Application of tetracycline drug in synchronous separation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells

Abstract

Provided is an application of a tetracycline drug in the synchronous separation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells. Bovine blastocysts or inner cell masses thereof are cultured in a medium containing a tetracycline drug, thereby synchronously separating and maintaining the dryness of bESCs and bTSCs in the same culture system. The bidirectional regulatory capability of the tetracycline drug is used to solve the technical problem of synchronous separation of bESCs and bTSCs, and an efficient and low-cost standardized platform is provided for artificial embryos, regenerative medicine and agricultural bioengineering.

Description

Application of tetracycline drugs in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2025114298052, filed on September 30, 2025, entitled "Application of Tetracycline Drugs in the Simultaneous Isolation and Maintenance of Bovine Embryonic Stem Cells and Bovine Trophoblast Stem Cells", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the fields of cell biology and animal embryo engineering technology, and more specifically to the application of tetracycline drugs in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells. Background Technology

[0004] Bovine embryonic stem cells (bESCs) originate from the inner cell mass (ICM) of the bovine blastocyst and are pluripotent, capable of differentiating into all cell types of the three germ layers. Bovine trophoblast stem cells (bTSCs) originate from the trophectoderm (TE) of the blastocyst and are key precursor cells for placental formation. These two types of stem cells are core biomaterials for studying embryonic development, organogenesis, and constructing synthetic embryos. However, the isolation and culture of bESCs and bTSCs face significant challenges in current technologies, as their culture conditions are often mutually exclusive, preventing their coexistence and simultaneous isolation within the same system.

[0005] The challenges of culturing bovine embryonic stem cells (bESCs): Traditional bESC culture relies on feeder cells (such as mouse embryonic fibroblasts, MEFs) and a combination of various cytokines, such as leukemia inhibitory factor (LIF) and fibroblast growth factor (FGF2). Although several improved culture media have been reported in recent years, such as FR1 / NBFR, LCDM, 3i / LAF, and TIFX, bESCs in these systems still generally suffer from problems such as easy spontaneous differentiation, unstable pluripotency, and difficulty in long-term stable passaging.

[0006] Culture limitations of bovine trophoblast stem cells (bTSCs): Reported conditions for establishing and maintaining bTSC culture lines require activation of the Wnt signaling pathway and inhibition of MEK / GSK3β (i.e., the 2i system, PD0325901, CHIR99021), or the use of LCDM containing LIF, CHIR, Minocycline hydrochloride (M), and (S)-(+)-Dimethindene maleate (DiM / D). However, these culture conditions, particularly the activation of the Wnt signaling pathway, often inhibit the self-renewal of human and bovine pluripotent stem cells, including bESCs, and may even promote their differentiation into other lineages. Therefore, bESC isolation and culture generally require Wnt inhibitors (IWR-1 or XAV939). Furthermore, although the LCDM culture system has been reported to successfully isolate either bESCs or bTSCs, there are currently no reports of simultaneously isolating both types of cells from the same embryo using LCDM.

[0007] Due to the fundamental conflict in the aforementioned culture conditions (Wnt signaling) and the unstable maintenance of pluripotency of bESCs in current culture systems, there are currently no publicly reported methods for simultaneously isolating and stably culturing bESCs and bTSCs from the same bovine blastocyst. Traditional methods can only employ sequential isolation (i.e., first isolating one type of cell from a batch of bovine blastocysts, then attempting to isolate the other type from another batch of bovine blastocysts). This method is inefficient, cumbersome, and makes it difficult to guarantee the original state and high purity of the two cell types.

[0008] Therefore, there is an urgent need in this field for an innovative technical system to overcome the technical bottleneck of mutually exclusive culture conditions for dual-lineage stem cells, and to achieve efficient and synchronous separation of bESCs and bTSCs while maintaining their stemness. Summary of the Invention

[0009] In view of this, this application provides the use of tetracycline drugs in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

[0010] The main purpose of this application is to solve the technical problem of being unable to simultaneously isolate and maintain bovine embryonic stem cells and bovine trophoblast stem cells in the existing technology, and to provide a new and efficient culture method and culture system.

