Application of IMR-1A in isolated culture of bovine embryonic stem cells
By adding IMR-1A to the bovine embryonic stem cell culture medium and optimizing the culture system, the problem of low isolation efficiency of bovine embryonic stem cells was solved, achieving efficient cell isolation and pluripotency maintenance, and promoting genetic progress.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-03-13
AI Technical Summary
Existing bovine embryonic stem cell isolation and culture techniques suffer from low isolation efficiency and a high rate of apoptosis, which affects the intensity of genetic screening and the speed of genetic progression.
IMR-1A, an inhibitor of the Notch signaling pathway, was added to the bovine embryonic stem cell culture medium at a concentration of 0.5 μM to 2.0 μM. The culture system was optimized by combining it with N2B27 medium, bovine serum albumin, and fibroblast growth factor 2.
It significantly improved the isolation efficiency of bovine embryonic stem cells to 80%, while maintaining the pluripotency of the isolated stem cells, thereby enhancing the intensity of genetic screening and the speed of genetic progress.
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Figure CN121653052A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of embryo engineering technology, specifically to the application of IMR-1A in the isolation and culture of bovine embryonic stem cells. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information has become prior art known to those skilled in the art.
[0003] Cattle are single-birth animals with long generation intervals, which is disadvantageous for the rapid breeding and propagation of superior germplasm. Since the advent of bovine embryonic stem cell isolation and culture technology in 2018, a surge of interest in in vitro breeding technologies based on embryonic stem cells has emerged. This involves an in vitro breeding cycle of embryo → embryonic stem cells → germ cells → embryo, during which genomic selection can be performed on the generated embryonic stem cells and the germ cell donors used. Scientists at home and abroad estimate that stem cell in vitro breeding technology can increase the rate of genetic progress in dairy cows by 10-30 times, and its application will break through the bottleneck in bovine germplasm breeding.
[0004] Establishing embryonic stem cell lines from embryos is the first step in in vitro breeding technology. Its efficiency directly affects the intensity of genetic screening and subsequent steps. Insufficient genetic screening can slow down genetic progress, thus diminishing the significance of in vitro stem cell breeding. Currently, few domestic and international research institutions possess this technology, and its efficiency needs improvement.
[0005] Using a culture system containing bFGF and the classic Wnt signaling pathway inhibitor IWR-1, bovine embryonic stem cells can be obtained from bovine in vitro fertilized embryos. The obtained bovine embryonic stem cells can maintain their morphology, transcriptome, karyotype, population doubling time, pluripotent marker gene expression and epigenetic characteristics under the culture system (CTFR).
[0006] Although breakthroughs were achieved in bovine embryonic stem cell isolation and culture technology in 2018, and the technology has undergone several generations of optimization, problems such as low isolation efficiency and high apoptosis rates still exist in actual experiments. Therefore, how to further improve the culture system to enhance the isolation efficiency of bovine embryonic stem cells is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0007] To address the aforementioned shortcomings in the prior art, this invention provides the application of IMR-1A in the isolation and culture of bovine embryonic stem cells, which can effectively improve the isolation efficiency and quality of bovine embryonic stem cells.
[0008] The technical solution of this invention is described in detail below: In a first aspect, the present invention provides the application of IMR-1A in the isolation and culture of bovine embryonic stem cells, which involves adding IMR-1A to a conventional bovine embryonic stem cell culture medium.
[0009] The conventional bovine embryonic stem cell culture medium can be any existing culture medium that can be used for bovine embryonic stem cell culture, including but not limited to serum-free culture medium, serum culture medium, and sequential culture medium. It can be purchased directly from commercial products or prepared by oneself.
