Sheep formation state pluripotent stem cell culture medium and application thereof

Abstract

The invention discloses a sheep formation state pluripotent stem cell culture medium and application thereof. The invention belongs to the technical field of biology, and particularly relates to a sheep formation state pluripotent stem cell culture medium and application thereof. The composition for culturing the sheep formation state pluripotent stem cells, disclosed by the invention, contains an N2B27 basic culture medium and cell factors, wherein the cell factors contain CHIR99021, IWR1, A419259, a recovery hub IL6, a recovery hub sIL6R, a recovery hub Activin A and a recovery hub FGF2 (Fibroblast Growth Factor 2). The culture medium prepared from the composition can obtain stable sfPSCs, and can be applied to construction of sheep gene editing model animals.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium for sheep pluripotent stem cells and applications thereof. Background Art

[0002] The embryonic epiblast is the source of pluripotent stem cells (PSCs), whose pluripotency states are a dynamic and continuous developmental process that transitions from naive, formative, to primed states. PSCs cultured in vitro can reflect the pluripotency states of embryonic epiblast development in vivo, and different culture conditions can also help to establish PSCs of different pluripotency states in vitro. To date, stable PSCs of different pluripotency states, including naive, formative, and primed, have been successfully established in mice, humans, and monkeys. These PSCs differ in signaling pathways, gene regulatory networks, epigenetic appearance, and metabolism. Due to ethical issues, the in vivo developmental potential of human naive PSCs cannot be verified.

[0003] The establishment of livestock embryonic stem cells (PSCs) is expected to promote biomedical development and accelerate livestock breeding. High-throughput single-cell transcriptomics sequencing can deepen researchers' understanding of early embryonic development and facilitate the establishment of PSCs.

[0004] Sheep are docile ruminants widely used for wool and meat production. Compared to other livestock species used for biological research, sheep and their fetuses are more similar in size to humans, making them ideal experimental animal models for studying human fetal diseases and improving therapeutic strategies. Sheep embryonic stem cells also have important application value in various fields. In recent years, studies have reported the ability to derive ovine PSCs using the CTFR and AFX culture systems. These PSCs maintain a clonal morphology and express classic pluripotency marker genes. However, the pluripotency state of these PSCs has not been fully characterized, and ovine PSCs that clearly resemble the pluripotency state of the embryonic epiblast have yet to be established. Summary of the Invention

[0005] The main problem to be solved by the present invention is how to culture ovine embryonic pluripotent stem cells.

[0006] In order to solve the above problems, the present invention provides a composition for culturing ovine pluripotent stem cells The present invention provides a composition for culturing ovine pluripotent stem cells, comprising N2B27 basal culture medium and cytokines, wherein the cytokines comprise CHIR99021, IWR1, A419259, recombinant human IL6, recombinant human sIL6R, recombinant human Activin A, and recombinant human FGF2.

[0007] In the above composition, the added concentration of CHIR99021 in the cytokines is 1 μM; the added concentration of IWR1 is 5 μM; the added concentration of A419259 is 0.3 μM; the added concentration of recombinant human IL6 is 10 ng / mL; the added concentration of recombinant human sIL6R is 10 ng / mL; the added concentration of recombinant human Activin A is 25 ng / mL; and the added concentration of recombinant human FGF2 is 12.5 ng / mL.

[0008] A419259 can also be replaced with WH-4-023, added at a final concentration of 1 μM.

[0009] The IWR1 can also be replaced with XAV939 or IWP2, and added at a final concentration of 5 μM.

[0010] In the above composition, the N2B27 basal medium (500 mL) contains 227 mL DMEM / F12 (Thermo Fisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2 supplement (Gibco, 17502-048), 5.0 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100× penicillin-streptomycin (Gibco, 15140-122), 5% knockout serum replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCKinhibitor Y27632 (Selleckchem, S1049).

