Bovine ectoderm stem cell line as well as establishment method and application thereof

Abstract

The invention discloses a bovine ectoderm stem cell line as well as an establishment method and application thereof. The bovine ectoderm stem cell line has the pluripotency of a bovine intermediate state stem cell line, expresses one or more pluripotency markers, can be stably passaged, still maintains pluripotency transcriptome characteristics and normal karyotype similar to those of an intermediate state (Formative) ectoderm cell after more than 112 generations, and has the potential of being differentiated into three-germ layers. The bovine intermediate-state stem cell line has the potential for myogenic differentiation, primordial germ cell-like cell differentiation and serving as somatic cell nuclear transplantation (SCNT) donor cells, which shows that the bovine intermediate-state stem cell line has wide application prospects in the aspects of promoting cell culture meat production and animal breeding.

Description

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 21, 2024, with application number 2024103257603 and application name “A bovine intermediate stem cell line and its in vitro preparation method and use”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of stem cell biology, and in particular to a bovine ectoderm stem cell line and an establishment method and application thereof. Background Art

[0003] Embryonic ectoderm cells can differentiate into a complete fetus and serve as an important source of pluripotent stem cells (PSCs). PSCs derived from the ectoderm at different developmental stages exhibit different pluripotent states, which can be specifically divided into primitive states, Human and mouse PSCs have been widely used in embryonic development, directed differentiation, disease modeling and other related fields.

[0004] Stable livestock PSCs not only contribute to understanding embryonic development and pluripotency in livestock but also represent an optimal cell source for animal breeding and cultured meat production. However, due to species-specificity, the establishment of stable pluripotent stem cell lines in large animals has been slow. Recently, the establishment of porcine pre-gastrulation epiblast stem cells has attracted widespread attention due to their potential applications in various fields. However, research on the molecular basis of bovine embryogenesis and PSCs lags far behind that of pigs, mice, humans, and non-human primates. Because the molecular basis of bovine embryonic development and pluripotent stem cell self-renewal remains unclear, the establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle is challenging. Therefore, further tracking of bovine embryonic stem cell lines at different developmental stages has important practical applications. Summary of the Invention

[0005] The technical problem addressed by this invention is to provide bovine ectodermal stem cells, their in vitro preparation methods, and uses. A stable bovine ectodermal stem cell line was successfully established using an optimized 3i / LAF culture system. This research provides valuable, high-quality seed cells and a new approach for studying the pluripotency of livestock stem cells, while also advancing research in stem cell-related breeding and cell-cultured meat production.

[0006] In a first aspect, the present invention provides a bovine ectodermal stem cell line, characterized in that the bovine ectodermal stem cell line has the pluripotency of bovine ectodermal stem cells, expresses one or more pluripotency markers, and can be stably passaged.

[0007] In certain embodiments, the ectodermal stem cell line is capable of stable inheritance at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 times or more.

[0008] In certain embodiments, the pluripotency marker is selected from one or more of POU5F1, NANOG, SOX2, CDH1, SSEA1, and SSEA4;

[0009] In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryos at E7-E14;

[0010] In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryos at E10-E12;

[0011] In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10;

[0012] In certain embodiments, said E7-E14 are of the EPI lineage.

[0013] In certain embodiments, the EPI lineage overexpresses one or more of TDGF1, POU5F1, and NANOG.

[0014] In certain embodiments, the bovine ectodermal stem cell line expresses one or more of the pluripotency-related genes ETV1, ETV5, ZIC2, LIN28B, NODAL, NANOG, and TDGF1 with high specificity.

[0015] In certain embodiments, the bovine ectodermal stem cell line overexpresses one or more of the NANOG, LEFTY2, and NODAL genes compared to primed bESCs;

[0016] In certain embodiments, the bovine ectodermal stem cell line underexpresses one or more of GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, and HES1 genes compared to primed bEDSCs.

[0017] In certain embodiments, compared to primed bESCs, the bovine ectodermal stem cell line overexpresses one or more of the following genes: FGF4, ZFP42, ETV3L, DPPA3, ACVRL1, TDGF1, and TBX3.

[0018] In certain embodiments, the bovine ectodermal stem cell line has upregulated expression of genes involved in stem cell proliferation and cell-cell adhesion regulation compared to bEDSCs;

[0019] In certain embodiments, the bovine ectodermal stem cell line further underexpresses one or more of the HAND1, GATA3, TGFB2, ZFP42, VMO1, MEIS2, BMP4, IGF1R, CCND2, and SPRY4 genes compared to bEDSCs.

[0020] In certain embodiments, the stem cell proliferation genes include ELL3 and / or FGF4;

[0021] In certain embodiments, the cell-cell adhesion regulating genes include one or more of NODAL, FOXA2 and EPCAM;

[0022] In certain embodiments, the bovine ectodermal stem cell line also overexpresses one or more of DPPA3, LMO1, ACVRL1, and WNT3A genes compared to bEDSCs.

[0023] In certain embodiments, the bovine ectodermal stem cell line exhibits higher expression levels of LIN28A / B and / or NODAL compared to biPSCs or bEPSCs;

[0024] In certain embodiments, compared to biPSCs, the bovine ectodermal stem cell line also overexpresses one or more of the following genes: NODAL, LIFR, IL6R, PDGFRA, ACVR1B, ETS1, IL6ST, SALL4;

[0025] In certain embodiments, the bovine ectodermal stem cell line further underexpresses one or more of the following genes: GDF15, BCL3, CD44, ETV2, FGFR4, KLF15, GDF1, BMP4, COX17, and MYC, compared to biPSCs. In certain embodiments, the bovine ectodermal stem cell line further overexpresses one or more of the following genes: LIFR, ESRRB, IL6R, ACVR1B, KLF5, SALL4, and CDH1, compared to bEPSCs.

[0026] In certain embodiments, the bovine ectodermal stem cell line also underexpresses one or more of the MSC, GCK, TGFB2, MAPK15, KLF15, GDF1, ID4, FOS, and COX17 genes compared to bEPSCs. In certain embodiments, the bovine ectodermal stem cell line relies on activation of the FGF / ERK and / or TGFβ / SMADs signaling pathways and inhibition of the WNT / β-catenin signaling pathway;

[0027] In certain embodiments, the FGF / ERK signaling pathway-related genes include one or more of MAPK1, MAPK14, FGF2, PDGFA, FGFR1, and FGFR2;

[0028] In certain embodiments, the TGFβ / SMADs signaling pathway-related genes include one or more of INHBA, NODAL, ACVR2A / 2B, BMP4, and BMPR1A / 1B;

[0029] In certain embodiments, the WNT / β-catenin signaling pathway-related genes include one or more of TCF7, APC, WNT11, CTNNB1, FZD2, and WNT3A.

[0030] In another aspect, the present invention discloses a culture medium for culturing a bovine ectodermal stem cell line, characterized in that the culture medium comprises a basal culture medium and an added component;

[0031] In certain embodiments, the basal culture medium comprises DMEM / F12 medium and / or Neurobasal medium;

[0032] In certain embodiments, the mass ratio or volume ratio of the DMEM / F12 medium to the Neurobasal medium is 1:1;

[0033] In certain embodiments, the basal culture medium further comprises one or more small molecule inhibitors or cytokines selected from the group consisting of: CHIR99021, IWR-1-endo (XAV939), WH-4-023, recombinant human LIF, recombinant human Activin A and recombinant human FGF-basic (154aa), ROCK inhibitor Y-27632, or any combination thereof;

[0034] In certain embodiments, the CHIR99021 is used at a concentration of 1 μM;

[0035] In certain embodiments, IWR-1-endo is used at a concentration of 0.5 μM;

[0036] In certain embodiments, the WH-4-023 is used at a concentration of 1 μM;

[0037] In certain embodiments, the recombinant human LIF is used at a concentration of 10 ng / mL;

[0038] In certain embodiments, the recombinant human Activin A is used at a concentration of 25 ng / mL;

[0039] In certain embodiments, the recombinant human FGF-basic is used at a concentration of 10 ng / mL;

[0040] In certain embodiments, the ROCK inhibitor Y-27632 is used at a concentration of 5 μM.

