Line establishment method and culture system of bovine trophoblast stem cells

Abstract

The invention discloses a line establishment method and a culture system of bovine trophoblast stem cells. The stem cell line is obtained by separation and induction from bovine blastocyst. The embryonic stem cell line has stability. The method can be applied to various life science and medical fields such as bovine animal breeding and breeding, gene editing models, animal cloned donor cells, medical musculoskeletal injury experiments, drug development carriers, vaccine production cell banks, embryo quality analysis and the like, and can be applied to large-scale production and application.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and molecular biology, specifically relating to a method for establishing and culturing bovine trophoblast stem cells. Background Technology

[0002] Trophoblastic cells play a crucial role in early pregnancy in cattle, mediating communication between the mother and the developing fetus. The establishment of pregnancy in cattle requires trophoblastic cell elongation, a unique physiological process for successful implantation and pregnancy establishment in ruminants. The differentiation of trophoblastic progenitor cells into mononuclear trophoblastic cells and subsequently into binuclear giant cells is a key step in embryonic elongation. These cells are responsible for fusing with uterine epithelial cells, forming the conditions necessary for maternal-fetal interaction. Early pregnancy loss in bovine reproductive production, often due to abnormal trophoblastic cell development and function, remains a significant challenge in cattle farming. The lack of accurate in vitro models simulating placental cell differentiation hinders our understanding of these processes. Currently, trophoblast stem cells (TSCs) have been successfully established in rodents and primates (Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, and Rossant J (1998). Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075.10.1126 / science.282.5396.2072.-DOI–PubMed; Okae H, Toh H, Sato T, Hiura H, Takahashi S, Shirane K, Kabayama Y, Suyama M, Sasaki H, and Arima T (2018). Derivation of human trophoblast stem cells. Cell Stem Cell 22, 50–63.e6.10.1016 / j.stem.2017.11.004.-DOI–PubMed; Matsumoto S, Porter CJ, Ogasawara N, Iwatani C, Tsuchiya H, Seita Y, Chang YW, Okamoto I, Saitou M, Ema M, et al. (2020). Establishment of macaque trophoblast stem cell lines derived from cynomolgusmonkey blastocysts. Sci. Rep. 10, 1–15. (PMC-PubMed). However, the long-term cultivation and sustainable derivation of bovine TSCs has remained elusive. This is an active research area, and the establishment of bovine TSCs would be a significant milestone, providing a powerful tool for studying bovine placental development and addressing issues related to bovine infertility.In 2023, Wu Jun's team established a bovine trophoblast stem cell line using a mouse fibroblast feeder layer and LCDM culture system (Wang Y, Ming H, Yu L, Li J, Zhu L, Sun HX, Pinzon-Arteaga CA, Wu J, Jiang Z. Establishment of bovine trophoblast stem cells. Cell Rep. 2023 May 30; 42(5):112439. doi:10.1016 / j.celrep.2023.112439.Epub2023May 4.PMID:37146606; PMCID:PMC10950030). We also rapidly and efficiently established a bovine TSC-like cell line using a bovine fibroblast feeder layer and LTSC culture system. This research provides a foundation for improving reproductive efficiency and understanding the basic biology of early bovine pregnancy. Summary of the Invention

[0003] One object of the present invention is to provide a method for establishing bovine trophoblast stem cells by isolating and inducing them from bovine blastocysts, and the stem cell lines obtained by the method have good stability.

[0004] Another object of the present invention is to provide a culture system used in the above-described establishment method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for establishing bovine trophoblast stem cells, the method comprising:

[0007] Bovine blastocysts hatched on the seventh or eighth day after fertilization were inoculated onto feeder cells of bovine fibroblasts, and the cells were cultured using a bovine trophoblast stem cell culture system (i.e., LTSC culture system) to construct a bovine trophoblast stem cell line.

[0008] The specific components of the bovine trophoblast stem cell culture system are: 100× penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021, 10 ng / ml LIF, 2 μM Dimethindene maleate, 2 μM Minocycline, and mTeSR. TM 1. Culture medium.

[0009] Furthermore, the feeder cells of the bovine fibroblasts are prepared by the following method: thawed bovine fibroblasts are cultured in bovine fibroblast culture medium until the cell confluence reaches more than 80%, and then passaged at a ratio of 1:16; when the cell confluence reaches 80% again, 10 μg / ml mitomycin is added and cultured in an incubator for 2.5-3 hours; after trypsin digestion, digestion is stopped with bovine fibroblast culture medium, centrifuged and the supernatant is removed, and the cells are resuspended in a culture medium containing 10% DMSO, 10% fetal bovine serum and 80% bovine fibroblast culture medium and then frozen.