[0010] IMR-1A is the acid metabolite of IMR-1, chemically named 2-(2-methoxy-4-((4-oxo-2-thiothiazolidin-5-ylidene)methyl)phenoxy)acetic acid. It is a small molecule compound with CAS number 331862-41-0, and its chemical structure is shown below: IMR-1A is a commercially available inhibitor of the Notch signaling pathway. Notch signaling relies on the binding of the NICD (Notch1 intracellular domain) to the transcription factor RBP-Jκ (also known as CSL) and other cofactors such as Mastermind to form a transcriptional activation complex. IMR-1A primarily inhibits downstream effects of Notch signaling by interfering with the formation of this complex, reducing transcriptional activation of the Notch signaling pathway. It is commonly used in research on specific pharmacological effects, particularly in studies related to stem cells, cancer, or cellular responses. Previously, IMR-1A was mainly used to investigate its regulatory effects on cell proliferation, differentiation, and related signaling pathways.
[0011] Optionally or preferably, the final concentration of the added IMR-1A is 0.5 μM to 2.0 μM.
[0012] Optionally or preferably, the basal culture medium for the conventional bovine embryonic stem cell culture medium is N2B27 medium.
[0013] Secondly, the present invention provides a bovine embryonic stem cell isolation culture medium, comprising N2B27 medium, bovine serum albumin, fibroblast growth factor 2 and IMR-1A; the final concentration of IMR-1A added is 0.5 μM to 2.0 μM.
[0014] Optionally or preferably, the N2B27 culture medium is composed of a culture medium consisting of DMEM / F12 and Neurobasal in a 1:1 volume ratio, with the addition of 0.5% N-2, 1% B-27, 1% GlutaMAX, 1% penicillin, 1% streptomycin, a 2 mM non-essential amino acid solution, 2.5 μM IWR1, and 0.1 mM 2-mercaptoethanol.
[0015] Optionally or preferably, the volume percentage of bovine serum albumin is 1%, and the final concentration of fibroblast growth factor 2 is 20 ng / ml.
[0016] Thirdly, the present invention provides a method for isolating and culturing bovine embryonic stem cells, comprising the following steps: (1) On day 7.5 of bovine embryonic development, collect all stages of bovine embryos except early blastocysts, rinse with HEPES-TALP medium (commercially available) and remove zona pellucida; preferably rinse 4 times, and remove zona pellucida using two 30Ga needles. (2) Mouse embryonic fibroblasts were inhibited by mitomycin C and washed with DPBS. Blastocysts with the zona pellucida removed were seeded onto mouse embryonic fibroblasts and cultured in conventional bovine embryonic stem cell culture medium containing Y-27632. On the second day, the medium was changed to conventional bovine embryonic stem cell culture medium without Y-27632. On the third day, the medium was changed to bovine embryonic stem cell isolation culture medium for culture. The bovine embryonic stem cell isolation culture medium contained IMR-1A at a final concentration of 0.5 μM to 2.0 μM. Y-27632 is a protein kinase (ROCK) inhibitor, and the final concentration added was 10 μM. (3) Change the bovine embryonic stem cell isolation culture medium every day and culture until the 7th day after inoculation, then pass the cells. (4) After culturing for another 7 days, perform the second subculture, and then subculture every 3 to 6 days depending on the degree of fusion.
[0017] Optionally or preferably, the early blastocysts in step (1) include unexpanded blastocysts, expanded blastocysts, and hatched blastocysts.
[0018] Optionally or preferably, the bovine embryonic stem cell isolation culture medium in steps (2) and (3) includes N2B27 medium, bovine serum albumin, fibroblast growth factor 2 and IMR-1A.
[0019] Compared with the prior art, the present invention has the following beneficial effects: Research on establishing cell lines from bovine embryonic stem cells is still in the exploratory stage, with a typical establishment efficiency of only 30% to 40%, which has not yet reached the ideal level. This invention has found that adding IMR-1A to conventional bovine embryonic stem cell culture medium can effectively increase the establishment efficiency of bovine embryonic stem cells to 80%, while effectively maintaining the pluripotency of the isolated stem cells. Attached Figure Description
[0020] Figure 1 These are microscopic images of bovine embryonic stem cell blastocysts in the embodiments. The left image is an image of an unexpanded blastocyst, and the right image is an image of an expanded blastocyst.
