. After 5 min of incubation, the cells were observed under a microscope until 70-80% of the cells floated, and 5 mL of R stop solution was immediately added. The cells were resuspended by blowing and finally collected in a 50 mL centrifuge tube. The cell pellet was centrifuged at 1300 rpm for 5 min to obtain the mitomycin-treated bovine fetal fibroblasts. 10 mL of M10 culture medium was added to resuspend the feeder layer cells, and 10 μL of the cell suspension was added to 990 μL of DPBS for cell counting.

[0052] One part was frozen at 4.2 x 10 6 cells per tube. The cells were transferred to a -80 °C refrigerator according to the programmed cooling method. The next day, the frozen BFF was thawed, and the cell density and growth were observed. If each step was normal, the frozen tube was transferred to liquid nitrogen for long-term storage.

[0053] Another part was inoculated at 4.2 x 10 6 cells per well in a 24-well plate (the 24-well plate was pre-coated with gelatin for 20 min), 2 ml of M10 culture solution was added, and the plate was incubated in a 38.5 °C CO2 incubator for 2 d. The M10 culture solution was removed with a pipette, and the bovine fetal fibroblast feeder layer (BFF-feeder) was obtained for use.

[0054] 2. Establishment of feeder layer stem cells and subculture: The 7.5-day blastocysts obtained from the oviduct of a cow were transferred to IVC culture solution containing 10% FBS for incubation. The blastocyst morphology was observed daily to ensure that the embryo had sufficient nutrition. After 4 days of in vitro culture, the blastocysts hatched from the zona pellucida were subjected to inner cell mass removal. The hatched blastocysts with removed inner cell mass were transferred to a 24-well plate pre-coated with BFF-feeder, and 2 ml of bovine feeder layer stem cell culture solution in Table 1 was added for induction culture (one hatched blastocyst with removed inner cell mass was added to each 24-well plate). The induction culture was performed at 37 °C in an environment with a CO2 volume concentration of 5%. After about 7 days, the cells began to expand on the feeder layer and form flat clones. The cell state was observed daily, and the culture solution was replaced every 48 h. When the cells grew to about 80%, they were subcultured.

[0055] Subculture method: the cells in the incubator were removed, the culture solution in the well was removed, 1 ml of DPBS buffer was slowly added to the well, the culture plate was gently shaken several times, and the well was washed to remove dead cells and impurities. After the DPBS was removed, 300 μL of preheated Tryple enzyme was immediately added, and after 3 min in the incubator, the cells were observed under a microscope to determine whether they were no longer adherent. Then, 700 μL of preheated R stop solution was added, and the mixture was repeatedly blown several times. The cell suspension was transferred to a 1.5 ml centrifuge tube, and centrifuged (1300 rpm, 3 min). After centrifugation, the bovine feeder layer stem cell culture solution in Table 1 was added, and the cells were subcultured on a 24-well plate at a density of 1 x 10 5 cells per ml at 37 °C in an environment with a CO2 volume concentration of 5%.

[0056] Figure 2bTSC obtained from the ACL system. From P0, P5 and P20 in the figure, which are the 0th, 5th and 20th passages, it can be seen that bTSC presents a classic flat clone and can be stably cultured in vitro for a long time.

[0057] Example 2 Karyotype analysis of bovine trophoblast stem cells The cell culture solution of bTSC stably cultured (passaged 5 times) in Example 1 was aspirated with a pipette, and a certain volume of colchicine was added according to the cell density for 3 h of treatment. After colchicine treatment, the cell culture solution was discarded, and the cells were digested with TrypLE for 3 min, then 10% KSR was added for termination, and then placed in a centrifuge at 1500 rpm for 3 min. After centrifugation, the supernatant was discarded, 8 mL of 0.075 mol / L preheated KCL solution was added to the cell precipitate, the cells were resuspended by blowing, and then 37°C water bath for 10 min for hypotonic treatment. 1 min before the end of hypotonic treatment, 1 mL of pre-cooled fixative was added for pre-fixation, and after mixing, centrifuged at 1000 rpm for 10 min. After centrifugation, the supernatant was removed, 8 mL of pre-cooled fixative was added, and the cell precipitate was resuspended, then fixed in a 37°C water bath for 30 min. After fixation, centrifuge at 1000 rpm for 10 min. This step was repeated twice. The last time the slide was briefly immersed in boiling pure water and then placed in a -20°C refrigerator for pre-cooling. After centrifugation, the supernatant was removed, the cells were resuspended with 300 µL of pre-cooled fixative, and the cell suspension was evenly and vertically dropped onto the pre-cooled slide. After dropping the slide, the slide was placed in a 70°C oven for 1 h. During the drying of the slide, the dried slide was placed in a preheated Giemsa staining solution and soaked in a 37°C water bath for 10 min to complete the staining. After staining, the back of the slide was rinsed with excess stain and then air-dried at room temperature. The number and morphology of chromosomes were observed under an inverted microscope.