[0010] Furthermore, the feeder cells of the bovine fibroblasts are thawed and inoculated into culture dishes covered with 0.1% gelatin before being cultured in bovine fibroblast culture medium.

[0011] According to a specific embodiment of this application, the bovine fibroblast culture medium is Knockout DMEM medium containing 10% FBS, 1×glutamine, 1×non-essential amino acids, and 1×penicillin.

[0012] Furthermore, the blastocysts hatched on the seventh or eighth day after bovine fertilization are grade A or B embryos that have undergone microscopic examination.

[0013] Furthermore, the cell culture conditions are as follows: cultured in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5%.

[0014] Furthermore, the cell culture time is 7-14 days.

[0015] Furthermore, after 7 days of cell culture, the bovine trophoblast stem cell culture medium was replaced every other day.

[0016] Secondly, the present invention provides a bovine trophoblast stem cell culture system used in the above-described method for establishing bovine trophoblast stem cells. The specific components of the bovine trophoblast stem cell culture system are: 100× penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021, 10 ng / ml LIF, 2 μM Dimethindene maleate, 2 μM Minocycline, and mTeSR. TM 1. Culture medium.

[0017] This invention also protects the application of the above-mentioned bovine trophoblast stem cell culture system in the establishment of bovine trophoblast stem cell lines.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The bovine trophoblast stem cell line establishment efficiency using the method of the present invention is as high as 83.33%, and it can be stably passaged in vitro.

[0020] (2) Bovine trophoblast stem cells prepared by this method can be used in a variety of life science and medical fields such as bovine breeding, gene editing models, animal cloning donor cells, medical musculoskeletal injury experiments, drug development vectors, vaccine production cell banks, and embryo quality analysis, and can be produced and applied on a large scale. Attached Figure Description

[0021] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0022] Figure 1 This is a morphological image of a bovine blastocyst graft onto feeder cells as described in this invention. The scale bar is 50 μm.

[0023] Figure 2 This is a morphological diagram of bovine trophoblast stem cells described in this invention, with a scale bar of 100 μm.

[0024] Figure 3 This is an image showing the detection of the marker genes of bovine trophoblast stem cells as described in this invention. Detailed Implementation

[0025] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1: Preparation and Culture of Feeder Layer Cells

[0027] 1.1 Preparation of feeder layer cells:

[0028] Thaw bovine fibroblasts (BEF) and culture them in bovine fibroblast culture medium, which is Knockout DMEM medium containing 10% FBS (BI), 1× glutamine, 1× non-essential amino acids, and 1× penicillin-streptomycin. When cell confluence reaches 80% or higher, passage the cells at a 1:16 ratio. When cell confluence again reaches 80%, add 10 μg / ml mitomycin C and incubate for 2.5-3 hours. After trypsin digestion, stop digestion with bovine fibroblast culture medium, centrifuge, discard the supernatant, resuspend in culture medium containing 10% DMSO, 10% fetal bovine serum, and 80% bovine fibroblast culture medium, and freeze.

[0029] 1.2 Culture of feeder cells:

[0030] The day before the experiment, feeder cells were thawed and inoculated into culture dishes with 0.1% gelatin and cultured in bovine fibroblast culture medium.

[0031] Example 2: Establishment, passage, cryopreservation, and thawing of bovine trophoblast stem cells

[0032] 2.1 Establishment of bovine trophoblast stem cell lines:

[0033] The day before the establishment experiment of bovine trophoblast stem cells, the feeder cells were thawed and seeded into culture dishes with 0.1% gelatin and cultured in bovine fibroblast culture medium for later use.

[0034] The following day, blastocysts hatched on the seventh or eighth day after bovine fertilization were collected and examined under a microscope. The embryos were graded according to the published embryo grading method (Demetrio DGB, Benedetti E, Demetrio CGB, Fonseca J, Oliveira M, Magalhaes A, Dos Santos RM. How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro (12 years of practical results at a California dairy farm). Anim Repord. 2020 Aug14;17(3):e20200053.doi:10.1590 / 1984-3143-AR2020-0053.PMID:33029219;PMCID:PMC7534552.). Grade A and B embryos were selected for the establishment of bovine trophoblast stem cell lines.