[0021] Figure 2 These are images of bovine embryonic stem cells stained with alkaline phosphatase in the examples. The left image shows the statistical results of alkaline phosphatase activity in cells isolated from different experimental groups; the middle image shows alkaline phosphatase staining in bovine embryonic stem cells in culture medium without IMR-1A; and the right image shows alkaline phosphatase staining in bovine embryonic stem cells in culture medium with 2 μM IMR-1A. Figure 3 These are fluorescence images of pluripotency-related proteins expressed in different groups of bovine embryonic stem cells in the examples. The top image is a fluorescence staining image of pluripotency-related protein OCT4, the middle image is a fluorescence staining image of pluripotency-related protein SOX2, and the bottom image is a fluorescence staining image of pluripotency-related protein DAPI.
[0022] Figure 4 This is a bar chart showing the expression levels of pluripotency-related genes in different groups of bovine embryonic stem cells in the examples. The left chart shows the pluripotency-related genes. NANOG Expression level statistics, the middle figure shows genes related to pluripotency. OCT4 Expression level statistics; the right figure shows genes related to pluripotency. SOX2 Statistical results of expression level. Detailed Implementation
[0023] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.
[0024] Example 1: Bovine embryonic stem cell isolation and culture by adding IMR-1A to conventional bovine embryonic stem cell culture medium. 1. Standard bovine embryonic stem cell culture medium formula The N2B27 medium was supplemented with 1% bovine serum albumin (0219989950, MP Biomedicals), 20 ng / ml fibroblast growth factor 2 (FGF2) (100-18B, PeproTech) and 2.5 μM IWR-1 (I0161, Sigma).
[0025] The specific formulation of N2B27 medium is as follows: DMEM / F12 (11320-033, Gibco) and Neurobasal (21103-049, Gibco) media with a volume ratio of 1:1 are supplemented with 0.5% N-2 (17502-048, Gibco), 1% B-27 (17504-044, Gibco), 1% GlutaMAX (35050-061, Gibco), 1% penicillin and 1% streptomycin (15140-122, Gibco), a 2 mM non-essential amino acid solution (M7145, Sigma), 2.5 μM IWR1, and 0.1 mM 2-mercaptoethanol (M6250, Sigma).
[0026] 2. Isolation and culture of bovine embryonic stem cells Bovine embryonic stem cell isolation culture medium: The conventional bovine embryonic stem cell culture medium in section 1 above is supplemented with IMR-1A at final concentrations of 0, 0.5 μM, 1 μM, 2 μM, and 5 μM, respectively, forming one group for each concentration, for a total of five groups.
[0027] Bovine embryonic stem cells were isolated and cultured using the bovine embryonic stem cell isolation and culture medium described above.
[0028] (1) Bovine embryonic cells were obtained from bovine in vitro fertilized embryos and cultured in conventional bovine embryonic stem cell culture medium until day 7.5. Blastocysts at all stages except the early blastocyst were collected. Early blastocysts included unexpanded blastocysts, expanded blastocysts, and hatched blastocysts. See also Figure 1 The image on the left is a microscopic photograph of an expanded blastocyst, and the image on the right is a microscopic photograph of an unexpanded blastocyst. The collected blastocysts were washed four times in HEPES-TALP medium, and the zona pellucida was removed in HEPES-TALP medium using two 30Ga needles under a stereomicroscope.
[0029] (2) On the day of blastocyst inoculation, mouse embryonic fibroblasts were first treated with mitomycin C inhibition, and then washed with Dulbecco phosphate-buffered saline (DPBS) to serve as feeder cells. Blastocysts with zona pellucida removed were inoculated onto feeder cells and evenly dispersed in five groups of conventional bovine embryonic stem cell culture medium containing the protein kinase (ROCK) inhibitor Y-27632 (final concentration 10 μM) for culture.