[0041] The basal medium also includes one or more small molecule inhibitors or cytokines selected from the group consisting of: CHIR99021 (1 μM, Selleckchem, S1263), XAV939 (1 μM, Selleckchem, S1180), WH-4-023 (1 μM, Selleckchem, S7565), recombinant human LIF (10 ng / mL, PeproTech, 300-05), recombinant human Activin A (25 ng / mL, PeproTech, 120-14E) and recombinant human FGF-basic (154aa) (10 ng / mL, PeproTech, 100-18B), ROCK inhibitor Y-27632 (5 μM, Selleckchem, S1049), or any combination thereof.

[0042] The supplementary components in the culture medium include 1×N2 supplement (Thermo Fisher Scientific, 17502-048), 1×B27 supplement (Thermo Fisher Scientific, 12587-010), 0.5% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% non-essential amino acids (Thermo Fisher Scientific, 11140-050), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985-023), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), 5% knockout serum replacement (KOSR, Thermo Fisher Scientific, A3181502, optional) and 50 μg / mL ascorbic acid (Sigma–Aldrich, A4544).

[0043] In another aspect, the present invention discloses an in vitro method for preparing a bovine ectodermal stem cell line, characterized in that the method comprises the steps of culturing single embryonic cells isolated from bovine E10-E14 embryos in the above-mentioned culture medium;

[0044] In certain embodiments, said E7-E14 are of the EPI lineage.

[0045] In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryos at E10-E12;

[0046] In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10.

[0047] In another aspect, the present invention discloses the use of any of the above-described bovine ectoderm stem cell lines or the bovine ectoderm stem cell lines prepared by the above-described method in inducing the generation of muscle cells or providing nuclear transplant donor cells.

[0048] In another aspect, the present invention discloses a method for preparing muscle cells, comprising the step of culturing any of the above-described bovine ectoderm stem cell lines or the bovine ectoderm stem cell line prepared by the above-described method in a myogenic culture medium to obtain muscle cells.

[0049] In another aspect, the present invention discloses a method for preparing bovine primordial germ cell-like cells, characterized in that the method comprises the steps of culturing the bovine ectoderm stem cell line as described above or the bovine ectoderm stem cell line prepared by the above method in a PGC induction culture system to obtain bovine primordial germ cell-like cells;

[0050] In certain embodiments, the bovine primordial germ cell-like cells highly express the TFAP2C gene and the PRDM1 gene compared to the bovine ectodermal stem cell line.

[0051] On the other hand, the present invention discloses a bovine nuclear transplantation method, which includes the steps of culturing the bovine ectoderm stem cell line as described above or the bovine ectoderm stem cell line prepared by the above method as a nuclear transplantation donor cell nucleus or nuclear transplantation donor cell to obtain bovine cells, tissues, organs, and complete individuals.

[0052] Beneficial effects:

[0053] This study used single-cell transcriptome sequencing to demonstrate the similarities between cattle and pigs in early embryonic development and pluripotency changes, and successfully established a stable bovine intermediate (Formative) ectodermal stem cell line using an optimized 3i / LAF culture system. The bovine intermediate ectodermal stem cell line established by this application still maintains pluripotent transcriptome characteristics and normal karyotype similar to those of intermediate (Formative) ectodermal cells after more than 112 generations, and has the potential to differentiate into three germ layers. This application also further evaluated the potential of bEpiSCs for myogenic differentiation, primordial germ cell-like cell differentiation, and as somatic cell nuclear transplantation (SCNT) donor cells. This study provides valuable high-quality seed cells and new approaches for studying the pluripotency of livestock stem cells, while advancing research on cell culture meat production and stem cell-related breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 For the generation and characterization of bEpiSCs.

[0055] A: Strategies for establishing bEpiSCs.

[0056] B: Growth efficiency of bovine embryos and cell lines at different stages in 3i / LAF medium.

[0057] C: Morphology of outgrowths (top) and bEpiSCs (bottom). White arrows indicate the morphology of selected and passaged bEpiSC colonies. Scale bar, 200 μm.

[0058] D: bEpiSCs population doubling time.

[0059] E: Single-cell cloning efficiency of bEpiSCs.

[0060] F: Alkaline phosphatase (AP) staining of EpiSCs. Scale bar, 100 μm.

[0061] G: Karyotype analysis of EpiSCs. For each cell line, 45 metaphase cells were tested.

[0062] H: Immunostaining of pluripotency markers POU5F1, NANOG, and SOX2 in bEpiSCs. DAPI was used for nuclear staining. Scale bar, 100 μm.

[0063] I: Immunostaining of the pluripotency surface markers CDH1, SSEA1, and SSEA4 in bEpiSCs. DAPI was used for nuclear staining. Scale bar, 50 μm.

[0064] J: In vitro EB differentiation assay. Immunostaining for the ectoderm neural marker β-III-tubulin, the mesoderm muscle marker α-SMA, and the endoderm marker SOX17. DAPI was used for nuclear staining. Scale bar, 100 μm.

[0065] K: In vivo teratoma formation assay. b Hematoxylin and eosin (H&E) staining of EpiSCs-derived teratomas. Scale bar, 50 μm.

[0066] For D and E, error bars represent ± SD (n = 3 independent experiments), ns. P ≥ 0.05. For (C) and (FK), similar results were obtained in three independent experiments.

[0067] Figure 2 3i / LAF is crucial for the long-term culture of bEpiSCs.

[0068] A: bEpiSCs were treated with factor reduction, using the 3i / LAF group as a control. Colony morphology and AP staining were observed. Scale bar, 200 μm. B: Colony size analysis of the bEpiSC culture system after factor reduction treatment. More than 30 colonies were counted for each treatment group. C: Real-time quantitative PCR analysis of gene expression differences between bEpiSCs in the 3i / LAF and I / F groups.

[0069] D, E: mRNA expression levels of pluripotency (D) and lineage marker genes (E) in the CHIR, IWR, and wh deletion groups were compared with those in the 3i / LAF group using qRT-PCR.

[0070] F: qRT-PCR comparison of mRNA expression levels of pluripotency and lineage marker genes between the FGF2-deficient group and the 3i / LAF group. G: qRT-PCR comparison of mRNA expression levels of pluripotency and BMP signaling pathway marker genes between the Activin A-deficient group and the 3i / LAF group.

[0071] H: qRT-PCR was used to compare the mRNA expression levels of pluripotency and JAK / STAT3 signaling pathway marker genes in the LIF-deficient group and the 3i / LAF group.

[0072] For (BH), error bars represent ± SD (n = 3 independent experiments), ns. P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For (AH), similar results were obtained in three independent experiments, see Figure 5 .

[0073] Figure 3Transcriptomic analysis of bEpiSCs.

[0074] A: PCA plot showing the distribution of scRNA-seq data obtained from bovine embryonic lineage cells and bEpiSCs, with color-coded clusters based on different embryonic lineages and bEpiSC lineages.

[0075] B: Spearman correlation analysis of bovine embryonic lineage cells and bEpiSCs. The color gradient from blue to red indicates increasing correlation from low to high.

[0076] C: Violin plots showing bovine embryonic lineages and bEpiSCs from different embryonic stages Formative and primed pluripotency represent the expression levels of genes.