[0011] To achieve the above objectives, this application adopts the following technical solution:

[0012] Application of tetracycline drugs in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

[0013] Furthermore, the tetracycline drug is doxycycline (DOX) or minocycline (MC).

[0014] Furthermore, the application of doxycycline in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

[0015] Beneficial effects achieved: Doxycycline, as a key bidirectional regulator, played an unprecedented dual role in this system, breaking down the barriers of culture conditions for bESCs and bTSCs.

[0016] First, doxycycline significantly enhanced the stability of bESCs during culture and inhibited their self-differentiation. This made it easier for bESCs initially isolated from the embryo to expand into stable cell lines. Second, doxycycline improved mitochondrial function and enhanced cell metabolism and activity, which may have enabled the rapid isolation and expansion of bTSCs even in culture systems containing Wnt inhibitors. These two mechanisms are likely the main reasons why doxycycline can promote the simultaneous isolation of both cell types from the same embryo.

[0017] Experiments revealed that bESCs readily undergo random differentiation in previously reported culture media, resulting in increasingly irregular clonal morphology and significant cell death. Further investigation involved adding doxycycline to previously reported culture media at concentrations ranging from 1 to 25 μg / mL. Observation of clonal morphology showed that doxycycline at concentrations of 5–10 μg / mL significantly improved clonal morphology, resulting in smoother clonal surfaces and edges, and a more three-dimensional appearance. Doxycycline was also found to inhibit random differentiation and apoptosis of bESCs in basal culture media and enhance stem cell self-expansion efficiency. Experiments demonstrated that doxycycline does not act by altering the expression of embryonic stem cell pluripotency markers, such as SSEA4, alkaline phosphatase, OCT4, NANOG, and SOX2. Experiments showed that inducing excessive nitric oxide (NO) production led to embryonic stem cell apoptosis, while doxycycline inhibited the expression of NO synthase (NOS) and nitric oxide production in bESCs, thereby inhibiting random differentiation and apoptosis of bESCs in basal culture media. Meanwhile, 5-10 μg / mL doxycycline significantly increased the mitochondrial membrane potential of bESCs and enhanced oxidative phosphorylation, thereby enhancing the metabolic activity of stem cells.

[0018] Furthermore, doxycycline was added to the previously reported culture medium to a final concentration of 10 μg / mL (based on observations of stem cell clonal morphology and growth).

[0019] The addition of doxycycline to the culture medium also led to the successful isolation and amplification of bTSCs. The specific reasons require further investigation. However, based on our experimental results on bESCs, it is suggested that doxycycline may stimulate in vitro cell growth by inhibiting NOS and increasing mitochondrial activity, thereby promoting the successful isolation and amplification of bTSCs from embryos.

[0020] Furthermore, minocycline was added to the previously reported culture medium to achieve a final concentration of 5–10 μg / mL.

[0021] Based on observations of bESC clonal morphology, similar to DOX, minocycline at concentrations of 5–10 μg / mL significantly improved clonal morphology, resulting in smoother clonal surfaces and edges with a more three-dimensional appearance. Further analysis revealed that 5 μg / mL minocycline significantly enhanced bESC mitochondrial activity, increased oxidative phosphorylation, and improved bESC's compensatory glycolysis capacity, thereby enhancing overall stem cell metabolic activity.

[0022] A bovine dual-potency stem cell culture medium, comprising tetracycline drugs.

[0023] Furthermore, it includes the following components: mTeSRTMPlus as the base culture medium, with the addition of 0.5–5 μM IWR-1, 0.5–3 μM PD184352, 1–6 μM SU5402, 1–6 μM EPZ-004777, 1–25 μM Forskolin, 1–10 μM CHIR99021, 5–25 ng / mL human leukemia inhibitory factor, and 5–10 μg / mL DOX / 2–10 μg / mL MC.

[0024] Furthermore, it includes the following components: mTeSRTMPlus-based culture medium supplemented with 2.5 μM IWR-1, 0.8 μM PD184352, 3 μM SU5402, 3.3 μM EPZ-004777, 10 μM Forskolin, 3 μM CHIR99021, 10 ng / mL human leukemia inhibitory factor, and 10 μg / mL DOX / 5 μg / mL MC.