[0030] The next day, the conventional bovine embryonic stem cell culture medium containing Y-27632 was removed and replaced with conventional bovine embryonic stem cell culture medium without Y-27632, and cultured for another day.
[0031] On the third day, the regular bovine embryonic stem cell culture medium without Y-27632 was removed and replaced with bovine embryonic stem cell isolation culture medium (the concentration of IMR-1A added to each group was different).
[0032] (3) Change the bovine embryonic stem cell isolation culture medium daily for each group and culture until day 7 after blastocyst inoculation. Then, passage the cells into 24-well plates of newly prepared feeder cells one day in advance. All cultures are passaged regardless of whether growth is observed in the observed blastocysts.
[0033] (4) After culturing bovine embryonic stem cells in culture medium for another 7 days, passage them for the second time. Then, passage them every 3 to 6 days depending on the degree of fusion.
[0034] 3. Performance evaluation of isolated and cultured bovine embryonic stem cells (1) Separation efficiency statistics Five groups of blastocysts were inoculated onto mouse embryonic fibroblasts and cultured for 3 days. The number of blastocysts adhering to the culture wall was observed, and the number of blastocysts expanding was observed after 5 days. After 7 days, the cells were passaged 1:1 into new mouse fibroblasts. The formation of densely structured cell clones was observed 3-7 days after passage. After three stable passages, the pluripotency of the isolated cells was tested. Cells with pluripotency were considered successfully isolated bovine embryonic stem cells. The number of cell lines was determined, and the isolation efficiency was calculated. Isolation efficiency = (number of cell lines / number of blastocysts) × 100%. The number of blastocysts adhering, expanding, and isolated cells were observed, and the statistical results are shown in Table 1. Table 1. Isolation efficiency of bovine embryonic stem cells in different groups The experimental results show that adding 0.5μM, 1μM, and 2μM IMR-1A can significantly improve the efficiency of bovine embryonic stem cell line establishment (i.e., isolation efficiency, where each isolated cell can be used to establish a cell line), increasing it to 80%.
[0035] (2) Differentiation status and self-renewal capacity of bovine embryonic stem cells Alkaline phosphatase activity assays were used to verify the undifferentiated state and self-renewal capacity of bovine embryonic stem cells. Undifferentiated bovine embryonic stem cells exhibit high alkaline phosphatase (ALP) activity, while differentiated cells show significantly reduced ALP activity. After staining, undifferentiated stem cells appear reddish-brown, contrasting with the pale yellow of the surrounding fibroblasts.
[0036] Vigorously growing bovine embryonic stem cells were collected, washed three times with DPBS, and fixed with 4% paraformaldehyde fixative at room temperature for 10 min. The staining procedure was performed according to the alkaline phosphatase kit (21101ES60, Yeasen). Simultaneously, a biochemical colorimetric analysis kit (AKFA018M, BOXBIO) was used to perform the experiments according to the kit instructions. To account for protein transformation during the experiments, the ratio of alkaline phosphatase activity to total protein was normalized using a BCA protein analysis kit (Thermo). The mean and standard deviation of the three experiments were calculated, and a t-test was used to confirm statistical significance (P≤0.05). Test results as follows Figure 2 The middle and right images show bovine embryonic stem cell clones, with the colored areas representing the cloning. The results indicate that, compared to the culture medium without IMR-1A in the middle image, the cells isolated from the culture medium with 2 μM IMR-1A in the right image exhibited high alkaline phosphatase activity; higher alkaline phosphatase activity indicates stronger cell stemness. Figure 2 The left and middle figures show the statistical results of alkaline phosphatase activity in cells isolated from different experimental groups, indicating that the addition of 2 μM IMR-1A positive clones significantly increased the number of clones, effectively improving the stemness of isolated bovine embryonic stem cells.