[0077] D: PCA plots were generated to compare bEpiSCs with publicly established primed bESCs 15 , bEDSCs 16 , bEPSCs, and biPSCs 18 , where each data point represents a different cell line.

[0078] E: Differentially expressed genes (DEGs) were identified between bEpiSCs and published bPSCs. Red bars indicate genes upregulated in bEpiSCs compared with published bPSCs, while green bars indicate genes downregulated in bEpiSCs by pairwise comparison.

[0079] F: Scatter plot showing the comparison of average gene expression levels between bEpiSCs and published bPSCs, with upregulated genes highlighted in orange and downregulated genes highlighted in blue. Key genes are appropriately annotated, see Figure 6 .

[0080] Figure 4 Potential applications of bEpiSCs.

[0081] A: Application prospects of bEpiSC in the model diagram.

[0082] B: Morphology of EpiSCs at different stages of myogenic differentiation. Scale bar, 200 μm.

[0083] C: qRT-PCR quantitative determination of mRNA expression of myogenic differentiation-related genes.

[0084] D: Immunostaining of myocyte-related proteins in myogenically differentiated bEpiSCs. Scale bar, 200 μm.

[0085] E: Embryoid body morphology of PGCLCs formed by bovine bEpiSCs.

[0086] F: Gene expression of PGCLCs formed by bovine bEpiSCs.

[0087] G: Clonal morphology of GFP-bEpiSCs, scale bar, 50 μm.

[0088] H: Morphology of GFP-bEpiSCs clone embryos, scale bar, 100 μm.

[0089] I: Statistics of bEpiSCs donor nuclear transfer cloning efficiency.

[0090] J: Clonal morphology of newly born GFP-bEpiSCs in bovine cloned embryos, scale bar, 200 μm.

[0091] For C, F, error bars represent ± SD (n = 3 independent experiments), ****, P < 0.0001. For (B, H), similar results were obtained in three independent experiments.

[0092] Figure 5 For the culture of bEpiSCs, the Figure 2 Related.

[0093] A: Clonal morphology and AP staining of bEpiSCs treated with different concentrations of CHIR (C) and IWR (I). Concentration unit: μM. Scale bar: 200 μM.

[0094] B: Quantification of expression of pluripotency and lineage marker genes, C (CHIR) and I (IWR), by qRT-PCR, concentration unit, μM.

[0095] C: Morphology of bEpiSC colonies treated with different concentrations of IWP2 and XAV939. Scale bar, 200 μm. D: Quantification of mRNA expression levels of pluripotent genes and lineage-specific marker genes in bEpiSCs cultured with different concentrations of IWP2 and XAV939. For B and D, error bars represent ±SD (n = 3 independent experiments), ns. P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For (A-D), similar results were obtained in three independent experiments.

[0096] Figure 6 For the comparative analysis of bEpiSC with published bPSC and porcine pgEpiSC, Figure 3 Related.

[0097] A: Gene Ontology and KEGG pathways identified by pairwise comparison of bPSCs.

[0098] B: Scatter plots were generated to compare the average gene expression levels between bEpiSCs and porcine pgEpiSCs, with upregulated genes highlighted in orange and downregulated genes highlighted in blue. Key genes were appropriately annotated.

[0099] C: Gene Ontology and KEGG pathway enrichment terms of differentially expressed genes in bEpiSCs and porcine pgEpiSCs. DETAILED DESCRIPTION

[0100] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0101] 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.

[0102] All mouse and cow experimental procedures involved in the following examples were approved in advance by the Laboratory Animal Welfare and Animal Experiment Ethics Review Committee of China Agricultural University, approval number AW10204202-3-1.

[0103] mice

[0104] (ICR) IGS and BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used for the isolation of mouse embryonic fibroblasts (MEFs) and the bEpiSCs teratoma formation assay. MEFs were treated with mitomycin C (Selleckchem, S8146) to prepare feeder cells for bEpiSCs.

[0105] ox

[0106] Bovine single-cell collection and single-cell transcriptome analysis were performed using Holstein-Friesian embryos at stages E5, E6, E7, E10, E12, and E14. Embryonic day n (E(n)) embryos were obtained at day n post-coitum. For the derivation of bEpiSCs, embryos at E7, E10, E12, and E14 were used.

[0107] Bovine embryo collection and embryonic single cell isolation

[0108] The embryonic cells used in this study were derived entirely from Holstein cow embryos. E5-E7 embryos were obtained by thawing and culturing frozen bovine embryos until the desired developmental stage was reached. The zona pellucida was treated with Pronase (Sigma, 10165921001) for 15-30 seconds and then cleaned and removed using a solution consisting of DPBS+0.1% BSA. Subsequently, the embryonic cells were mechanically separated and transferred to a lysis buffer. E10, E12, and E14 embryos were obtained by in vivo transplantation and subsequent rinsing, and single embryonic cells were separated and collected by a combination of enzyme treatment and mechanical manipulation.

[0109] Bovine Ectodermal Stem Cell Culture Medium

[0110] The composition of the bovine ectoderm stem cell culture system was the same as that of the previously reported 3i / LAF culture system, and the content of each component was optimized accordingly. Specifically, basal medium (BM) was a 1:1 mixture of DMEM / F12 medium (Thermo Fisher Scientific, 10565-018) and Neurobasal medium (Thermo Fisher Scientific, 21103-049), supplemented with 1×N2 supplement (Thermo Fisher Scientific, 17502-048), 1×B27 supplement (Thermo Fisher Scientific, 12587-010), 0.5% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% non-essential amino acids (Thermo Fisher Scientific, 11140-050), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985-023), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), 5% knockout serum replacement (KOSR, Thermo Fisher Scientific). Scientific, A3181502, optional) and 50 μg / mL ascorbic acid (Sigma–Aldrich, A4544). To prepare the 3i / LAF culture system, the following small molecule inhibitors or cytokines were added to the BM: CHIR99021 (1 μM, Selleckchem, S1263), IWR-1-endo (0.5 μM, Selleckchem, S7086), WH-4-023 (1 μM, Selleckchem, S7565), recombinant human LIF (10 ng / mL, PeproTech, 300-05), recombinant human Activin A (25 ng / mL, PeproTech, 120-14E), and recombinant human FGF-basic (154aa) (10 ng / mL, PeproTech, 100-18B). After optimization, the ROCK inhibitor Y-27632 (5 μM, Selleckchem, S1049) was added. Alternatively, XAV939 (1 μM, Selleckchem, S1180) can be substituted for IWR-1-endo. bEpiSCs were cultured with mouse fibroblasts treated with mitomycin C.

[0111] In this application, E refers to the Embryonic Day, which is the number of days a fertilized egg develops. Generally, the day of fertilization is E0, and so on. That is, E7, E10, E12, and E14 refer to the 7th, 10th, 12th, and 14th days of development of the fertilized egg, respectively.

[0112] Experimental model and study participant details

[0113] Material availability

[0114] All bEpiSCs generated in this study are available from the primary contact with a complete material transfer agreement.

[0115] Data and code availability

[0116] The scRNA-seq and RNA-seq datasets generated during this study are available on Gene Expression Omnibus (GEO) with the following accession code: GSE256201.

[0117] Animal treatment and ethics statement

[0118] All experimental procedures for mice and dairy cows were approved in advance by the Laboratory Animal Welfare and Animal Experimentation Ethics Review Committee of China Agricultural University, approval number AW10204202-3-1.

[0119] mice

[0120] (ICR) IGS and BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used for the isolation of mouse embryonic fibroblasts (MEFs) and the bEpiSCs teratoma formation assay. MEFs were treated with mitomycin C (Selleckchem, S8146) to prepare feeder cells for bEpiSCs.