[0025] The above-mentioned culture medium is used in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

[0026] A method for simultaneously isolating and maintaining bovine embryonic stem cells and bovine trophoblast stem cells involves seeding bovine blastocysts or their inner cell masses into a culture plate pre-coated with feeder cells and adding the aforementioned culture medium for culturing.

[0027] Furthermore, based on the mouse fibroblast feeder layer, bESCs and bTSCs were isolated, digested, and stably passaged using the above-mentioned culture medium according to conventional methods, achieving synchronous isolation and maintenance of stemness within the same culture system, which facilitates subsequent specialized culture operations and applications.

[0028] Furthermore, the vertically growing, dense, multi-layered oval structure represents bovine embryonic stem cells, while the flat, spread-out monolayer of cells at the bottom of the dish represents bovine trophoblast stem cells.

[0029] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by this application are as follows:

[0030] (1) Achieved simultaneous separation of two spectral systems:

[0031] This application demonstrates for the first time that by adding DOX, bESCs and bTSCs cell lines can be successfully and synchronously isolated from the same bovine blastocyst in a single culture system, greatly simplifying the operation process and improving the isolation efficiency.

[0032] (2) It reveals the dual regulatory function of DOX:

[0033] For bESCs: DOX can effectively promote the stable proliferation of bESCs by inhibiting the nitric oxide synthase (NOS) / nitric oxide (NO) signaling pathway, while significantly inhibiting apoptosis and lineage differentiation, and maintaining the stability of their clonal morphology in the long term.

[0034] Regarding bovine embryonic stem cells (bTSCs): DOX treatment can produce bTSCs that can be stably passaged in vitro from bovine embryos. The specific mechanism of DOX on bTSCs needs further confirmation. However, based on our above research on bovine embryonic stem cells, the possible mechanisms by which DOX acts on bTSCs include inhibiting NOS / NO signaling and promoting mitochondrial and metabolic activity.

[0035] (3) It provides a high-quality cell source:

[0036] The bESCs and bTSCs acquired simultaneously have a clear source consistency, making them ideal materials for constructing bovine synthetic embryos (blastocysts), providing unprecedented convenience for studying the interaction between the embryo and the trophoblast and simulating the embryo implantation process.

[0037] (4) Broad application prospects:

[0038] This application system is efficient, stable, and relatively low-cost, providing a standardized platform and key technical support for research in embryonic developmental biology, drug reproductive toxicity screening, livestock genetic improvement (transgenic technology), and regenerative medicine. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0040] Figure 1 shows that the DOX-containing culture medium in Example 2 of this application can support the simultaneous isolation and passage culture of bTSC and bESC. In this figure, A is the initial morphology of bTSC growing on feeder cells, scale bar: 500 μm; B is the SSEA4 staining results of primary embryonic stem cells and trophoblast stem cells, where primary embryonic stem cells are strongly positive and trophoblast stem cells are negative, scale bar: 200 μm; C is the clonal morphology of bESC at P80; D is the clonal morphology of bTSC at P20.

[0041] Figure 2 shows the identification results of embryonic stem cells in Experiment 1 of this application. In Figure 2, A shows the staining results of SSEA4, a marker on the surface of bovine embryonic stem cells treated with DOX for 72 hours and those treated with DOX for 72 hours; B shows the staining results of alkaline phosphatase (AP), a marker protein of bovine embryonic stem cells treated with DOX for 72 hours and those treated with DOX for 72 hours, and C shows the immunofluorescence identification of SOX2, OCT4, and NANOG, marker proteins of bovine embryonic stem cells treated with DOX for 72 hours and those treated with DOX for 200 μm. Scale bar: 200 μm.

[0042] Figure 3 shows the expression of pluripotency genes OCT4, NANOG, and SOX2 in bESCs after untreated and treated DOX for 72 h, as detected by PCR in Experiment 1 of this application.