[0011] The present invention also provides a culture medium for culturing ovine pluripotent stem cells, wherein the active ingredient of the culture medium is the composition according to any one of claims 1 to 4.

[0012] The present invention also provides a method for culturing ovine pluripotent stem cells, comprising the following steps: 1) Selecting sheep embryos at developmental day 8-11, mechanically removing the hypoblast and trophoblast cells, and digesting the epiblast of the embryo to obtain epiblast cell clusters; 2) Prepare a four-well plate with feeder cells 12 hours in advance and inoculate the digested epiblast cell clusters in the culture medium described above. After culturing for 4-6 days, obtain primary clones. 3) Digesting the primary clones obtained in step 2), re-seeding them in the culture medium described above, and performing subculture to obtain the ovine pluripotent stem cells.

[0013] Furthermore, the digestion in step 1) is performed using TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes.

[0014] Furthermore, in step 3), the digestion is performed using Accutase (Gibco, A11105-01) at 37°C for 5 minutes; and the incubation temperature is 37°C.

[0015] The present invention also provides a method for constructing a sheep gene-edited model animal, which comprises fluorescently labeling the sheep embryonic pluripotent stem cells obtained by the method described above, and using the fluorescently labeled sheep embryonic pluripotent stem cells as donor cells to obtain the model.

[0016] The present invention also provides use of the aforementioned composition and culture medium in preparing a sheep embryonic pluripotent stem cell product, or in preparing a product that increases the cell density of sheep embryonic pluripotent stem cells.

[0017] The present invention also provides the use of the method described above in preparing an ovine embryonic pluripotent stem cell product, or in preparing a product that increases the cell density of ovine embryonic pluripotent stem cells.

[0018] Compared with the prior art, the advantages of the present invention are as follows: 1. sfPSCs can be stably passaged for more than 100 generations; 2. Molecular analysis showed that the transcriptome characteristics of sfPSCs were close to those of the embryonic E10 epiblast, with clear formative pluripotency characteristics; 3. sfPSCs have a certain embryonic chimerism capacity, but the chimerism capacity at the individual level is low; 4. sfPSCs have gene editing capabilities and can be used as donor cells for somatic cell nuclear transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This figure illustrates the regulatory mechanisms of pluripotency in the epiblast of early goat embryos. A shows a cluster of single cells from the epiblast at different developmental stages (E4-E14) of early goat embryos; B shows the changes in pluripotency of epiblast cells in early goat embryos; C shows the expression of marker genes for naïve, formative, and primed pluripotency states in the epiblast of early goat embryos; D shows the metabolic profile of epiblast cells at different developmental stages; and E shows the expression levels of key genes in the signaling pathways regulating pluripotency in the epiblast of early goat embryos.

[0020] Figure 2The following figure shows the establishment and pluripotency characterization of sfPSCs. A shows the morphology and alkaline phosphatase staining of sfPSCs (scale bar, 100 μm); B shows the karyotype analysis of sfPSCs; C shows the cell proliferation curve of sfPSCs; D shows the cell doubling time of sfPSCs; E shows the single-cell clone formation efficiency of sfPSCs; F shows the immunofluorescence staining of pluripotency marker proteins in sfPSCs (scale bar, 50 μm); G shows the transcriptome correlation analysis between sfPSCs and epiblast cells at different stages of the early embryo.

[0021] Figure 3 This figure shows the functional analysis of cytokines and small molecules in sfPSC culture systems. A shows the morphology of goat embryonic stem cells in different culture systems. Medium 1 is sfPSC culture medium, Medium 2 is basal medium without any small molecules or cytokines, Medium 3 is cultured without the small molecule CHIR99021, Medium 4 is cultured without the small molecule IWR1, Medium 5 is cultured without the small molecule A419259, Medium 6 is cultured without the cytokine IL6 / sIL6R, Medium 7 is cultured without the cytokine Activin a, and Medium 8 is cultured without the cytokine FGF2. B shows alkaline phosphatase staining of cells in different culture systems. C shows the expression of the pluripotency protein POU5F1 in different culture systems. D is the expression level of pluripotent marker genes after withdrawal of CHIR99021, IWR1, and A419259; E is the quantitative analysis of mesendoderm marker gene expression after removal of IWR1; F is the quantitative analysis of pluripotent marker gene expression after withdrawal of IL6 / sIL6R; G is the expression level of p-STAT3 protein after withdrawal of IL6 / sIL6R; H is the quantitative analysis of marker gene expression after withdrawal of FGF2 and Activin A.