[0037] (3) Detection of expression of bovine embryonic stem cell pluripotency-related proteins The expression levels of pluripotency-related proteins in bovine embryonic stem cells were detected using immunofluorescence assay.
[0038] Circular glass slides were placed in 4-well plates, coated with collagen at 37°C for 1 hour, and then the collagen was aspirated and seeded into feeder cells (MEF, mouse embryonic fibroblasts). After overnight culture, bovine embryonic stem cells were seeded and cultured in standard bovine embryonic stem cell culture medium supplemented with Y-27632 for one day. Then, they were cultured in the corresponding bovine embryonic stem cell isolation culture medium (containing different concentrations of IMR-1A) for each group. When the cell clone confluence was about 80%, the culture medium was aspirated and washed 2-3 times with DPBS.
[0039] Fixation: Add 4% paraformaldehyde and fix at room temperature (10-15 min), wash 3 times with DPBS (with calcium and magnesium), 5 min each time; Blocking and permeabilization: Add blocking buffer (5% BSA + 0.3% Triton) and block at room temperature for 2 hours; Incubate primary antibody: Dilute Oct4 antibody, Sox2 antibody, and Nanog antibody 1:50 with antibody dilution buffer (5% BSA + 0.1% Triton) and incubate overnight at 4°C; Wash primary antibody: Wash 3 times with DPBS (with calcium and magnesium), 5 min each time; Incubate secondary antibody: Dilute secondary antibody 1:1000 with antibody dilution buffer (5% BSA + 0.1% Triton) and incubate at room temperature for 1 hour; Wash secondary antibody: Wash 3 times with DPBS (with calcium and magnesium), 5 min each time; Drop 7 μL of anti-fluorescence quencher onto a glass slide, remove the slide and place it on a glass slide, seal the slide, and observe.
[0040] OCT4, SOX2, and NANOG are marker proteins of bovine embryonic stem cell pluripotency and are important criteria for measuring whether cells possess pluripotency. (See results below.) Figure 3 The image shows the fluorescence staining results of related proteins in bovine embryonic stem cells cultured in a medium containing 2 μM IMR-1A. The results indicate that all three pluripotency-related proteins were expressed in the isolated cells, demonstrating the successful isolation of bovine embryonic stem cells.
[0041] (4) Detection of expression of genes related to pluripotency of bovine embryonic stem cells RNA extraction and reverse transcription Bovine embryonic stem cells, after three stable passages and with approximately 80% clonal confluence, were used to extract tissue RNA using a rapid tissue RNA extraction kit (Shandong Cisco Biotechnology Co., Ltd.). The obtained RNA was then reverse transcribed using a two-step method with a reverse transcription kit (Takara) to obtain cDNA.
[0042] ① DNA removal step: Thaw the RNA on ice, add less than 1 μg of RNA, 1 μL of gDNA Eraser, and RNaseFree H2O to a 200 μL centrifuge tube, make up to 10 μL, gently mix with a pipette, and incubate with a PCR instrument at 42 ℃ for 5 min. After the reaction is complete, place on ice.
[0043] ②cDNA synthesis steps: Add 10 μL of 2×SPARKscript IIRT Plus to the solution from the previous step where gDNA was removed, gently mix with a pipette, incubate at 50 ℃ for 20 min in a PCR instrument, heat at 85 ℃ for 5 min to inactivate the enzyme, store at -20 ℃ for later use, and store at -80 ℃ for long-term storage.
[0044] Primer design Primer 5 and NCBI online primer design software (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) were used to design quantitative real-time primers and an internal control β-actin primer based on the reference sequences of the bovine oct4 gene (accession number: NC_037350.1), sox2 gene (accession number: NC_037328.1), and nanog gene (accession number: NC_037332.1) published by NCBI. All primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. Primer sequences and annealing temperatures are shown in Table 2, and the qRT-PCR amplification system is shown in Table 3. The expression differences of oct4, sox2, and nanog in bovine embryonic stem cells cultured in different culture media were analyzed.