[0121] ox

[0122] Bovine single-cell collection and single-cell transcriptome analysis were performed using Holstein-Friesian embryos at stages E5, E6, E7, E10, E12, and E14. Embryonic day n (E(n)) embryos were obtained at day n post-coitum. For the derivation of bEpiSCs, embryos at E7, E10, E12, and E14 were used.

[0123] Bovine embryo collection and embryonic single cell isolation

[0124] The embryonic cells used in this study were derived entirely from Holstein cow embryos. E5-E7 embryos were obtained by thawing and culturing frozen bovine embryos until the desired developmental stage was reached. The zona pellucida was treated with Pronase (Protease Pronase, Sigma, 10165921001) for 15-30 seconds and then washed and removed using a solution consisting of DPBS+0.1% BSA. Subsequently, the embryonic cells were mechanically separated and transferred to a lysis buffer. E10, E12, and E14 embryos were obtained by in vivo transplantation and subsequent rinsing, and single embryonic cells were separated and collected by a combination of enzyme treatment and mechanical manipulation.

[0125] Bovine Ectodermal Stem Cell Culture Medium

[0126] The composition of the bovine ectoderm stem cell culture system is similar to the previously reported 3i / LAF culture system 2The content of each component was optimized accordingly. Specifically, 3i / LAF culture medium: basal medium (BM) is a 1:1 mixture of DMEM / F12 medium (Thermo Fisher Scientific, 10565-018) and Neurobasal medium (Thermo Fisher Scientific, 21103-049), supplemented with 1×N2 supplement (Thermo Fisher Scientific, 17502-048), 1×B27 supplement (Thermo Fisher Scientific, 12587-010), 0.5% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% non-essential amino acids (Thermo Fisher Scientific, 11140-050), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985-023), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), 5% knockout serum replacement (KOSR, Thermo Fisher Scientific). Scientific, A3181502, optional) and 50 μg / mL ascorbic acid (Sigma–Aldrich, A4544). To prepare the 3i / LAF culture system, the following small molecule inhibitors or cytokines were added to the BM: CHIR99021 (1 μM, Selleckchem, S1263), IWR-1-endo (0.5 μM, Selleckchem, S7086), WH-4-023 (1 μM, Selleckchem, S7565), recombinant human LIF (10 ng / mL, PeproTech, 300-05), recombinant human Activin A (25 ng / mL, PeproTech, 120-14E), and recombinant human FGF-basic (154aa) (10 ng / mL, PeproTech, 100-18B). After optimization, the ROCK inhibitor Y-27632 (5 μM, Selleckchem, S1049) was added. Alternatively, XAV939 (1 μM, Selleckchem, S1180) can be substituted for IWR-1-endo. bEpiSCs were cultured with mouse fibroblasts treated with mitomycin C.

[0127] Derivation of bovine ICMs, epiblast, and epiblast bEpiSCs

[0128] Bovine ICMs, epiblast, and ectoderm were separated by mechanical dissociation and stained with TrypLE TM The cells were treated with 5% FcGen Express (an animal-derived recombinant enzyme primarily used to dissociate various adherent mammalian cells, Gibco, 12605010) for 3 minutes and then plated onto feeder cells supplemented with 3i / LAF medium. The cultures were incubated at 37°C in 5% O₂ and 5% CO₂. Spherical outgrowths were harvested and digested using Accutase Cell Dissociation Reagent (Gibco, A11105-01) and passaged every 3 days at a 1:4 ratio.

[0129] Experimental methods

[0130] Single-cell RNA library preparation and sequencing

[0131] As previously studied 7,8 Single-cell RNA-seq libraries were prepared using a modified Smart-seq2 protocol as described in . Briefly, single embryonic cells were transferred to lysis buffer containing an 8 bp barcode. Subsequently, first-strand cDNA was reverse synthesized and amplified in a reverse transcription (RT) mix consisting of 4 U RNase inhibitor, 100 U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1 mM dNTPs (TAKARA, 4019), 60 mM MgCl2, and 3 μM RT primer with 10 μM TSO primer. After PCR amplification, the product was purified using 0.8× Beckman's AMPure XP beads (A63882). Biotin PCR enrichment was then performed to further improve library quality. Finally, single-cell RNA-seq libraries were constructed according to the instructions provided by the KAPA PCR Library Amplification / Illumina Series (KAPA KK8054). High-quality libraries were sequenced using the Illumina HiSeq Xten platform (Novogene), with paired-end reads of 150 bp in length. The primers used in the experiments are listed in the Key Resources table.

[0132] Cell population doubling time

[0133] bEpiSCs were collected at 3×10 5 The cells were seeded at a density of 100 μg / mL in 12-well plates and the growth curve of bEpiSCs was plotted. TM An automated cell counter was used to digest and count cells at intervals of 12, 24, 36, 48, and 60 hours. Three replicates were performed at each time point. Doubling time (DT) was calculated using the following formula: Doubling time (DT) = 12 × [log2 / (logN t-1gN0)], where 12 is the cell culture time (hours); N t is the number of cells at 48 hours of culture; N0 is the number of cells recorded at 36 hours.

[0134] Single-cell cloning efficiency analysis

[0135] Using TrypLE TM Bovine EpiSCs were digested with ELISA Express (an animal-derived recombinase primarily used to dissociate various adherent mammalian cells, Gibco, 12605010) and filtered through a 40 μm cell strainer. The cells were seeded in 6-well plates at a density of 100, 500, and 1000 cells. After 3 days of culture, the number of colonies formed was counted, and the single-cell clonogenicity rate was calculated and averaged.

[0136] Karyotype analysis

[0137] Before karyotyping, 1% KaryoMAX Colcemid solution (mitotic inhibitor, mainly used in cell culture experiments to arrest cells in metaphase, Gibco, 15212012) was added to the bEpiSCs culture medium and incubated for 1 hour. TM bEpiSCs were dissociated into single cells using ELISA Express (an animal-derived recombinant enzyme primarily used to dissociate various adherent mammalian cells, Gibco, 12605010) and collected by centrifugation. Subsequently, bEpiSCs were suspended in a hypotonic solution of 0.075 M KCl (Sigma, P5405) and incubated at 37°C for 15 minutes. Following this step, bEpiSCs were fixed with methanol and acetic acid in a 3:1 ratio; this process was repeated three times. The resulting bEpiSC suspension was dropped onto a pre-chilled glass slide, dried thoroughly at room temperature, and stained with 10% Giemsa stain (Sangon, E6073140001) for 30 minutes. More than 45 metaphase cells were assayed for each cell line.

[0138] Alkaline phosphatase (AP) staining

[0139] For detailed information on the AP staining procedure and precautions, please refer to the Alkaline Phosphatase Detection Kit (Millipore, SCR004). AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic germ cells (EGCs), which have the ability to self-renew and differentiate into all three germ layers (ectoderm, mesoderm, and endoderm).

[0140] Immunofluorescence analysis

[0141] The cells were washed with DPBS for immunofluorescence (IF) analysis and then fixed in 4% paraformaldehyde (PFA) for 30 minutes at room temperature. Subsequently, the cells were rinsed with DPBS and infiltrated with 0.1% Triton X-100 for 20 minutes. After another round of DPBS washing, the cells were blocked with 3% BSA for 1 hour at room temperature. The primary antibody was incubated overnight at 4°C and then washed three times with a cleaning solution (DPBS containing 0.1% Triton X-100 and 0.1% Tween 20). The secondary antibody was incubated at room temperature for 1 hour and then washed three times with the same cleaning solution. Finally, DAPI (nuclear dye DAPI) was used to stain the nuclei to visualize the cells, thereby enabling direct observation and photography. The antibodies involved in this application are as shown in Table 4.