[0022] Figure 4 This is a transcriptomic analysis of the WNT signaling pathway after depletion of CHIR99021, A419259, and IWR1. Figure A shows principal component analysis (PCA) comparing transcriptional differences between sfPSCs, -CHIR, -A419259, and -IWR1 samples; Figure B shows a Venn diagram comparing differentially expressed genes between different groups (CHIR vs. sfPSCs, A419259 vs. sfPSCs, and IWR1 vs. sfPSCs) using the criteria of |log² FC| > 1 and FDR < 0.05); Figure C shows enrichment analysis of representative upregulated and downregulated genes and signaling pathways after depletion of CHIR99021, A419259, and IWR1, respectively, compared to sfPSCs; Figure F shows a heatmap of expression patterns of genes related to pluripotency, differentiation, and the WNT signaling pathway among different groups.

[0023] Figure 5Detection of embryoid bodies (EBs) formed by in vitro differentiation of sfPSCs. A shows immunofluorescence staining of marker genes for the inner, middle, and outer germ layers of EBs, with DAPI staining of cell nuclei. Scale bar: 50 μm. B shows quantitative expression of marker genes for the three germ layers in EBs.

[0024] Figure 6 This image shows the in vivo differentiation of sfPSCs into teratomas. A shows H&E staining of teratoma tissue, representing the histological structures of the ectoderm (neural rosette), mesoderm (cartilage), and endoderm (epithelium), respectively. Scale bar, 100 μm. B shows immunofluorescence staining of endoderm, mesodermal, and ectoderm marker genes, with DAPI staining of cell nuclei. Scale bar, 100 μm. C shows the quantitative expression of three germ layer marker genes in teratoma tissue.

[0025] Figure 7 Figure 1: Embryonic chimerism assay for sfPSCs. A shows the clonal morphology of GFP-sfPSCs (scale bar, 100 μm); B shows the developmental status and chimerism of embryos injected with GFP-sfPSCs (scale bar, 100 μm); C shows immunofluorescence staining of chimeric embryos for SOX2 (red), CDH1 (magenta), DAPI (blue), and sfPSCs (green); upper scale bar, 100 μm; lower scale bar, 25 μm; D shows a statistical table of the number of chimeric embryos transplanted; E shows GFP-positive signals from sfPSCs in chimeric fetuses. The right panel shows a magnified image; arrows indicate representative GFP-sfPSC cells. DAPI stains the nuclei. Scale bar, 200 μm. F shows the detection of GFP DNA inserts in various tissues of chimeric fetuses.

[0026] Figure 8 This image shows the ability of sfPSCs to serve as donor cells for somatic cell nuclear transfer. A shows the morphology of cloned embryos obtained using GFP-sfPSCs. Scale bar: 100 μm. B shows the efficiency of parthenogenetic embryo formation, the efficiency of cloned embryos from sfPSCs, and the efficiency of cloned embryos from sheep fibroblasts. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0028] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0029] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0030] The sheep used in the following examples were purchased from Tianjin Academy of Agricultural Sciences.

[0031] Animal Handling and Ethics Statement: All animal experiments involving mice and sheep described in the following examples were approved in advance by the Institutional Animal Care and Ethics Committee (IACUC) of China Agricultural University and the IACUC of Beijing Agricultural College. CD-1® (ICR) IGS mice and BALB / c nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and used for isolation of mouse embryonic fibroblasts and teratoma formation experiments. All sheep were in natural estrus and mated for the collection of early embryonic single cells and establishment of embryonic pluripotent stem cells.

