An epigenetic regulation inhibitor, a culture medium and application thereof
By introducing epigenetic regulatory inhibitors into bovine embryonic stem cell culture, the problem of pluripotency instability in bovine embryonic stem cells was solved, achieving higher pluripotency and differentiation potential, and establishing a stable culture system suitable for research on early bovine embryonic development mechanisms and pluripotency maintenance mechanisms.
Patent Information
- Application Number
- CN202610504616.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-16
- Publication Date
- 2026-07-10
Smart Images
Figure CN122357434A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to an epigenetic regulation inhibitor, a culture medium, and their applications. Background Technology
[0002] Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass (ICM) of early blastocysts. They possess long-term self-renewal capacity and can differentiate into various cell types derived from the three germ layers in vitro. The isolation, establishment, and pluripotency regulation mechanisms of mouse and human ESCs have been extensively studied, leading to the development of a series of mature in vitro maintenance systems and culture medium formulations. These systems have enabled ESCs to play a crucial role in developmental biology, disease model construction, bio-breeding, and regenerative medicine.
[0003] In contrast, research on bovine embryonic stem cells (bESCs) started later and is technically more challenging. Current reports indicate that while ESC-like cell lines can be obtained from bovine blastocysts, their pluripotency is unstable, their self-renewal capacity is limited, and they generally exhibit low differentiation potential, showing a significant difference compared to human and mouse ESCs. This disparity severely impacts the application potential of bovine ESCs in genetic improvement, embryo engineering, early embryonic development research, and the protection of superior germplasm resources. Therefore, developing a more effective culture system for maintaining the pluripotency and self-renewal capacity of bovine ESCs has become an urgent technical challenge.
[0004] Current reports on bovine ESC culture systems primarily maintain pluripotency by regulating signaling pathways (such as FGF / ERK and TGF-β / SMAD) and subsequently influencing transcription factor networks, with the core mechanism mainly focused on transcriptome regulation. However, the fundamental role of epigenetic modifications (such as DNA methylation and histone acetylation / methylation) in the establishment and maintenance of pluripotency has been confirmed in various mammals. Significant differences exist among different species in developmental stage characteristics, genome activation timing, and epigenetic regulation and maintenance mechanisms. In particular, bovine embryonic stem cells are highly sensitive to epigenetic homeostasis during in vitro culture, making the combined use of multiple epigenetic regulatory inhibitors more likely to induce gene expression imbalance and cell death. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides an epigenetic regulation inhibitor, a culture medium, and their applications.
[0006] An epigenetic regulation inhibitor for improving the pluripotency of bovine embryonic stem cells, the epigenetic regulation inhibitor being composed of 1 nM to 10 nM of 3-Deazaneplanocin A, 1 nM to 10 nM of Trichostatin A, and 10 nM to 100 nM of 5-Azacytidine.
[0007] 3-Deazaneplanocin A inhibits EZH2-mediated repressive histone methylation levels, such as H3K27me3, thus activating some pluripotent genes from a silenced state. Trichostatin A, as a histone deacetylase inhibitor, increases histone acetylation levels, making chromatin structure more open and facilitating the binding of pluripotency-related transcription factors. 5-Azacytidine reduces genomic DNA methylation levels by inhibiting DNA methyltransferases, thereby relieving methylation repression in pluripotent gene promoter regions. The synergistic effect of these three agents simultaneously reduces repressive methylation, increases acetylation levels, and decreases DNA methylation, improving chromatin openness and gene expression activity. This creates an epigenetic environment more conducive to the expression of pluripotent genes such as OCT4, SOX2, and NANOG, promoting higher pluripotency and differentiation potential in bovine embryonic stem cells.
[0008] A culture medium for improving the pluripotency of bovine embryonic stem cells, comprising the epigenetic regulation inhibitor and a bovine embryonic stem cell culture medium.
[0009] Preferably, the bovine embryonic stem cell culture medium consists of a basal culture medium and additives, wherein the additives and their concentrations in the basal culture medium are as follows: 1% penicillin-streptomycin (v / v), 0.1 mM β-mercaptoethanol, 1 μM~3 μM CHIR99021, 0.1 μM~0.5 μM WH4-023, 1 μM~5 μM IWR-1-endo, 1 μM~5 μM XAV939, 10 ng / ml~30 ng / ml activator A, 5 ng / ml~15 ng / ml LIF, and 30 μg / ml~80 μg / ml vitamin C.
[0010] Preferably, the basal culture medium is mTeSR Plus.
[0011] The application of the epigenetic regulation inhibitor or the culture medium in improving the pluripotency of bovine embryonic stem cells.
[0012] Preferably, bovine embryonic stem cells are added to the culture medium for culture, thereby improving the pluripotency of bovine embryonic stem cells.