[0043] Figure 4 shows the immunofluorescence staining of trophoblast stem cell markers (KRT8 and CDX2) in Experiment 2 of this application. Scale bar: 200 μm.

[0044] Figure 5 shows the cell states of bESCs after 72 h of untreated and treated cells with DOX (1-25 μg / mL) in Experiment 3 of this application (first row is 4X, scale bar: 500 μm; second row is 10X, scale bar: 200 μm).

[0045] Figure 6 shows the cell growth curves of untreated and 4d treated bESCs in Experiment 3 of this application. The data are mean ± SD, *p≤0.05; **p≤0.01, n=3.

[0046] Figure 7 shows the expression of differentiation genes in bESCs after 72 h of untreated and treated DOX in Experiment 3 of this application, including ectoderm marker genes (GFAP, TUBB3, MAP2), mesoderm marker genes (T (Brachyury), ACTA2 (SMA)), and endoderm marker genes (FOXA2, GATA4, GATA6). Data are mean ± SD, *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns: no significant difference, n=3.

[0047] Figure 8 shows the TUNEL detection of apoptosis of bESCs after DOX treatment (untreated and treated for 72 h) in Experiment 3 of this application. The left figure shows the fluorescence identification results (scale bar: 100 μm), and the right figure shows the data statistics (mean ± SD, *p ≤ 0.05, n = 3).

[0048] Figure 9 shows the gene expression of nitric oxide synthase NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS) in bESCs after DOX treatment for 72 h in Experiment 3 of this application. The data are mean ± SD, *p≤0.05; **p≤0.01, n=3.

[0049] Figure 10 shows the NO secretion in bESCs after 72 hours of DOX treatment and before DOX treatment in Experiment 3 of this application. The data are mean ± SD, **p≤0.01, n=3.

[0050] Figure 11 shows TUNEL staining of bESCs cells after 72 h of untreated, separately treated, or treated together with DOX and nitric oxide (NO) donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) in Experiment 3 of this application. Scale bar: 100 μm and TUNEL. Data are mean ± SD, *p≤0.05, n=3.

[0051] Figure 12 shows the gene expression of NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS) after 72 h of untreated, separately treated, or jointly treated DOX and nitric oxide (NO) donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) in Experiment 3 of this application. Data are mean ± SD, *p≤0.05; **p≤0.01; ns: no significant difference, n=3.

[0052] Figure 13 shows the staining (top) and statistical results (bottom) of mitochondrial membrane potential indicator TMER (tetramethylrhodamine methylester) on untreated and treated bESCs in Experiment 3 of this application. Scale bar: 200 μm, data are mean ± SD, *: p < 0.05, n = 3.

[0053] Figure 14 shows the changes in basal and compensated glycolysis of bESCs after DOX treatment and before DOX treatment, as measured by the Seahorse metabolometer in Experiment 3 of this application. The data are mean ± SD, *p≤0.05; ***p≤0.001; ****p≤0.0001; n=3.

[0054] Figure 15 shows the proton efflux rates of glycolysis in bESCs before and after DOX treatment, as measured by the Seahorse metabolometer in Experiment 3 of this application.

[0055] Figure 16 shows the changes in mitochondrial basal respiration of bESCs before and after DOX treatment, as measured by the Seahorse metabolometer in Experiment 3 of this application. The data are mean ± SD, **p≤0.01, n=3.

[0056] Figure 17 shows the proliferation and maintenance of typical morphology of bTSC in DOX-containing medium in Experiment 4 of this application. In this figure, A shows the typical morphology of bTSC at different growth times in DOX-containing medium, D3: Day 3 of culture; D5: Day 5 of culture; D7: Day 7 of culture; D9: Day 9 of culture, scale bar: 500 μm; B shows the cell growth curve of bTSC cultured in DOX-containing medium for 9 days.

[0057] Figure 18 shows the clonal state of bESCs cells after untreated and treated with Minocycline (MC, 5-10 μg / mL) for 72 h in Experiment 5 of this application (first row is 4X, scale bar: 500 μm; second row is 10X, scale bar: 200 μm).