[0032] The GFP-NLS plasmid in the following examples has been described in: Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Zhi M, Zhang J, Tang Q, et al. Cell Res. 2022 Apr;32(4):383-400. doi: 10.1038 / s41422-021-00592-9. Epub2021 Nov 30. The public can obtain the biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.

[0033] All experiments in the following examples were performed with three biological and technical replicates. Graphical presentation and statistical analysis were performed using GraphPad Prism 8.0. The values ​​in the graphs are expressed as mean ± standard deviation, and statistical significance was calculated using Student's two-tailed t-test. P Values ​​less than 0.05 were considered statistically significant and are shown as * P <0.05,** P <0.01, and *** P <0.001.

[0034] Example 1: Study on the Culture Medium and Culture Method of Sheep Fibroblastic Pluripotent Stem Cells (sfPSCs) 1. Establishment of a culture system for sheep fetal pluripotent stem cells (sfPSCs) Single-cell transcriptome data analysis showed that ( Figure 1 In Figures (Figure 5A), the pluripotency of the early sheep embryonic epiblast undergoes a transition from naive to formative and primed states. During the first lineage separation, naive pluripotency rapidly decreases, entering the relatively stable formative and primed states. Therefore, it is considered possible to establish PSCs of different pluripotency states from the embryonic epiblast.

[0035] To optimize the culture medium, we focused on the JAK / STAT3, WNT, FGF, and TGF-β signaling pathways associated with pluripotency ( Figure 1 Middle E). Analysis revealed that IL6 and IL6R were highly expressed in ICMs and pre-EPIs, so the cytokines IL6 and sIL6R were added to the culture medium.

[0036] The WNT signaling pathway regulates early embryonic development, including cell fate determination, tissue specification, and organogenesis. WNT-related genes begin to express at E8 and are significantly upregulated starting at E10, highlighting the importance of WNT signaling inhibition.

[0037] Co-inhibition of IWR1 and GSK3β can stabilize the expression of β-catenin in the cytoplasm and promote the homogeneity of PSCs, so IWR1 and CHIR99021 were chosen for combined use.

[0038] In addition, the receptors of Activin A and FGF2 are highly expressed in epiblast cells starting from E8, which is consistent with the previous report that embryonic epiblast pluripotency requires activation of FGF and TGF-β pathways.

[0039] LCK, a member of the non-receptor protein tyrosine kinase family, is highly expressed in epiblast cells. It binds to many growth factor receptors and regulates cell survival, proliferation, and epithelial-mesenchymal transition. This suggests that non-receptor protein tyrosine kinase signaling pathways may regulate the self-renewal of ovine epiblast stem cells.

[0040] In summary, a serum-free N2B27 basal culture system was developed, supplemented with cytokines IL6, sIL6R, FGF2, Activin A, and small molecule inhibitors of GSK3β, WNT, and LCK. To enhance cell viability, ascorbic acid and the ROCK inhibitor Y-27632 were also added.

[0041] N2B27 basal medium (500 mL) was prepared according to the following recipe: 227 mL DMEM / F12 (ThermoFisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2supplement (Gibco, 17502-048), 5.0 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100× penicillin-streptomycin (Gibco, 15140-122), 5% knockout serum replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCKinhibitor Y27632 (Selleck, S1049).

[0042] Small molecules and cytokines were added at the following concentrations: CHIR99021 (1 μM, Selleck, S1263), IWR1 (5 μM, Selleck, S7086), A419259 (0.3 μM, MedChemExpress, HY-15764A), recombinant human IL6 (10 ng / mL, PeproTech, AF200-06), recombinant human sIL6R (10 ng / mL, PeproTech, 200-06RC), recombinant human Activin A (25 ng / mL, PeproTech, 120-14E), and recombinant human FGF2 (12.5 ng / mL, PeproTech, 100-18B). A419259 can also be replaced by WH-4-023 (1 μM, Selleck, S7565). IWR1 can also be replaced by XAV939 (5 ​​μM, Selleck, S1180) or IWP2 (5 μM, Selleck, S7085).