[0142] Table 4

[0143]

[0144] Embryoid body differentiation

[0145] After digestion, bEpiSCs were plated at 1 × 10 per well. 6 The cells were seeded at a density of 100 cells and cultured in MEF medium on 35 mm low attachment plates for 5-7 days. MEF medium: DMEM (Gibco, 11960-044), supplemented with 10% FBS (Gibco, 16000-044), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122) and 1% GlutaMAX (ThermoFisher Scientific, 35050-061), cultured on a horizontal shaker at 70 rpm. Subsequently, embryoid bodies (EBs) were transferred to 12-well plates and incubated in the same medium for one week, with the medium changed twice a day. Immunofluorescence staining was then performed using adherent cells.

[0146] Teratoma formation

[0147] After digestion, 1×10 7 bEpiSCs were resuspended in 50 μL of BM and injected subcutaneously into the neck of BALB / c nude mice. After 4–5 weeks, teratomas were harvested and subsequently subjected to H&E analysis.

[0148] H&E analysis

[0149] Teratomas were washed twice in DPBS and fixed with 4% PFA at 4°C for 2 days. Subsequently, teratoma tissue was dehydrated using a gradient of alcohol (70%, 80%, 90%, 95%, and finally 100% every hour), transferred to xylene, and embedded in paraffin. Samples were sectioned to 5 μm thickness, deparaffinized in xylene, and rehydrated using decreasing concentrations of ethanol. Finally, samples were stained with hematoxylin (which stains nuclear chromatin and cytoplasmic nucleic acids purple-blue, Sigma–Aldrich, MHS16) and eosin (which stains components of the cytoplasm and extracellular matrix red, Sigma-Aldrich, HT110116) and observed under a microscope (Leica, DM5500B).

[0150] RT–qPCR

[0151] Total RNA was extracted from bEpiSCs using the RNA prep Pure Cell / Bacteria kit (TIANGEN, DP430) and then reverse transcribed into cDNA using 5× All-In-One RT Master Mix (Abm, G490). PCR amplification was then performed on the Archimed Real Time System (ROCGENE) using 2× RealStar Green Power Mixture (GenStar, A311-05). CT (2 -ΔΔCT ) method. ΔC values ​​were calculated using GAPDH as an internal control. All experiments were performed in triplicate. Primer sequences used for real-time PCR can be found in the Key Resources table.

[0152] Myogenic differentiation of bEpiSCs

[0153] This experimental protocol was similar to that of porcine pgEpiSCs. 1The same. Briefly, myogenic differentiation basal medium (MDBM) consists of DMEM / F12, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR, and 200 μM ascorbic acid. In the first stage, bEpiSCs were separated into small pieces and cultured for 3 days in MDBM supplemented with 1% B27 supplement, 3 μM CHIR99021, and 2 μM SB431542 (Selleckchem, S1067). In the second stage, from day 4 to day 6, the medium was replaced with a combination containing 3 μM CHIR99021, 2 μM SB431542, 500 nm LDN193189 (Stemgent, 04-0074), and 20 ng / mL recombinant human FGF-basic (154 a.a.). In the third stage, the culture medium was replaced with 10 ng / mL HGF (Peprotech, 100-39H), 10 ng / mL IGF-1 (Peprotech, 100-11), 20 ng / mL recombinant human FGF-basic (154a.a.), and 0.5 μM LDN193189 for 2 days. In the fourth stage, bEpiSCs began to differentiate into muscle precursor cells. bEpiSC-MPCs were treated with 10 ng / mL IGF-1 for 4 days. In the fifth stage, bEpiSC-MPCs were treated with a combination of 10 ng / mL HGF and 10 ng / mL IGF-1 for 20 to 25 days to promote skeletal muscle maturation. For skeletal muscle maturation, cells were treated with N2 medium consisting of DMEM / F12 supplemented with 15% KOSR, 1% N2 supplement, 1% penicillin-streptomycin, and 1% non-essential amino acids.

[0154] RNA-seq with rRNA depletion (rRNA accounts for a high proportion of total RNA and usually does not contain useful transcriptome information. Removing rRNA can improve sequencing efficiency, reduce sequencing costs, and improve data quality)

[0155] Total RNA was extracted from four bEpiSCs samples using the RNeasy Mini kit (Qiagen, 74106). To construct strand-specific RNA-seq libraries, we used an rRNA removal protocol (Globin-Zero Gold rRNARemoval Kit, Illumina, GZG1224) combined with Ultra TM Directional RNA Library PrepKit for All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen, Life Technologies, Q32851) and sequenced on the Illumina HiSeq4000 platform.

[0156] Vector construction

[0157] The GFP plasmid was kept in the laboratory. In summary, we generated the PB-CMV-EF1A-GFP-NLS plasmid by modifying the PB-CAG-MCS vector (provided by Professor Wu Sen). Specifically, we replaced the chicken β-actin promoter with the human elongation factor 1α (EF1A) promoter and integrated the GFP-NLS downstream of the EF1A promoter.

[0158] bEpiSCs transfection

[0159] Using Lipofectamine TM Bovine EpiSCs were transfected with Opti-MEM 3000 reagent (Invitrogen, L3000008). Specifically, normal cells were passaged in a 24-well plate for 16 hours before transfection. First, 25 μL of Opti-MEM TM culture medium, and then add 0.75 μL Lipofectamine TM 3000 reagent and mix thoroughly. Then, take another centrifuge tube and add 25μL Opti-MEM TM culture medium, add 0.5 μg DNA and 1 μL P3000 TM Reagents, mix thoroughly. Then the DNA mixture is mixed with Lipofectamine TM The reagents were mixed at a 1:1 ratio, incubated for 10-15 minutes, and then added to the cells for culture.

[0160] Generation of bEpiSCs cloned embryos

[0161] Ovaries were obtained from cattle farms around Beijing, and oocytes were extracted from 3-8 mm follicles. Oocytes with three layers of cumulus cells were transferred to maturation medium and matured at 38.5°C and 5% CO2 for 18 hours. The maturation medium was based on TCM199 (Gibco, 12340-030) supplemented with 10% FBS (Gibco, 16000-044), 0.01 IU / mL follicle-stimulating hormone (FSH, Sigma, F4021), 0.01 IU / mL luteinizing hormone (LH, Sigma, L6420), and 1 μg / mL estradiol (Sigma, E2257). After 18 hours of oocyte maturation, 0.1% hyaluronidase (Sigma, H4272) was used to remove excess cumulus, and the oocytes with polar bodies were placed in HM medium containing 7.5μg / mL cytochalasin (Sigma, C6762) for 10 minutes and then transferred to HM medium containing 10% FBS for enucleation. The enucleated oocytes were transferred to maturation medium until the injection of the cell nucleus. bEpiSCs were differentiated for more than 1 week in a basal medium containing 10ng / mL BMP4 (PeproTech, 315-27), 5μM SB431542 and 10ng / mL FGF2, and then used as donor cells for nuclear transplantation. Transparent round donor cells were selected and injected into the perivitelline space, so that the cells were as close to the cytoplasm as possible to improve fusion efficiency. The reconstructed embryo was placed between the two electrodes of the fused cells and aligned with the microneedle so that the somatic cells faced one of the two electrodes. Fusion conditions were: double direct current pulses of 2.5 kV / cm, 10 μs, with 1-s intervals; the fusion solution consisted of 0.3 mmol / L mannitol (Sigma, 1375105), 0.15 mmol / L CaCl2 (Sigma, C7902), and 0.15 mmol / L MgCl2 (Sigma, M2393). The fusion rate was assessed under a stereomicroscope. Reconstructed embryos were then transferred to IVC medium containing 5 μM ionomycin (Sigma, 407950) and cultured for 4 minutes, followed by transfer to IVC medium containing 2 mM 6-DMAP (Sigma, D2629) and cultured for 4 hours. Activated embryos were washed three times in IVC medium and transferred to IVC medium for culture.