[0058] Figure 19 shows the changes in basal and compensated glycolysis of bESCs in untreated and treated MCs as measured by the Seahorse metabolometer in Experiment 5 of this application. The data are mean ± SD, **p≤0.01; n=3.

[0059] Figure 20 shows the proton efflux rates of glycolysis in bESCs before and after treatment, measured by the Seahorse metabolometer in Experiment 5 of this application.

[0060] Figure 21 shows the changes in basal mitochondrial respiration of bESCs before and after MC treatment, as measured by the Seahorse metabolometer in Experiment 5 of this application. The data are mean ± SD, **p≤0.01, n=3. Detailed Implementation

[0061] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0062] The reagents required for this application are standard laboratory reagents, purchased from commercially available channels; the experimental methods not mentioned are standard experimental methods, and will not be described in detail here.

[0063] Example 1

[0064] Preparation of bovine dual-potency stem cell culture medium

[0065] The culture medium was based on mTeSRTMPlus and supplemented with 2.5 μM IWR-1, 0.8 μM PD184352, 3 μM SU5402, 3.3 μM iDOT1L (EPZ-004777), 10 μM Forskolin, 3 μM CHIR99021, 10 ng / mL human leukemia inhibitory factor (LIF) and 10 μg / mL DOX.

[0066] Example 2

[0067] A method for simultaneously isolating and maintaining bovine embryonic stem cells and bovine trophoblast stem cells

[0068] (1) Mouse embryonic fibroblasts were inoculated into 24-well plates one day in advance, and 300 μL of bovine dual-potency stem cell culture medium prepared in Example 1 was replaced 1 hour before inoculation.

[0069] (2) Place the 7-10 day old bovine blastocysts in pronase and, under a stereomicroscope, repeatedly blow the embryos with a pipette to remove the zona pellucida.

[0070] (3) Transfer the blastocysts with the zona pellucida removed to a dish containing preheated DMEM / F12 culture medium and wash 2-3 times; then transfer the blastocysts to the culture medium in step (1) and incubate at 37°C and 5% CO2 for 48 hours, adding 200 μL of medium; then change 250 μL of medium every other day (partial replacement) until the embryos adhere to the wall.

[0071] (4) The medium was changed every day thereafter. The embryonic cells proliferated and grew into dense oval cylindrical structures (bESCs colonies) that grew vertically upward and spread out as a single layer of cells on the bottom of the dish (bTSCs colonies) (Figure 1A, Figure 1B).

[0072] (5) Under a microscope, the above oval cylindrical structures were picked out with a fine glass needle, digested and passaged by TrypLE, and embryonic stem cell-like colonies were obtained.

[0073] (6) After digesting the trophoblast stem cells with collagenase for 5 minutes, they were cut into small pieces under a microscope and passaged.

[0074] Note: When passaged individually, the bovine dual-potency stem cell culture medium prepared in Example 1 of this application should still be used.

[0075] bESCs can be cultured to at least 80 generations in bovine dual-potency stem cell culture medium (Figure 1C), with a self-replication time of 19.1 h. bTSCs have a relatively slower self-replication time of 55.2 h, and have currently been cultured to 20 generations (Figure 1D).

[0076] Using the above method, this application successfully obtained stable passageable bESCs and bTSCs cell lines simultaneously from a single bovine blastocyst under the same culture conditions, verifying the feasibility and efficiency of the technical solution of this application.

[0077] Comparative Example 1

[0078] No DOX was added to the bovine dextrose stem cell culture medium, and the rest of the operation was the same as in Example 2.

[0079] The results showed that bovine embryonic stem cells could be isolated from the culture medium without the addition of DOX, but bovine trophoblast stem cells could not be isolated.

[0080] Experiment 1

[0081] Bovine embryonic stem cells were isolated using the method described in Comparative Example 1. They were then cultured with and without DOX (10 μg / mL). The resulting embryonic stem cell colonies were identified using AP staining, immunofluorescence staining with SSEA4, OCT4, NANOG, and SOX2 antibodies, and qPCR identification.

[0082] The results are shown in Figures 2 and 3.