[0043] The culture method of sheep fetal pluripotent stem cells (sfPSCs) is as follows: 1) Epiblast cells were isolated from sheep embryos at E8-E11 (mechanically removing the hypoblast and trophoblast cells, and digesting them with TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes to separate the epiblast into small cell clusters). Single cells were then inoculated onto feeder cells. Primary colonies formed 4-5 days after inoculation with 100% efficiency.

[0044] 2) Primary clones were digested with Accutase to single cells and seeded onto new feeder cells for subculture. Subculture was performed every 3-4 days at a ratio of 1:4 to 1:5. Before subculturing, cells were washed once with DPBS and then digested with Accutase (Gibco, A11105-01) at 37°C for 5 minutes. After digestion, cells were dissociated into single cells by pipetting, and the cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, sfPSCs were resuspended and seeded in the culture medium described above. Cultured in a 37°C incubator with 20% O2 and 5% CO2.

[0045] Medium No. 1 in Table 1 was the optimal medium. Finally, stable sfPSC cell lines were established. These cell lines exhibited dome-shaped colonies with smooth edges and positive AP staining (Figure 2A). They could be subcultured for over 100 generations while maintaining a normal karyotype (Figure 2B). The cells proliferated vigorously, with passages every 3 days (a 1:4 passage ratio). The doubling time was approximately 15 hours (Figure 2C and D). The single-cell clone formation efficiency was approximately 30% (Figure 2B). Figure 2 Middle E). Expression of pluripotency markers POU5F1, SOX2, NANOG, SSEA1, SSEA4, TRA-1-60, and TRA-1-81 ( Figure 2 At the transcriptional level, Spearman correlation analysis further demonstrated that sfPSCs have a strong correlation with the pluripotency of the E10 epiblast in vivo, and possess formative pluripotency characteristics ( Figure 2 Middle G).

[0046] 2. Study the effects of different small molecule inhibitors and cytokines on the maintenance of sfPSCs pluripotency in the culture system Table 1 shows the grouping of each culture medium component individually removed. The culture system was divided into eight groups: sfPSCs culture medium No. 1, basal culture medium without any small molecules or cytokines, basal culture medium No. 2 without the small molecule CHIR99021, basal culture medium No. 3 without the small molecule CHIR99021, basal culture medium No. 4 without the small molecule IWR1, basal culture medium No. 5 without the small molecule A419259, basal culture medium No. 6 without the cytokine IL6 / sIL6R, basal culture medium No. 7 without the cytokine Activin A, and basal culture medium No. 8 without the cytokine FGF2. SfPSCs culture medium No. 1 served as a control. The other groups included groups without any small molecules or cytokines and groups with either a small molecule or cytokine removed. The morphological characteristics of the cells in each group were observed, and the expression of alkaline phosphatase and pluripotency proteins and genes in each group was measured.

[0047] Withdrawal of CHIR99021 resulted in differentiated clones, heterogeneous expression of POU5F1, and weakened AP staining signals ( Figure 3 IWR1 is a typical tankyase inhibitor of the WNT / β-catenin signaling pathway. Ablation of IWR1 leads to a rapid loss of clonal morphology and the expression of pluripotency genes such as POU5F1 , SOX2 , NANOG , SALL4 and OTX2 A significant downregulation of Figure 3 At the same time, mesoderm and endoderm differentiation marker genes CER1 、 VIM 、 BMP4 and FOXA2 Up-regulated expression ( Figure 3 Middle E). When A419259 was withdrawn from the culture medium, the colonies became flattened ( Figure 3 We further determined the function of growth factors and found that the withdrawal of IL6 / sIL6R did not affect the cell morphology and pluripotency of sfPSCs, but the downstream transcription factors of JAK / STAT signaling pathway were STAT3 and p-STAT3 protein expression levels were downregulated ( Figure 3 AC, F, G). It was observed that the withdrawal of FGF2 or Activin A reduced the pluripotency of cells and the expression of ectoderm marker genes. PAX6 The upregulation of expression, the withdrawal of FGF2 seriously affects cell proliferation, and the withdrawal of Activin A leads to the upregulation of the expression of differentiation-related genes downstream of SMAD signaling ( Figure 3 These results indicate that long-term stable culture of sfPSCs requires the addition of small molecules and cytokines including CHIR99021, IWR1, A419259, IL6 / sIL6R, Activin A, and FGF2.