[0035] Add 1 ml of bovine trophoblast stem cell culture medium, resuspend and mix well, and then inoculate onto feeder cells that have been thawed the day before. The morphology of the blastocysts inoculated onto feeder cells is shown in the image. Figure 1 After shaking well, place in a cell culture incubator at 37°C and 5% CO2 concentration for incubation.

[0036] Bovine trophoblast stem cell culture medium, the specific components of which are: 100× penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021 (Tocris, cat.no.4423), 10 ng / ml LIF (Millipore), 2 μM M methithindene maleate (Tocris, cat.no.1425), 2 μM M nonocycline (Santa Cruz Biotechnology, cat.no.sc-203339), and mTeSR TM 1. (STEMCELL) medium.

[0037] Bovine trophoblast stem cells were observed to grow after day 7. The culture medium for bovine trophoblast stem cells was changed every other day, and the bovine trophoblast stem cell line was successfully established between days 7 and 14. Cell line morphology results are shown below. Figure 2 The bovine trophoblast stem cells described in this invention exhibit good clonal morphology and clear boundaries. The cell line establishment efficiency results are shown in Table 1, with the Holstein bovine trophoblast stem cell establishment efficiency reaching 83.33%. The rapid and efficient establishment of bovine trophoblast stem cells provides strong support for the industrial application of bovine stem cells.

[0038] Table 1. Results of bovine trophoblast stem cell line establishment efficiency described in this invention.

[0039] Embryo varieties Number of blastocysts bovine trophoblast stem cell construction coefficient Bovine trophoblast stem cell establishment rate Holstein cattle 72 60 83.33％

[0040] 2.2 Passage culture of bovine trophoblast stem cells:

[0041] The day before the passage culture experiment, feeder cells were thawed and inoculated into culture dishes with 0.1% gelatin and cultured in bovine fibroblast culture medium.

[0042] When the bovine trophoblast stem cells reached a growth density of 80%, they were digested with TrypLE and digestion was stopped with KSR10 medium, which is DMEM / F12 medium containing 10% KSR, 1×glutamine, 1×non-essential amino acids, and 1×penicillin. After centrifugation at 1300 rpm for 3 min, the supernatant was discarded, and the cells were passaged every other day at a ratio of 1:2 or 1:4. 1 ml of bovine trophoblast stem cell culture medium was added to resuspend the cells, mix well, and inoculate them onto pre-thawed feeder cells. After mixing, the cells were placed in a cell culture incubator at 37°C and 5% CO2.

[0043] 2.3 Cryopreservation of bovine trophoblast stem cells:

[0044] When the bovine trophoblast stem cells reached a growth density of 80%, they were digested with TrypLE and digestion was stopped with KSR10 medium. The cells were centrifuged and the supernatant was discarded. The cells were then resuspended in 10% DMSO and 90% FBS and frozen.

[0045] 2.4 Resuscitation and culture of bovine trophoblast stem cells:

[0046] The day before the resuscitation culture experiment, feeder cells were thawed and seeded into culture dishes lined with 0.1% gelatin (Sigma, G7765) and cultured in bovine fibroblast culture medium.

[0047] After removing the bovine feeder cell stem cells from the cryopreservation tubes from liquid nitrogen, they were quickly placed in a 37°C water bath to thaw. Once thawed, the liquid was pipetted from the cryopreservation tubes into 15ml centrifuge tubes, and 5ml of preheated 37°C KSR10 cell culture medium was added. After centrifugation at 1300rpm for 3min, the supernatant was discarded, and 1ml of LTSC culture medium was added to resuspend and mix well. The mixture was then inoculated onto the pre-thawed feeder cells, agitated, and placed in a 37°C, 5% CO2 cell culture incubator for culture.