[0058] Figure 3 The karyotype analysis result of bTSC is consistent with the distribution of bovine (2n=60) chromosomes, indicating that bTSC is a normal diploid karyotype.

[0059] Example 3 Flow cytometry analysis of trophoblast stem cells bTSC and BFF-feeder stably cultured (passaged for 5 times) in Example 1 were collected in ice PBS solution, centrifuged at 4 °C, 250 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in fresh ice PBS solution and centrifuged under the same conditions. The cells were blocked with blocking buffer (2.5 g BSA dissolved in 50 mL PBS solution) at room temperature for 15 min, and then terminated by adding appropriate amount of PBS buffer and divided into different groups according to experimental requirements. The blank control group and the experimental group were prepared. After being fixed with 4% paraformaldehyde at room temperature for 15 min in the dark, anhydrous methanol was added for incubation in the dark for 10 min. The supernatant was removed by centrifugation at 4 °C, 250 rpm for 15 min. In the experimental group, Anti-CDX2 and Anti-GATA3 antibodies were diluted at a ratio of 1:500 μL in 100 μL flow cytometry staining buffer and incubated with the antibodies at room temperature in the dark for 1 h. The eluent (0.05% Tween-20 in PBS buffer) was used for centrifugation under the above conditions. In the experimental group, DyLight 549 dye and DyLight 488 dye were diluted at a ratio of 1:1000 μL in 100 μL flow cytometry staining buffer and incubated with the antibodies at room temperature in the dark for 30 min. Non-single cell clusters were filtered using a 300-mesh screen, and the samples were analyzed using a cytoFLEX S flow cytometer.

[0060] Figure 4 Flow cytometry analysis of bTSC using the trophoblast cell markers CDX2 and GATA3. The proportion of positive cells in the figure is close to 50%.

[0061] Example 4 Detection of related genes and protein levels CDX2, Gata3, and Tfap2c are commonly used for the identification of trophoblast stem cells, and together reflect the trophoblast properties of the cells. Oct4 is a pluripotent stem cell marker. After bTSC in Example 1 was stably cultured, sampling was performed, and the cells were detected at the mRNA and protein levels.

[0062] Gene level detection: After removing the culture solution, add TRIzol reagent in the culture dish, use micropipette to blow and react fully. After the adherent cells are completely lysed, collect them into a centrifuge tube. After standing at room temperature for 5 min, add chloroform according to the volume of one-fifth of the previous TRIzol reagent. After standing at room temperature for 10 min, centrifuge at 4°C, 12000 rpm for 15 min. After centrifugation, carefully tilt the centrifuge tube at 45 degrees and take it out, collect the RNA in the supernatant into a new centrifuge tube. Add isopropanol. After standing at room temperature for 10 min, centrifuge at 4°C, 12000 rpm for 10 min. The collected precipitate is vortexed and resuspended in freshly prepared 75% ethanol, and then centrifuged at 4°C, 7500 rpm for 5 min. After removing the supernatant, invert it on the clean bench and wait until it is ensured that there is no ethanol residue in the centrifuge tube. Resuspend the precipitate with a micropipette by adding enzyme-free water, and then place the sample in a water bath at 55°C for 10 min. Detect the RNA concentration by ultraviolet spectrophotometer and perform reverse transcription. Prepare the following reaction mixture with a total volume of 10 μL in a PCR tube: 7 μL of RNase Free ddH2O, 1 μL of dNTP Mixture, 1 μL of Oligo dT Primer, and 1 μg of RNA template. Incubate the above reaction mixture at 55°C for 5 min, and then immediately cool on ice. Prepare the following reaction mixture with a total volume of 20 μL: 10 μL of prepared RNA reaction substrate, 5 μL of RNase Free ddH2O, 4 μL of 5× Prime Script Buffer, 1 μL of RNase Inhibitor, and 1 μL of Prime Script RNase. After gentle mixing, incubate the reaction mixture at 37°C for 1 h. Then incubate at 95°C for 5 min to inactivate the enzyme, and then immediately cool on ice to obtain cDNA.

[0063] Under light-proof conditions, prepare the following reaction mixture with a total volume of 20 μL in an opaque PCR tube: 10 μL of SYBR Premix Ex Taq, 2 μL of primers (1 μL of forward primer and 1 μL of reverse primer), 2 μg of cDNA template, and 6 μL of RNase Free ddH2O. The primer sequence information is shown in Table 5, and the primers are synthesized by a biological engineering company.