[0048] To further analyze the regulatory targets of the WNT signaling pathway, transcriptome sequencing was performed on sfPSCs and cell samples in which WNT signaling pathway-related small molecules CHIR99021, IWR1, and A419459 were individually removed. PCA analysis showed a clear separation trend between the overall transcriptome levels of the four samples ( Figure 4 Middle A). Veen plot results show the number of genes commonly and specifically expressed in pairwise differential expression analysis ( Figure 4 Middle B). After the withdrawal of the small molecule CHIR99021, compared with sfPSCs, the downregulated gene functions were enriched in cell proliferation, cell growth and cell cycle, indicating that the addition of CHIR99021 is required for cell proliferation ( Figure 4 Middle C). After the small molecule A419259 was withdrawn, compared with sfPSCs, the upregulated gene functions were enriched in epithelial cell proliferation and epithelial to mesenchymal transition ( Figure 4 (D) After the removal of the small molecule IWR1, compared with sfPSCs, the upregulated differentially expressed genes were enriched in areas related to cell morphogenesis, cell fate determination, endoderm-mesoderm development, etc., and pluripotency genes such as LIN28B 、 EZH2 、 PRDM14 、 ZIC2 、 POU5F1 、 SOX2 、 UTF1 The expression of mesoderm and endoderm differentiation genes such as LGR5 、 KRT19 、 GATA4 、 DAB2 、 HNF4A 、 FOXA1 、 EOMES 、 SOX17 In addition, the expression of WNT ligand genes was significantly upregulated. WNT11 、 WNT9A and WNT2B , receptor gene LRP5 , and regulatory factors DKK1 、 SFRP5 High expression after IWR1 removal indicates that sfPSCs differentiated ( Figure 4 (E, F).

[0049] Table 1. Grouping of culture medium components by reduction factor

[0050] Alkaline phosphatase (AP) staining: Remove the culture medium from sfPSCs, rinse once with DPBS, and fix in 4% paraformaldehyde (Sangon Biotech, 3053589-4) at room temperature for 3 minutes. Rinse once with DPBS, incubate in AP staining solution (Millipore, SCR004) at 37°C in the dark for 10 minutes, and then rinse once with DPBS. Observe the cells under a microscope for staining. sfPSCs were fixed with 4% paraformaldehyde for 30 minutes at room temperature and washed with DPBS. Subsequently, sfPSCs were permeabilized in 0.5% Triton X-100 for 20 minutes and blocked with 3% BSA (Sigma, A1470) for 1 hour at room temperature. The cells were then incubated with primary antibodies overnight at 4°C. Rinsed with DPBS three times for 5 minutes each. The cells were incubated with appropriate secondary antibodies, depending on the species of the primary antibody, for 1 hour at room temperature, followed by three washes with DPBS. Nuclei were stained with DAPI (Roche Life Science, 10236276001) for 3 minutes, and the cells were observed and photographed under a fluorescence microscope.

[0051] RT-PCR: Total RNA was extracted using an RNA extraction kit (TIANGEN, DP430) according to the manufacturer's instructions. cDNA was reverse transcribed using 5× All-In-One RT Master Mix (Abm, G490). RT-qPCR reactions were performed using 2× RealStar Green Power Mixture (GenStar, A311-05) on a LightCycler 480 II Real Time System (Roche). -ΔΔCt The expression levels of genes relative to GAPDH were calculated using the PCR method. All experiments were performed in triplicate, and significance analysis was expressed as Mean ± SD.