[0045] Table 2 RT-qPCR experimental primers Table 3 qRT-PCR amplification system The amplification conditions are as follows: Each sample was added three times. The relative expression levels of oct4, sox2, and nanog genes were calculated using the 2-ΔΔCt method. The final data were expressed as mean ± SD. Two-way ANOVA and Bonferroni post-hoc test were used to analyze the differences between groups. A p-value < 0.05 was considered statistically significant.
[0046] See results Figure 4 , OCT4, SOX2, NANOOG It is a gene related to pluripotency; adding an appropriate amount of IMR-1A can effectively improve... OCT4, NANOOG The expression level indicates that appropriate addition of IMR-1A can improve the stemness of bovine embryonic stem cells.
[0047] The experimental results above show that adding IMR-1A to the culture medium can effectively improve the efficiency of bovine embryonic stem cell isolation and establishment. Compared with conventional embryonic stem cell culture medium, adding IMR-1A can also significantly enhance the expression of pluripotency-related genes and proteins.
[0048] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.
Claims
1. The application of IMR-1A in the isolation and culture of bovine embryonic stem cells, characterized in that, IMR-1A was added to the standard bovine embryonic stem cell culture medium.
2. The application according to claim 1, characterized in that, The final concentration of IMR-1A added is 0.5 μM to 2.0 μM.
3. The application according to claim 1, characterized in that, The basal culture medium for the conventional bovine embryonic stem cell culture medium is N2B27 medium.
4. A bovine embryonic stem cell isolation and culture medium, characterized in that, The medium included N2B27, bovine serum albumin, fibroblast growth factor 2, and IMR-1A; the final concentration of IMR-1A was 0.5 μM to 2.0 μM.
5. The bovine embryonic stem cell isolation and culture medium according to claim 4, characterized in that, The N2B27 medium is composed of DMEM / F12 and Neurobasal in a 1:1 volume ratio, with the addition of 0.5% N-2, 1% B-27, 1% GlutaMAX, 1% penicillin, 1% streptomycin, a 2mM non-essential amino acid solution, 2.5μM IWR1, and 0.1mM 2-mercaptoethanol.
6. The bovine embryonic stem cell isolation and culture medium according to claim 4, characterized in that, The volume percentage of bovine serum albumin was 1%, and the final concentration of fibroblast growth factor 2 was 20 ng / ml.
7. A method for isolating and culturing bovine embryonic stem cells, characterized in that, Includes the following steps: (1) On day 7.5 of development, collect all stages of bovine embryos except the early blastocysts, rinse them with HEPES-TALP medium, and remove the zona pellucida with a needle. (2) Mouse embryonic fibroblasts were inhibited by mitomycin C and washed with DPBS. Blastocysts with the zona pellucida removed were seeded onto mouse embryonic fibroblasts and cultured in conventional bovine embryonic stem cell culture medium containing Y-27632. On the second day, the medium was changed to conventional bovine embryonic stem cell culture medium without Y-27632. On the third day, the medium was changed to bovine embryonic stem cell isolation culture medium for culture. The bovine embryonic stem cell isolation culture medium contained IMR-1A at a final concentration of 0.5 μM to 2.0 μM. (3) Change the bovine embryonic stem cell isolation culture medium every day and culture until the 7th day after inoculation, then pass the cells. (4) After culturing for another 7 days, perform the second subculture, and then subculture every 3 to 6 days depending on the degree of fusion.
8. The method for isolating and culturing bovine embryonic stem cells according to claim 7, characterized in that, In step (1), the early blastocysts include unexpanded blastocysts, expanded blastocysts, and hatched blastocysts.
9. The method for isolating and culturing bovine embryonic stem cells according to claim 7, characterized in that, The bovine embryonic stem cell isolation culture medium described in steps (2) and (3) includes N2B27 medium, bovine serum albumin, fibroblast growth factor 2 and IMR-1A.