[0162] Quantitative statistical analysis

[0163] Single-cell RNA-seq low-level processing and filtering

[0164] For the STRT-seq dataset, raw reads were segmented by the 8-bp cell barcode located on Read 2, allowing for two mismatches. In addition, the 8-bp unique molecular identifiers (UMIs) located on Read 2 were switched to the identifier line of the paired Read 1. Read 1 was then processed to remove template switching oligonucleotide (TSO) primers, low-quality bases, and polyA sequences. 51 The trimmed reads were aligned to their respective reference genomes (bovine: Bos_taurus.ARS_UCD.12; porcine: Sscrofa1.1). Unique molecular identifiers (UMIs) were generated using kallisto (v-0.46.0). 3 count.

[0165] Identification of differentially expressed genes at embryonic stages across different lineages

[0166] Based on the differentiation process during bovine embryonic development, we divided bovine embryonic cells into three major lineages: the embryonic lineage, including the pre-ICM (inner cell mass) at E5, the ICM (inner cell mass) at E6 and E7, the epiblast at E10 and E12, and the epiblast at E14; the TE (trophectoderm) lineage, including the pre-TE at E5 (pre-trophectoderm at E5 (late morula)), the TE at E6, E7, E10, E12, and E14; and the hypoblast lineage, including the hypoblast at E10 and E12, and the definitive endoderm ( Figure 1 Middle C).

[0167] Construction of expression tendency

[0168] To track the dynamic changes of DEGs during embryonic development (differentially expressed genes (DEGs) in the trophectoderm (TE) lineage exhibit distinct expression trends, and the expression patterns of these genes at different developmental stages reflect the differentiation and functional changes of the trophectoderm), we constructed the expression trends of DEGs in the ectoderm lineage. We first calculated the average expression level of each gene in each lineage at a specific embryonic developmental time point. The average expression levels of the embryonic lineages were rescaled and analyzed using the k-means clustering method with parameters k = 10 and iter.max = 100. DEGs with similar trends during embryonic development were grouped into separate clusters.

[0169] Cross-species comparative analysis

[0170] We downloaded the orthologous gene lists of the four species using the BioMart tool in the genome browser 105 (http: / / dec2021.archive.ensembl.org / index.html) and retained 16,841 genes, all of which were 1:1 orthologous genes. We then retained 1:1 orthologous genes in the bovine and porcine embryonic lineage datasets. For the integrated analysis between the porcine and bovine embryonic datasets, we used the Seurat CCA method for anchoring and dataset alignment. The top 2,000 features with repeated variables in the dataset were selected, and the “FindIntegrationAnchors” function with the following parameters was used to identify anchors: “reduction = 'cca, k, anchor = 5, normalization. Method = 'SCT'”. Then, based on the identified anchors, the dataset was integrated using the “IntegrateData” function with the following parameters: “dims = 1:30, k.weight = 50, normalization. Method = 'SCT'”. The “AverageExpression” function was used to obtain the average expression value of genes in the comprehensive determination, and the “cor” function was used to calculate the Spearman correlation coefficient between different cell types of the two species. The Wilcoxon rank sum test was used to determine the expression of genes in the two species using the “FindMarkers” function. Fold changes in gene expression levels between the formative state and the primed state, and between the formative state and the primed state. Only those with |'avg_logFC'|>0.25 and 'p_val'<0.05 were considered DEGs.

[0171] Pseudo-time analysis

[0172] Using the R package Monocle3 (v-1.3.1) 4 The developmental trajectory of embryonic cells was reconstructed. The UMI matrix was used as input, and the cells were pseudo-timed using the variable genes obtained from Seurat. Destiny (v-2.14.0) R package was used to further analyze the cells. 5 The developmental order of individual cells from the two species was determined according to the differentially expressed genes (DEGs) between all embryonic day cells of pigs calculated by the “FindAllMarkers” function.

[0173] RNA-seq data processing and analysis

[0174] The expression levels of protein-coding genes (from the gene annotation file [GTF] of Ensembl-Bos_taurus.ARS_UCD.12) were quantified as transcripts per million (TPM) using kallisto (v-0.46.0). 6 To identify DEGs between different cell types, we used a Benjamini-Hochberg-adjusted false discovery rate (FDR) < 0.05 and an absolute log2 (fold change) > 2 as cutoffs for statistical significance.

[0175] Correlation analysis of transcriptomes between bEpiSCs and embryonic cells

[0176] For the integrated analysis between the bEpiSC and bovine embryo datasets, we used the Seurat RPCA method for anchoring and dataset alignment. We selected the top 2,000 features with reproducible variability across the datasets, and identified anchors using the `FindIntegrationAnchors` function with the following parameters: `reduction = 'rpca', k, anchor = 15`. The datasets were then integrated based on the identified anchors using the `IntegrateData` function with the following parameters: `dims = 1:30, k.weight = 50`. Scaling and PCA were applied to the combined dataset, and the integrated PCA coordinates were used as input for the clustering and t-SNE visualization workflow with the following parameters: `dims = 1:5`. Key markers were visualized using the `FeaturePlot` function.

[0177] Functional enrichment analysis

[0178] Using Metascape http: / / metascape.org ) Functional enrichment analysis was performed on the selected genes. Bovine genes were mapped to their human orthologs, with humans (Homo sapiens) being the target species for the analysis. Enrichment analysis was performed using all genes in the genome as a background set, with Gene Ontology (GO)-Biological Process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways as ontology sources. Terms with a minimum count ≥ 3, adjusted P < 0.01, and enrichment factor ≥ 1.5 were considered significant, and similar terms were grouped into clusters. Using -log 10 (P value) Histogram depicts key pathways.

[0179] Statistical analysis

[0180] for Figure 2 CH, Figure 5 Middle B, Figure 5 The RT-qPCR data in D were subjected to two-way ANOVA and the Figure 2 The data in panels 1B, 1D, and 1E were analyzed. Figure 2 B, 2 D, 2 E and Figure 6 Dunnett's multiple comparison test was used in B. Figure 2 Figure 2C, Figure 2F-2H used Bonferroni's multiple comparison test. Figure 2 Tukey's multiple comparison test was used to compare cell population doubling time and single-cell cloning efficiency in Figure 2D and Figure 2E.

[0181] Table 3 Key resources

[0182]

[0183]

[0184]

[0185] Steps for selecting clones:

[0186] During the process of establishing stem cell lines, the adherent growth characteristics of embryonic cells are a key step. However, in addition to the epiblast cells that can adhere normally, other germ layer cells such as hypoblast cells and trophectoderm cells may also adhere, which can adversely affect the purity and quality of stem cell clones. Therefore, selecting clones with typical dome-shaped morphology is a key step in ensuring the success of stem cell line establishment. To this end, we have established the following clone screening criteria:

[0187] 1. The clones must present a three-dimensional dome-shaped structure, which is the hallmark morphology of typical embryonic stem cell clones.

[0188] 2. Clones should have clear and distinct boundaries to facilitate differentiation and selection.

[0189] 3. During the growth process, there should be no depression in the center of the clone, and the whole should not appear as a single layer attached to the wall to ensure that the three-dimensional structure of the clone is maintained.