[0052] Example 2: Study on the Differentiation Ability of Sheep Fibroblastic Pluripotent Stem Cells (sfPSCs) 1. Detection of sfPSCs differentiation ability in vitro The specific detection methods are as follows: Disintegrate sfPSCs into single cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in 2 mL of fibroblast culture medium and transfer them to a 3.5 cm dish. Incubate the cells in a 37°C cell culture incubator on a shaker at 70 rpm / min for approximately 3-4 days to allow the cells to aggregate and form EB spheres. Select well-preserved EB spheres and seed them into 12-well plates. Approximately 6-7 days after the EB spheres have adhered and differentiated, fix the cells and perform immunofluorescence staining.

[0053] Analysis revealed that sfPSCs were able to form embryoid bodies (EBs) in vitro. EBs spontaneously differentiated into the three germ layers after adherent culture. Immunofluorescence analysis revealed the expression of NESTIN (an ectoderm marker), α-SMA (a mesoderm marker), and GATA6 (an endoderm marker) (Figure 5, A). RT-PCR analysis further validated these results (Figure 5, B).

[0054] 2. Detection of sfPSCs differentiation ability in vivo The specific detection methods are as follows: Embryonic stem cells were digested into single cells and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in 100 µL of culture medium and placed on ice. The cell suspension was aspirated using an insulin syringe, and after expelling all air from the syringe, the cells were injected subcutaneously into the neck or foreleg of immunodeficient mice (BALB / c nude mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). Approximately 1 × 10 cells were injected at each site. 7 The mice were carefully raised and the growth of subcutaneous teratomas in the immunodeficient mice was examined after 4-6 weeks. The mice were killed by cervical dislocation and the teratoma tissue was removed and sampled for testing.

[0055] H&E staining showed that the teratoma formed contained cells of the ectodermal lineage (neural rosette), mesodermal lineage (cartilage), and endoderm lineage (epithelium) ( Figure 6 Representative germ layer differentiation genes can also be detected by RT-PCR ( Figure 6 Middle C).

[0056] 3. Testing whether sfPSCs have the ability to integrate into early embryos to form chimeras Preparation of GFP-labeled sfPSCs: The GFP plasmid vector GFP-NLS (constructed by inserting the GFP-NLS fragment into the backbone vector PB-CAG-MCS) was transfected into sfPSCs via lipofection. GFP-labeled cells were then sorted by flow cytometry. Lipofectamine™ 3000 Transfection Reagent (Invitrogen™, L3000015) was used for lipofection.

[0057] GFP-labeled sfPSCs were injected into E5.5 early blastocysts ( Figure 7 The chimeric blastocysts were transplanted into the uterus of recipient ewes in estrus at the same time, and 11 chimeric fetuses were obtained from the uterus of 5 pregnant recipient sheep ( Figure 7 Middle D).

[0058] Analysis of various tissues of the chimeric fetus using frozen sections revealed that, despite the low chimerism efficiency of donor cells, GFP-positive signals were found in multiple tissues and organs ( Figure 7 Genomic DNA PCR assay further confirmed the presence of GFP DNA fragments in these chimeras ( Figure 7 Middle F).

[0059] 4. Nuclear transfer experiments using GFP-sfPSCs as nuclear donor cells The specific experimental method is as follows: After the sheep oocytes were cultured and matured, the cumulus cells were digested with 0.1% hyaluronidase (Sigma, H4272), and GFP-labeled sfPSCs were injected into the enucleated oocytes. The embryos were equilibrated in the incubator for 20 minutes before electrofusion (1300v / cm, 25 μs, 1 second interval, double pulse). The fused embryos were activated with 5 μM calcium ionomycin (Sigma, I3909) for 5 minutes and then treated with 2 mM 6-DMAP (Sigma, D2629) for 4 hours. Then, the cloned embryos were cultured with IVC medium (BOIVC2404). GFP fluorescence was detected in the cloned blastocysts, and the cloning efficiency was approximately 20% ( Figure 8 Figures (A and B) indicate that sfPSCs can be used in the construction of ovine gene-edited model animals.