[0013] Preferably, the steps for culturing bovine embryonic stem cells are as follows: Bovine embryonic stem cells were digested and centrifuged to obtain bovine embryonic stem cell precipitate and resuspended in bovine embryonic stem cell culture medium. The resuspended cells were seeded on culture dishes with bovine feeder cells, and cultured in the culture medium. 10 μM Y27632 was added to the bovine embryonic stem cell culture medium. After the cells adhered, Y27632 was removed.
[0014] Preferably, the amount of culture medium added is 0.15~0.30 mL per square centimeter of culture area.
[0015] Preferably, the culture conditions are 38.5℃ and 5% CO2.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to introduce an epigenetic regulatory inhibitor composed of DZNep (3-Deazaneplanocin A), TSA (Trichostatin A), and 5-Aza (5-Azacytidine) into a bovine embryonic stem cell culture system. By simultaneously reducing repressive histone methylation levels, increasing histone acetylation levels, and decreasing DNA methylation levels, it achieves synergistic regulation of the epigenetic state of bovine embryonic stem cells. This epigenetic regulatory inhibitor not only improves the chromatin openness of bovine embryonic stem cells but also significantly enhances the transcriptional activity of the pluripotency gene network, thereby effectively improving the pluripotency and in vitro culture stability of bovine embryonic stem cells.
[0017] Compared to traditional culture systems that rely solely on signaling pathway regulation, this invention yields bovine embryonic stem cells with higher expression levels of pluripotency genes (such as OCT4, SOX2, and NANOG) and more stable morphology. The epigenetic regulation strategy established in this invention provides a novel culture method for obtaining high-quality bovine embryonic stem cells, which can be widely applied in various fields such as research on early bovine embryonic development mechanisms, pluripotency maintenance mechanisms, functional gene screening, cell model construction, and gene-edited animal breeding. Attached Figure Description
[0018] Figure 1 The results show the cell morphology observation and alkaline phosphatase staining results of bovine embryonic stem cells obtained in Example 1 and Comparative Example 1.
[0019] Figure 2 The results of qRT-PCR detection of mRNA expression of pluripotency-related genes (including but not limited to OCT4, SOX2, Nanog, SALL4 and MYC) in bovine embryonic stem cells obtained in Example 1 and Comparative Example 1 are shown.
[0020] Figure 3The results of immunofluorescence staining of bovine embryonic stem cells obtained in Example 1 and Comparative Example 1 include the protein expression of OCT4 and SOX2 and their quantitative analysis of fluorescence intensity.
[0021] Figure 4 The results are from a cell survival experiment. Detailed Implementation
[0022] 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.
[0023] Culture media and small molecules from the following companies and catalog numbers: mTeSR Plus (STEM CELL, 100-0276), penicillin-streptomycin (Gibco, 15070063), β-mercaptoethanol (Sigma, 444203), CHIR99021 (Selleck, S1263), WH4-023 (Selleck, S7565), IWR-1-endo (Selleck, S7086), XAV939 (Sigma, X3004), activin A (Peprotech, 120-14E), LIF (Peprotech, 300-05), Vitamin C (Sigma, V-047), 3-Deazaneplanocin A (Sigma, S7120), Trichostatin A (Selleck, S1045), 5-Azacytidine (MCE, HY-10586), Y27632 (Selleck, S6390).
[0024] The bovine embryonic stem cell culture medium (bESCM medium) described in this invention consists of mTeSR Plus and additives added to mTeSR Plus. The additives and their concentrations in mTeSR Plus are as follows: 1% penicillin-streptomycin, 0.1mM β-mercaptoethanol, 2μM CHIR99021, 0.3μM WH4-023, 2.5μM IWR-1-endo, 2.5μM XAV939, 20ng / ml activin A, 10ng / ml LIF, and 50μg / ml vitamin C.
[0025] The concentrations of 3-Deazaneplanocin A, Trichostatin A, and 5-Azacytidine in the bovine embryonic stem cell culture medium were 1 nM to 10 nM, 1 nM to 10 nM, and 10 nM to 100 nM, respectively.