[0048] Example 3: Detection of bovine trophoblast stem cells

[0049] 3.1 Detection of marker genes in bovine trophoblast stem cells:

[0050] Total RNA was extracted from bovine trophoblast stem cells using the RNA 220011 extraction kit from Fejet Biotechnology and dissolved in RNase-free water. 1.8 μl of the extracted RNA was used to measure concentration and purity. 1 μg of RNA was reverse transcribed into 20 μl (50 ng / μl) cDNA using the Vazyme HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. This cDNA was used for quantitative PCR detection of the expression of the bovine trophoblast stem cell marker gene CDX2 (Zhao L, Gao X, Zheng Y, et al. Establishment of bovine expanded potential stem cells[J]. Proceedings of the National Academy of Sciences, 2021, 118(15):e2018505118.DOI:10.1073 / pnas.2018505118.). ChamQ Universal SYBR qPCR MasterMix was used for real-time quantitative PCR (Q-PCR) with the ChamQ Universal SYBR qPCR Master Mix enzyme from Vazyme. The Q-PCR reaction conditions were as follows: pre-denaturation: 95℃ for 30 s; cycling reaction: 95℃ for 5 s, then 60℃ for 30 s, 40 cycles; melting curve: 95℃ for 15 s, 60℃ for 60 s, then 95℃ for 15 s to terminate the experiment. The TaqMan Probe (Assay ID: Mm01232884_ml) and the quantitative PCR instrument (9700HTFast Real-Time PCR System) used in the Q-PCR experiment were purchased from Applied Biosciences. Gene expression analysis was performed using the ΔCt algorithm, with GAPDH as the internal control. The detection results are shown below. Figure 3 As shown in the figure, the expression of pluripotency genes in bovine trophoblast stem cells was higher than that in bovine fibroblasts.

[0051] This invention provides a method and culture system for establishing bovine trophoblast stem cells, which are illustrated in detail with reference to specific embodiments. All raw materials described in the embodiments are commercially available. Those skilled in the art can make appropriate modifications, alterations, and combinations to the method provided by this invention to achieve the same result. It should be specifically noted that all such modifications, alterations, and recombinations made to the system provided by this invention are considered to be within the scope and content of this invention.

Claims

1. A method for establishing bovine trophoblast stem cell lines, characterized in that, The method includes: Bovine blastocysts hatched on the seventh or eighth day after fertilization were inoculated onto feeder cells of bovine fibroblasts, and the cells were cultured using a bovine trophoblast stem cell culture system to construct a bovine trophoblast stem cell line. The specific components of the bovine trophoblast stem cell culture system are: 100× penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021, 10 ng / ml LIF, 2 μM Dimethindene maleate, 2 μM Minocycline, and mTeSR. TM 1. Culture medium.

2. The method for establishing a system according to claim 1, characterized in that, The feeder cells of the bovine fibroblasts were prepared by the following method: thawed bovine fibroblasts were cultured in bovine fibroblast culture medium until the cell confluence reached more than 80%, and then passaged at a ratio of 1:16; when the cell confluence reached 80% again, 10 μg / ml mitomycin C was added and cultured in an incubator for 2.5-3 hours; after trypsin digestion, digestion was stopped with bovine fibroblast culture medium, centrifuged and the supernatant was discarded, and the cells were resuspended in a culture medium containing 10% DMSO, 10% fetal bovine serum and 80% bovine fibroblast culture medium and then frozen.

3. The method for establishing a system according to claim 1, characterized in that, Before establishing the lineage, the feeder cells of the bovine fibroblasts were thawed and inoculated into culture dishes covered with 0.1% gelatin and cultured in bovine fibroblast culture medium.

4. The method for establishing a system according to claim 2 or 3, characterized in that, The bovine fibroblast culture medium was Knockout DMEM medium containing 10% FBS, 1×glutamine, 1×non-essential amino acids, and 1×penicillin.

5. The method for establishing a system according to claim 1, characterized in that, The blastocysts hatched on the seventh or eighth day after bovine fertilization are grade A or B embryos that have undergone microscopic examination.

6. The method for establishing a system according to claim 1, characterized in that, The cell culture conditions are as follows: cultured in a cell culture incubator at a temperature of 37°C and a CO2 concentration of 5%.

7. The method for establishing a system according to claim 1, characterized in that, The cell culture time is 7-14 days.

8. The method for establishing a system according to claim 1, characterized in that, After 7 days of cell culture, the bovine trophoblast stem cell culture medium was replaced every other day.

9. A bovine trophoblast stem cell culture system used in the method for establishing bovine trophoblast stem cells according to any one of claims 1-8, characterized in that, The specific components of the bovine trophoblast stem cell culture system are: 100× penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021, 10 ng / ml LIF, 2 μM Dimethindene maleate, 2 μM Minocycline, and mTeSR. TM 1. Culture medium.

10. The application of the bovine trophoblast stem cell culture system according to claim 9 in the establishment of bovine trophoblast stem cell lines.

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