[0060] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A composition for culturing ovine pluripotent stem cells, characterized in that: The composition contains N2B27 basal medium and cytokines, wherein the cytokines contain CHIR99021, IWR1, A419259, recombinant human IL6, recombinant human sIL6R, recombinant human Activin A and recombinant human FGF2.

2. The composition according to claim 1, wherein: The added concentration of CHIR99021 in the cytokines is 1 μM; the added concentration of IWR1 is 5 μM; the added concentration of A419259 is 0.3 μM; the added concentration of recombinant human IL6 is 10 ng / mL; the added concentration of recombinant human sIL6R is 10 ng / mL; the added concentration of recombinant human Activin A is 25 ng / mL; and the added concentration of recombinant human FGF2 is 12.5 ng / mL.

3. The composition according to claim 1 or 2, characterized in that: The N2B27 basal medium (500 mL) contains 227 mL DMEM / F12 (Thermo Fisher Scientific, 10565-018), 227 mL Neurobasal (Gibco, 21103-049), 2.5 mL N2 supplement (Gibco, 17502-048), 5.0 mL B27 supplement (Gibco, 12587-010), 2.5 mL 100× GlutaMAX (Gibco, 35050-061), 5.0 mL 100× nonessential amino acids (Gibco, 11140-050), 0.1 mM β-mercaptoethanol (Gibco, 21985-023), 5.0 mL 100× penicillin-streptomycin (Gibco, 15140-122), and 5% knockout serum. replacement (KOSR, Gibco, A3181502), 50 μg / mL ascorbic acid (Sigma, A4544) and 2.5 μM ROCK inhibitor Y27632 (Selleckchem, S1049).

4. A culture medium for culturing ovine pluripotent stem cells, characterized in that: The active ingredient of the culture medium is the composition according to any one of claims 1 to 3.

5. A method for culturing ovine pluripotent stem cells, characterized by: The following steps are involved: 1) Selecting sheep embryos at developmental day 8-11, mechanically removing the hypoblast and trophoblast cells, and digesting the epiblast of the embryo to obtain epiblast cell clusters; 2) Prepare a four-well plate with feeder cells 12 hours in advance, inoculate the digested epiblast cell clusters in the culture medium of claim 4, and culture for 4-6 days to obtain primary clones; 3) Digesting the primary clones obtained in step 2), re-seeding them in the culture medium according to claim 4, and performing subculture to obtain the ovine pluripotent stem cells.

6. The method according to claim 5, characterized in that: In step 1), the digestion was performed using TrypLE™ Express (Gibco, 12605010) at 37°C for 3 minutes.

7. The method according to claim 5 or 6, characterized in that: Step 3) The digestion was performed using Accutase (Gibco, A11105-01) at 37°C for 5 minutes; the incubation temperature was 37°C.

8. A method for constructing a sheep gene-edited model animal, characterized by: The method comprises gene editing the sheep embryonic pluripotent stem cells obtained by the method according to any one of claims 5 to 7, and performing somatic cell nuclear transplantation using the gene-edited sheep embryonic pluripotent stem cells as donor cells to obtain the model.

9. Use of the composition according to any one of claims 1 to 3 or the culture medium according to claim 4 in preparing an ovine embryonic pluripotent stem cell product, or in preparing a product for increasing the cell density of ovine embryonic pluripotent stem cells.

10. Use of the method according to claims 5 to 7 in preparing a product of ovine embryonic pluripotent stem cells, or in preparing a product with increased cell density of ovine embryonic pluripotent stem cells.

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