[0064] Table 5 Primer sequence information

[0065] The above reactants were placed in a PCR instrument according to the induction days and the accurate arrangement of genes. The following cycles were set: 95°C pre-denaturation for 2 min; 95°C high-temperature denaturation for 30 s, 58°C low-temperature annealing for 45 s, 72°C extension for 30 s, a total of 38 cycles, and then 4°C storage.

[0066] Immunofluorescence detection: remove the cell culture fluid, and wash with ice PBS solution for 3 times. Fix the cells with 4% paraformaldehyde solution at room temperature for 15 min. Remove the fixing solution, and wash with 1x PBS solution for 3 times. Permeabilize the cells with permeabilization solution (0.01 mL Triton-X-100 in 10 mL PBS solution) at room temperature for 10 min. Remove the permeabilization solution, and wash with 1x PBS solution for 3 times. Add blocking buffer (0.2 g BSA, 1.126 g Glysine and 0.05 Tween20 in 25 mL 1x PBS solution) and block at room temperature for 1 h. Dilute the primary antibody (CDX2, GATA3) in 500 μL blocking buffer at a ratio of 1:500, and incubate at 4°C overnight in the dark. Remove the primary antibody solution, and wash with 1x PBS solution for 3 times. Dilute the secondary antibody (Alexflour 488 Goat anti-mouse IgG, Alex Flour 549 Goat anti-rabbit IgG) in 500 μL blocking buffer at a ratio of 1:1000, and incubate at room temperature on a shaker in the dark for 1 h. Remove the secondary antibody (Alex flour 488 Goat anti-mouse IgG, Alex Flour 549 Goat anti-rabbit IgG) solution, and wash with 1x PBS solution for 3 times. Dilute 0.1 μg / mL DAPI in 500 μL blocking buffer, and incubate at room temperature on a shaker in the dark for 5 min. Remove the DAPI solution, and wash with 1x PBS solution for 3 times. Mount the slide with gum, and perform confocal imaging using a scanning laser confocal microscope (NIS-Elements Viewer 4.20).

[0067] Figure 5 is the detection result of the gene expression level of bTSC, P5 and P20 are 5 times and 20 times of passage, respectively. Compared with BFF-feeder, the pluripotency gene Oct4 of bTSC cell has almost no obvious expression, while the trophoblast cell markers CDX2, Tfap2c and Gata3 have obvious expression.

[0068] Figure 6 is the immunofluorescence staining result of bTSC with 5 times of passage. The results show that bTSC expresses trophoblast cell marker proteins CDX2 and GATA3.

[0069] After the hatched blastocyst is cultured by the method, the bTSC can be obtained in vitro, and the bTSC can be cultured in vitro for a long time.

[0070] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity according to the present embodiment, and these embodiments all belong to the protection scope of the present application.

Claims

1. A composition, characterized in that, It includes activin A, CHIR99021, and recombinant human leukemia inhibitory factor.

2. The composition according to claim 1, characterized in that, The molar ratio of the activator A to CHIR99021 is (40~60μg): (2×10⁻⁶μg). -5 ~4×10 -5 mol); The molar ratio of CHIR99021 to recombinant human leukemia inhibitory factor is 6:(0.5~1.5).

3. A bovine trophoblast stem cell culture medium, characterized in that, The cell culture medium is an mTeSR containing the composition of claim 1 or 2.

1. Pluripotent stem cell culture medium; In the cell culture medium, the concentration of activin A is 40-60 μg / ml; the concentration of CHIR99021 is 20-40 mM; and the concentration of recombinant human leukemia inhibitory factor is 4-6 mM.

4. The cell culture medium according to claim 3, characterized in that, The concentration of activator A is 45-55 μg / ml; the concentration of CHIR99021 is 24-35 mM; and the concentration of recombinant human leukemia inhibitory factor is 4.5-5.5 mM.

5. A culture medium composition, characterized in that, Includes the cell culture medium and bovine fetal fibroblasts as described in claim 3 or 4.

6. The use of the composition of claim 1 or 2, the cell culture medium of claim 3 or 4, or the culture medium combination of claim 5 in the establishment of bovine trophoblast stem cells.

7. A method for establishing bovine trophoblast stem cells, characterized in that, Includes the following steps: Hatched blastocysts are inoculated into a feeder layer of bovine fetal fibroblasts treated with mitomycin C, induced and cultured using the cell culture medium described in claim 3 or 4, and after forming an expanded structure, they are passaged to obtain trophoblast stem cells.

8. The method according to claim 7, characterized in that, The concentration of mitomycin is 0.3~0.7 mg / mL; The treatment time for mitomycin is 2-3 hours.

9. The method according to claim 7, characterized in that, The induction culture time is 3-10 days.

10. The method according to any one of claims 7 to 9, characterized in that, The duration of each subculture is 3-5 days.

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