[0190] 4. For flat and non-monolayer clones, you can perform whole-cell digestion and subculture to observe whether dome-shaped clones can be formed in the secondary cells. Usually, after 1-2 rounds of screening, the clones will show a uniform dome-shaped morphology, and the growth rate and size of the clones will also tend to be consistent. At this point, it can be considered that the stem cell line has been successfully established. By strictly following these screening criteria, the success rate of stem cell line establishment can be effectively improved, and the established stem cell line can be ensured to have good purity and stability.

[0191] Optimization of embryo line establishment methods at different stages:

[0192] For embryos on days E6-8, taking E7 embryos as an example, whole embryo inoculation is used. During inoculation, a glass needle is used to split the embryo and expose the inner cell mass to the feeder layer cells to facilitate the adherent growth of the epiblast stem cells.

[0193] For E9-E14 embryos, the embryonic blastoderm needs to be separated before inoculation, and then the blastoderm needs to be enzymatically digested into cell clusters, which are then inoculated onto the feeder layer cells.

[0194] Example 1. Establishment and characteristics of bovine epiblast stem cells (bEpiSCs)

[0195] 1. If Figure 1 As shown in A, the epiblast was separated from bovine embryos at different developmental stages (including E7, E10, E12 and E14) by mechanical separation (Epiblast isolation), and the ectoderm was isolated using TrypLE TM The cells were treated with Express (Gibco, 12605010) for 3 minutes and then inoculated onto feeder cells (mouse fetal fibroblasts) supplemented with 3i / LAF medium. The cultures were incubated at 37°C in an environment containing 5% O2 and 5% CO2. The spherical growths were collected and digested using Accutase cell dissociation reagent (Gibco, A11105-01) and passaged every 3 days at a ratio of 1:4. After passage, the initial spherical growths (also called outgrowth, which is the primary clone formed after the embryonic cells adhere to the wall) were obtained. After the outgrowth was successfully subcultured, the E7bEpiSCs cell line (also referred to as E7bEpiSCs line or E7bEpiSCs in this application), E10bEp iSCs cell line (also referred to as E10bEpiSCs line or E10bEpiSCs in this application), E12bEpiSCs cell line (also referred to as E12bEpiSCs line or E12bEpiSCs in this application) and E14bEpiSCs cell line (also referred to as E14bEpiSCs line or E14bEpiSCs in this application) (cell line refers to a cell line that can be passaged and has a certain number and stability, and bEpiSCs refers to bovine epiblast stem cells).

[0196] 2. Statistics on the efficiency of establishing lines of embryos at different developmental stages are as follows: Figure 2 As shown in (B).

[0197] Table 1

[0198] Bovine embryonic development stages Number of embryos Proportion of primary derivatives cell lines E7 20 6 / 20 2 E10 4 4 / 4 4 E12 3 3 / 3 3 E14 3 3 / 3 3

[0199] 3. Selection and subculture of bovine epiblast stem cells (bEpiSCs):

[0200] The above bEpiSCs (E7bEpiSCs, E10bEpiSCs, E12bEpiSCs, E14bEpiSCs) were cultured on mitomycin C (Selleckchem, S8146) treated mouse embryonic fibroblast (MEF) feeder cells (5×10 4 cells / cm 2 ) and cultured on fresh 3i / LAF medium every 12 hours. To maintain the undifferentiated state of bEpiSCs, the following three points are important: (a) freshly prepared 3i / LAF medium should be stored at 4°C for no more than one week and should not be frozen; (b) the subculture density must be appropriate: the seeding density of bEpiSCs is approximately 3-5×10 4 cells / cm 2 (c) Fresh feeder cells and appropriate density (3-4×10 4 cells / cm 2 bEpiSCs were passaged every 2-3 days using Accutase cell separation reagent (Gibco, A11105-01) at a ratio of 1:3-1:5. The specific passage days and ratio should be adjusted according to the actual situation.

[0201] When subcultured to Figure 1 After the passage time shown in the lower half of Figure C, the cells were placed under a microscope for observation. The results were as follows: Figure 1 As shown in C (outgrowth refers to the primary clone formed after embryonic cells adhere to the wall, and 19\14\11\15 in bEpiSCs-P19\P14\P11\P15 refers to the number of passages (P=passages)), it can be seen that all bEpiSCs lines have a dome morphology.

[0202] 4. Analyze the cell proliferation capacity and single-cell colony formation efficiency of bEpiSCs derived from embryos at different developmental stages:

[0203] Doubleing time:

[0204] The above bEpiSCs (E7bEpiSCs, E10bEpiSCs, E12bEpiSCs, E14bEpiSCs) were cultured at 3×10 5 The cells were seeded at a density of 100 μg / mL in 12-well plates and the growth curve of bEpiSCs was plotted. TMThe cells were digested and counted using an automated cell counter every 12 hours, 24 hours, 36 hours, 48 ​​hours, and 60 hours. Each time point was repeated three times. The doubling time was calculated as follows: doubling time (DT) = 12 × [log2 / (logNt-logN0)], where 12 is the cell culture time (hours); Nt is the number of cells cultured for 48 hours; and N0 is the number of cells recorded at 36 hours. The results are shown in Figure 2. Figure 1 As shown in D.

[0205] Single cell colony formation efficiency:

[0206] The above bEpiSCs were respectively TM The cells were digested with 1% Glutamax (Gibco, 12,605,010) and filtered through a 40 μm cell strainer. The cells were seeded in 6-well plates at 100, 500, and 1000 cells, respectively. After 3 days of culture, the number of colonies formed was counted, and the single-cell colony formation rate was calculated and averaged. The results are shown in Figure 2. Figure 1 As shown in E.

[0207] The results are as follows Figure 1 As shown in D and E (in the figure, X in bEpiSCs-PX refers to the number of passages (P=passages)), it can be seen that there is no significant difference in the cell proliferation ability of bEpiSCs derived from different embryonic ectoderm layers, the doubling time is 12 hours, and the single-cell colony formation efficiency is about 23%, indicating that the cell lines derived from different embryonic ectoderm layers have good consistency.

[0208] 5. Considering that E10EPI is the key stage of pluripotency formation, E10bEpiSCs were selected for pluripotency determination. Specifically, E10bEpiSCs were stained using an alkaline phosphatase detection kit (Millipore, SCR004). The staining results are shown in Figure 5. Figure 1 As shown in middle F, positive alkaline phosphatase staining was observed in the colonies, which confirmed their pluripotency.

[0209] Karyotype analysis of E10bEpiSCs:

[0210] 1% KaryoMAX Colcemid solution (Gibco, 15212012) was added to the bEpiSCs culture medium and incubated for 1 hour. TMExpress (Gibco, 12605010) was used to dissociate bEpiSCs into single cells and collected by centrifugation. Subsequently, bEpiSCs were suspended in a hypotonic solution of 0.075M KCl (Sigma, P5405) and incubated at 37°C for 15 minutes. After this step, bEpiSCs were fixed with methanol and acetic acid in a ratio of 3:1; this process was repeated three times. The resulting bEpiSCs suspension was dropped onto a pre-cooled slide, dried thoroughly at room temperature, and stained with 10% Giemsa staining solution (Sangon, E6073140001) for 30 minutes. More than 45 metaphase cells were detected for each cell line. The results are shown in Figure 2. Figure 1 As shown in G, karyotype analysis showed that the normal chromosome count of bEpiSCs was 60, indicating that they had the normal chromosome karyotype of the bovine species.