[0083] Figure 2A shows the staining results of SSEA4, a marker on the surface of bovine embryonic stem cells (BECs) treated with DOX for 72 hours, and both results were positive. Figure 2B shows the staining results of alkaline phosphatase (AP), a marker protein of BECs treated with DOX for 72 hours, and both results were positive. Figure 2C shows the immunofluorescence identification of SOX2, OCT4, and NANOG, marker proteins of BECs treated with DOX for 72 hours, and the results showed no significant difference in positive signals, indicating that DOX does not maintain the stemness of bESCs by regulating the expression of pluripotency markers.

[0084] Figure 3 shows that there was no significant difference in the expression of NANOG, OCT4, and SOX2, indicating that DOX does not maintain the stemness of bESCs by regulating the expression of pluripotency markers.

[0085] Experiment 2

[0086] The trophoblast stem cells prepared in step (6) of Example 2 were identified, including KRT8 and CDX2 antibody immunofluorescence staining.

[0087] The result is shown in Figure 4, and the result is positive.

[0088] Experiment 3

[0089] DOX treatment of bESCs promoted proliferation, inhibited apoptosis and differentiation, maintained stemness, and enhanced mitochondrial activity. After DOX treatment, clonal morphology of untreated bESCs was observed, growth rates were compared by cell counting, gene expression was compared by qRT-PCR, apoptosis was detected by TUNEL assay, NO secretion was detected using Griess Reagent assay, mitochondrial membrane potential changes were detected using tetramethylrhodamine methylester (TMRE), and glycolysis and mitochondrial respiration were detected using a SEAHORSE energy metabolism analyzer.

[0090] The results are shown in Figures 5 to 16.

[0091] Figure 5 shows the clonal state of bESCs cells after 72 h of untreated and treated cells with DOX (1–25 μg / mL). The results show that the clonal state became more regular under DOX treatment of 5–10 μg / mL.

[0092] Except for glycolysis and mitochondrial respiration assays (Figures 14–16) which used 5–10 μg / mL, all other experiments below used 10 μg / mL Dox.

[0093] Figure 6 shows the growth curves of bESCs under DOX treatment and after 4 days of treatment. The results show that DOX treatment significantly promoted the proliferation of bESCs.

[0094] Figure 7 shows the expression of differentiation genes in bESCs after 72 hours of DOX treatment and before treatment. The results show that the random differentiation genes of bESCs are suppressed by DOX.

[0095] Figure 8 shows the apoptosis of bESCs after TUNEL detection of DOX-untreated and DOX-treated cells for 72 h. The results show that DOX treatment significantly inhibited cell apoptosis.

[0096] Figure 9 shows the gene expression of nitric oxide synthases NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS) in bESCs after DOX treatment and 72 h of treatment. The results show that DOX treatment significantly inhibited the mRNA expression of NOS in cells.

[0097] Figure 10 shows the NO secretion in bESCs after 72 hours of DOX treatment and before treatment. The results show that DOX treatment significantly inhibited NO production in cells.

[0098] Figure 11 shows the status of bESCs cells after 72 h of untreated, separately treated, or treated together with DOX and nitric oxide donor SNAP, as well as TUNEL staining. The results show that DOX treatment significantly inhibited apoptosis and SNAP-induced enhanced apoptosis.

[0099] Figure 12 shows the expression of NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS) genes after 72 h of untreated, separately treated, or treated together with DOX and nitric oxide donor SNAP. The results show that DOX treatment significantly inhibited SNAP-induced enhancement of NOS expression.

[0100] Figures 13–16 show the changes in mitochondrial membrane potential of bESCs after DOX treatment and 24 h of treatment, as well as the changes in glycolysis and basal mitochondrial respiration in bESCs as measured by the SEAHORSE energy metabolism analyzer. The results show that DOX treatment significantly increased mitochondrial membrane potential, inhibited glycolysis, and significantly increased basal mitochondrial respiration, i.e., oxidative phosphorylation activity.