[0026] Example 1 Methods to improve the pluripotency of bovine embryonic stem cells include the following steps: Preparation of bovine feeder cells: When bovine fetal fibroblasts reached a cell confluence of more than 75%, they were passaged at a ratio of 1:3. After the cells were confluent again, the cell culture medium was replaced with a culture medium containing 22 μg / ml mitomycin C. The cells were treated for 3 hours, the liquid containing mitomycin C was discarded, and the cells were washed and digested with 0.05% trypsin to obtain a cell suspension. The suspension was then centrifuged to obtain bovine feeder cells. Passaging of bovine embryonic stem cells: Bovine embryonic stem cells were digested using TryplE Express at 38.5°C for 2 minutes. Digestion was terminated using bESCM medium. After centrifugation, the cell pellet was obtained and resuspended in bovine embryonic stem cell culture medium. The stem cells were then passaged at approximately 3.0 × 10⁻⁶ cells / mL. 4 Cells / cm² were seeded at a density of approximately 3.0 × 10⁻⁶ cells / cm² in culture dishes pre-coated with bovine feeder cells (35 mm culture dish, total seeding volume approximately 3.0 × 10⁻⁶). 5 Bovine ESCs were cultured and maintained by adding bESCM medium containing epigenetic regulation inhibitors to the cells / plate. 10 μM Y27632 was added to the bESCM medium. Y27632 was removed within 24 hours after cell attachment. Culture conditions were 38.5℃ and 5% CO2.
[0027] When adding bESCM medium containing epigenetic regulation inhibitors, the amount of 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 medium added is approximately 0.15–0.30 mL per square centimeter of culture area. In this example, approximately 0.20 mL of bESCM medium is added per square centimeter of culture area. This example uses a 35 mm culture dish, and the added culture medium volume is 2 ml.
[0028] Culture and maintenance of bovine ESCs: The cells were enzymatically digested and passaged every 3-4 days on feeder cells of bovine embryonic fibroblasts treated with mitomycin C.
[0029] The specific enzymatic digestion and passage were 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 parts according to the total volume. One part was seeded into bESCM medium containing epigenetic regulation inhibitors, and the cells were cultured at 38.5℃ and 5% CO2.
[0030] In bESCM medium containing epigenetic regulation inhibitors, the concentrations of 3-Deazaneplanocin A were 8 nM, Trichostatin A was 5 nM, and 5-Azacytidine was 50 nM.
[0031] Comparative Example 1 Passaging of bovine embryonic stem cells: Bovine embryonic stem cells were digested using TryplE Express at 38.5°C for 2 minutes. Digestion was terminated with bESCM medium. After centrifugation, cell pellet was obtained, and an appropriate amount of cells was seeded onto culture dishes containing bovine feeder cells. bESCM medium was added, along with 10 μM Y27632. Y27632 was removed within 24 hours after cell attachment. The culture conditions were 38.5°C and 5% CO2. Subsequent culture and maintenance of bovine ESCs were then carried out.
[0032] Culture and maintenance of bovine ESCs: The cells were enzymatically digested and passaged every 3-4 days on feeder cells of bovine embryonic fibroblasts treated with mitomycin C.
[0033] Enzymatic digestion and passage: Cells were dissociated into single cells using TryplE Express (38.5℃, 3 min), centrifuged (250g, 5 min), and resuspended. The resuspended cell suspension was divided into 6 equal parts according to the total volume. One part was seeded into bESCM medium and cultured at 38.5℃ and 5% CO2.
[0034] The bovine ESCs of Example 1 and Comparative Example 1 were identified.
[0035] Alkaline phosphatase staining: An alkaline phosphatase staining kit (Sigma, SCR004) was used, following the prescribed staining procedure. Bovine ESCs stained a purplish-red color, indicating alkaline phosphatase expression and suggesting that bovine ESCs are pluripotent (see [link to kit]). Figure 1 ).
[0036] Depend on Figure 1It was found that in bESCM, some cells at the edge of the bovine embryonic stem cell clone did not express alkaline phosphatase and had irregular clonal morphology, while in bESCM+DZNep+TSA+5-Aza, all cells expressed alkaline phosphatase and the clones were dense and raised with clear edges. Adding epigenetic regulation inhibitors helps maintain the undifferentiated state and stability of bovine ESCs.
[0037] qRT-PCR identification: RNA was extracted according to the instructions of the RNA extraction kit (Kangwei Century, CW0580S), and cDNA was obtained by reverse transcription according to the instructions of the reverse transcription kit (abm, G592). Following the instructions of the qRT-PCR 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 fluorescence quantitative detection system (SYBR Green). 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 2 ).
[0038] Depend on Figure 2 It can be seen that compared with bESCM, bESCM+DZNep+TSA+5-Aza has higher mRNA expression levels of bovine ESC pluripotency genes, such as OCT4, SOX2, KLF2, SALL4, and SSEA1.