[0101] Experiment 4

[0102] 2.5 × 10⁴ bTSCs / well were seeded into 24-well plates on day D0. The medium was changed daily and the cells were counted on days D3, D5, D7, and D9.

[0103] The results are shown in Figure 17, indicating that bTSC can proliferate and maintain its typical morphology in DOX-containing medium.

[0104] Experiment 5

[0105] Untreated bovine embryonic stem cell (bESC) were treated with minocycline (MC), a member of the tetracycline family (similar to DOX), for 72 hours. Clonal morphology was observed, and cellular glycolysis and mitochondrial respiration were assessed using a SEAHORSE energy metabolism analyzer. The results showed that treatment with 5–10 μg / mL MC significantly improved the clonal morphology of bESCs, and 5 μg / mL MC significantly enhanced mitochondrial respiration. Furthermore, unlike DOX, MC did not inhibit stem cell glycolysis, and 5 μg / mL MC significantly improved compensatory glycolysis in stem cells. These results suggest that other members of the tetracycline family (such as MC) or other small molecules with molecular structures similar to DOX may have similar effects on the separation and maintenance of stemness in bESCs and bTSCs, and may even be more effective in improving the cells' stress-resistant metabolic capacity.

[0106] The results are shown in Figures 18 to 21.

[0107] Figure 18 shows the clonal state of bESCs cells after 72 h of untreated and treated cells with MC (5–10 μg / mL). The results show that the clonal state became more regular under MC treatment of 5–10 μg / mL.

[0108] Figures 19–21 show the changes in glycolysis and mitochondrial basal respiration in bESCs measured by a SEAHORSE energy metabolism analyzer before and after 24 hours of MC treatment. The results showed that 5–10 μg / mL MC treatment did not inhibit glycolysis, and 5 μg / mL MC significantly improved cellular compensatory glycolysis. Simultaneously, similar to DOX treatment, 5 μg / mL MC significantly increased the mitochondrial basal respiration, i.e., oxidative phosphorylation activity, in bESCs.

[0109] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0110] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of tetracycline drugs in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

2. The application as described in claim 1, characterized in that, Add tetracycline drugs to the basal culture medium to achieve a final concentration of 1–25 μg / mL.

3. The application as described in claim 1, characterized in that, Add tetracycline drugs to the basal culture medium to achieve a final concentration of 5–10 μg / mL.

4. The application as described in any one of claims 1 to 3, characterized in that, The tetracycline drug is either doxycycline or minocycline.

5. A bovine dual-potency stem cell culture medium, characterized in that, Including tetracycline drugs.

6. The culture medium as described in claim 5, characterized in that, It includes the following components: mTeSRTMPlus as the base culture medium, with the addition of 0.5–5 μM IWR-1, 0.5–3 μM PD184352, 1–6 μM SU5402, 1–6 μM EPZ-004777, 1–25 μM Forskolin, 1–10 μM CHIR99021, 5–25 ng / mL human leukemia inhibitory factor, and 5–10 μg / mL DOX / 2–10 μg / mL MC.

7. The culture medium as described in claim 5, characterized in that, The medium consists of the following components: mTeSRTMPlus as the base medium, supplemented with 2.5 μM IWR-1, 0.8 μM PD184352, 3 μM SU5402, 3.3 μM EPZ-004777, 10 μM Forskolin, 3 μM CHIR99021, 10 ng / mL human leukemia inhibitory factor, and 10 μg / mL DOX / 5 μg / mL MC.

8. The use of the culture medium according to any one of claims 5 to 7 in the simultaneous isolation and maintenance of bovine embryonic stem cells and bovine trophoblast stem cells.

9. A method for simultaneously isolating and maintaining bovine embryonic stem cells and bovine trophoblast stem cells, characterized in that, Bovine blastocysts or their inner cell masses are inoculated into culture plates pre-coated with feeder cells and cultured in the culture medium described in any one of claims 5 to 7.

10. The method as described in claim 9, characterized in that, The vertically growing, dense, multi-layered oval structure represents bovine embryonic stem cells, while the flat, spread-out monolayer of cells at the bottom of the dish represents bovine trophoblast stem cells.

Images