[0039] 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 for 1 hour at room temperature. Primary antibodies were diluted in primary antibody dilution buffer; the primary antibodies were anti-OCT4 (Abcam, ab181557) and anti-SOX2 (R&D, AF2018). Cells were incubated with the primary antibody at room temperature in the dark for 1 hour, washed three times with DPBS, soaking for 5 min each time. Secondary antibodies, including goat anti-rabbit Alexa Fluor 488 (Beyotime, A0423) and donkey anti-goat Alexa Fluor 647 (PBM, DG2005), were incubated with the secondary antibody diluted in secondary antibody dilution buffer at room temperature in the dark for 1 hour. Samples were washed three times with DPBS, soaking for 5 min each time. Finally, the samples were stained with DAPI at room temperature in the dark for 15 minutes, the DAPI staining solution was discarded, and the samples were washed twice with DPBS, soaking for 5 minutes each time. The stained cells were then mounted on slides and photographed under a Zeiss inverted microscope (see...). Figure 3 ).
[0040] Depend on Figure 3It can be seen that, compared with bESCM, bESCM+DZNep+TSA+5-Aza has higher expression levels of bovine ESC pluripotency gene proteins, such as OCT4 and SOX2. Quantitative fluorescence intensity analysis also shows the same result.
[0041] In this invention, the culture system includes basic nutrients (mTeSR Plus, penicillin-streptomycin, β-mercaptoethanol), signaling pathway regulators, and epigenetic regulatory inhibitors (DZNep, TSA, 5-Aza). The signaling pathway regulators are used to maintain the homeostasis of cell pluripotency-related signaling pathways, and optionally include, but are not limited to, WNT signaling pathway regulators, TGF-β signaling pathway regulators, and JAK-STAT signaling pathway regulators, such as CHIR99021, WH4-023, IWR-1-endo, XAV939, Activin A, and LIF. The epigenetic regulatory inhibitors... Figure 4 These are called epigenetic regulatory factors, used to regulate the epigenetic state of cells.
[0042] Cell survival experiment results as follows Figure 4 The results showed that directly adding epigenetic regulatory factors to a culture system containing only basic nutrients led to a significant decrease in cell survival. However, after introducing signaling pathway regulators to establish a stable signaling pathway background, adding the epigenetic regulatory factors allowed cells to maintain a high survival rate and stable growth state. Furthermore, it could further improve the uniformity, clear margins, and pluripotency of stem cell clones.
[0043] Therefore, the technical effect of this invention does not come from the optimization of a single component or a specific concentration, but from the synergistic effect of signaling pathway regulators and epigenetic regulators, thereby solving the technical problem that epigenetic regulators are difficult to apply directly in embryonic stem cell culture, and further improving the pluripotency of bovine embryonic stem cells.
[0044] 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.
[0045] 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 both the preferred embodiments and all changes and modifications falling within the scope of the invention.
[0046] 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. An epigenetic regulatory inhibitor for improving the pluripotency of bovine embryonic stem cells, characterized in that, The epigenetic regulation inhibitor consists of 1 nM to 10 nM of 3-Deazaneplanocin A, 1 nM to 10 nM of Trichostatin A, and 10 nM to 100 nM of 5-Azacytidine.
2. A culture medium for improving the pluripotency of bovine embryonic stem cells, characterized in that, It consists of the epigenetic regulation inhibitor described in claim 1 and bovine embryonic stem cell culture medium.
3. The culture medium according to claim 2, characterized in that, The bovine embryonic stem cell culture medium consists of a basal culture medium and additives. The additives and their concentrations in the basal culture medium are as follows: 1% penicillin-streptomycin (v / v), 0.1mM β-mercaptoethanol, 1μM~3μM CHIR99021, 0.1μM~0.5μM WH4-023, 1μM~5μM WR-1-endo, 1μM~5μM XAV939, 10ng / ml~30ng / ml activator A, 5ng / ml~15ng / ml LIF, and 30μg / ml~80μg / ml vitamin C.
4. The culture medium according to claim 3, characterized in that, The basal culture medium is mTeSR Plus.
5. The use of the epigenetic regulation inhibitor of claim 1 or the culture medium of claim 2 in improving the pluripotency of bovine embryonic stem cells.
6. The application according to claim 5, characterized in that, Bovine embryonic stem cells are added to the culture medium for culture, thereby improving the pluripotency of bovine embryonic stem cells.
7. The application according to claim 6, characterized in that, The steps for culturing bovine embryonic stem cells are as follows: Bovine embryonic stem cells were digested and centrifuged to obtain bovine embryonic stem cell precipitate and resuspended in bovine embryonic stem cell culture medium. The resuspended cells were seeded on culture dishes with bovine feeder cells, and cultured in the culture medium. 10 μM Y27632 was added to the bovine embryonic stem cell culture medium. After the cells adhered, Y27632 was removed.
8. The application according to claim 6, characterized in that, The amount of culture medium added is 0.15~0.30 mL per square centimeter of culture area.
9. The application according to claim 6, characterized in that, The culture conditions were 38.5℃ and 5% CO2.