[0211] 6. Wash the cells with DPBS for immunofluorescence (IF) analysis and then fix them in 4% paraformaldehyde (PFA) at room temperature for 30 minutes. Subsequently, rinse the cells with DPBS and permeabilize with 0.1% Triton X-100 for 20 minutes. After another round of DPBS wash, block the cells with 3% BSA for 1 hour at room temperature. The primary antibody was incubated overnight at 4°C and then washed three times with a washing solution (DPBS containing 0.1% Triton X-100 and 0.1% Tween 20). The secondary antibody was incubated at room temperature for 1 hour and then washed three times with the same washing solution. Finally, DAPI staining was performed to visualize the cell nucleus for direct observation and photography (as shown in Figure HI). The results are shown in Figure HI. Figure 1 As shown in Figure HI, pluripotency analysis demonstrated high expression of POU5F1, NANOG, and SOX2. In addition, pluripotency-associated surface markers such as CDH1, SSEA1, and SSEA4 were also highly expressed.

[0212] After E10bEpiSCs were digested, 1×10 6 The cells were seeded at a density of 100 cells per well and cultured for 5-7 days in MEF medium on 35 mm low attachment plates: DMEM (Gibco, 11960-044), supplemented with 10% FBS (Gibco, 16000-044), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122) and 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), and cultured on a horizontal shaker at 70 rpm. Subsequently, embryoid bodies (EBs) were transferred to 12-well plates and incubated in the same medium for one week, with the medium changed twice a day. Immunofluorescence staining was then performed using adherent cells, and the results were shown in Figure 2. Figure 1As shown in middle J, in vitro differentiation assays confirmed that E10bEpiSCs had the ability to form embryoid bodies and differentiate into the three germ layers.

[0213] After digestion, 1×10 7 E10bEpiSCs were resuspended in 50 μL BM and then injected subcutaneously into the neck of BALB / c nude mice. After 4-5 weeks, teratomas were collected and subjected to subsequent H&E analysis. Figure 1 In vivo teratoma experiments demonstrated that these cells were able to generate teratomas with organized structures representing all three germ layers, as shown in middle K. In summary, E10bEpiSCs possess essential PSC characteristics.

[0214] Example 2: 3i / LAF factors are crucial for the long-term maintenance of bEpiSCs

[0215] CHIR99021 (1 μM, Selleckchem, S1263), WH-4-023 (1 μM, Selleckchem, S7565), recombinant human LIF (10 ng / mL, PeproTech, 300-05), recombinant human Activin A (25 ng / mL, PeproTech, 120-14E) and recombinant human FGF-basic (154aa) (10 ng / mL, PeproTech, 100-18B), IWR-1-endo (0.5 μM, Selleckchem, S7086).

[0216] To investigate the effects of different cytokines and small molecule inhibitors incorporated into the 3i / LAF culture system on the pluripotency and self-renewal capacity of bEpiSCs, we performed single factor reduction to evaluate the function of these molecules. Using E10bEpiSCs cultured in 3i / LAF as a control group, we replaced the factor-reduced medium 12 hours after the E10bEpiSCs passage experiment in Example 1. Cells were cultured in factor-free medium for 48 hours and the cell morphology and AP staining were observed. The results are shown in Figure 2. Figure 2As shown in the AB (-CHIR99021, -WH-4-023, -IWR-1-endo, -LIF (10 ng / mL, PeproTech, 300-05), -Activin A, and -FGF-basic, respectively, which lack the corresponding factors), it can be seen that in E10bEpiSCs from the IWR-1-endo (IWR) factor-free group, clones were significantly flattened, with unclear boundaries and differentiation. In contrast, bEpiSCs clones from the FGF2 factor-free group exhibited a significantly slower proliferation rate, smaller clone size, and more severe cell apoptosis. The key role of IWR and FGF2 in the self-renewal of E10bEpiSCs is obvious, confirming previous reports on bovine primed PSCs.

[0217] By comparing the gene expression profiles of E10bEpiSCs cultured under 3i / LAF and IWR / FGF2 (I / F) conditions (with IWR and FGF2 added alone), we found that I / F alone resulted in downregulation of pluripotency-related genes and upregulation of development-related genes ( Figure 2 (C). Thus, the combination of IWR and FGF2 can effectively maintain the pluripotency of primed bESCs, but this combination is insufficient to maintain the pluripotency of E10 bEpiSCs.

[0218] To investigate the functions of three small molecule inhibitors related to the WNT / β-catenin signaling pathway in the pluripotency and self-renewal of bEpiSCs, we collected the third generation E10 bEpiSCs that were minus CHIR99021 (CHIR) / IWR / WH4023 (WH) (representing the removal of CHIR99021, IWR-1-endo, and WH-4-023 factors, respectively) and performed differentially expressed gene (DEG) analysis. The analysis showed that the removal of any WNT-related small molecules resulted in abnormal expression of core pluripotency genes POU5F1, SOX2, and NANOG in bEpiSCs, as well as a significant decrease in the expression of ZFP42, UTF1, and TDGF1 ( Figure 2 In addition, genes involved in mesoderm differentiation and embryonic gastrulation were upregulated, such as BMP4, CDH2, GATA6, WNT5A, LEF1, and CTNNB1 ( Figure 2 Middle E) shows that the removal of WNT-related small molecules directly or indirectly triggers the differentiation process of E10bEpiSCs.

[0219] The combination of CHIR and IWR was shown to maintain the self-renewal of mouse EpiSCs and human ESCs. Figure 5 The effects of different ratios of CHIR and IWR on the pluripotency of E10bEpiSCs were studied. The concentrations of IWR and CHIR in 3i / LAF medium were adjusted to Figure 5 The concentrations shown in A in Figure 1 (where C is CHIR and I is IWR; in xC / yI, x is the concentration of CHIR in μM and y is the concentration of IWR in μM) were configured and grouped. We replaced the reduced factor medium after 12 hours of the E10bEpiSCs passage experiment in Example 1. The cells were cultured in a factor-free medium for 48 hours and subjected to clone morphology, AP staining, and gene expression analysis. The results showed that Figure 5 As shown in Figures A and B, the presence of low concentrations of IWR (>0.5 μM) can offset the promoting effect of high CHIR concentrations (<5 μM) on EpiSCs differentiation.

[0220] Using the above method, we further Figure 5 The concentrations in C and D of 7 were used to explore whether there is a concentration range of IWP2 or XAV939 that can replace IWR in 3i / LAF culture medium. The effects of IWP2 and XAV939 (alternative factors for IWR) on the morphology and maintenance of pluripotency of E10bEpiSCs were studied. It was found that the concentration range of 1-2.5 μM XAV939 can be used to effectively replace IWR ( Figure 5 Middle C and 7 Middle D, Figure 5 In D, 3i / LAF is 3i / LAF medium, and x and y in xIWP2 and yXAV939 represent concentrations (unit: μM).

[0221] The concentrations of FGF2, Activin A and LIF in 3i / LAF culture medium were adjusted according to Figure 2 We replaced the factor-reduced medium with that of E10bEpiSCs in Example 1 after 12 hours of passage, cultured them in factor-free medium for 48 hours, and investigated the effects of FGF2, activin A, and LIF on the pluripotency of bEpiSCs. We found that the depletion of FGF2 led to the dysregulation of the core pluripotency regulatory network and the multi-lineage differentiation of bEpiSCs ( Figure 2 F, -FGF2, -Action A, and -LIF represent the subtraction of the corresponding small molecules, and the other concentrations remain unchanged). Removal of activin A resulted in a significant decrease in the expression of POU5F1 and NANOG, while the expression of mesendoderm-related genes (such as BMP2 gene, BMP4 gene, and IDs gene) was significantly increased ( Figure 2(G), -FGF2, -Action A, and -LIF represent the subtraction of the corresponding small molecules, and the other concentrations remain unchanged. Activation of the JAK / STAT3 signaling pathway is crucial for maintaining stem cell pluripotency. 38 However, the expression of the core JAK / STAT3 signaling gene STAT3 is downregulated, while the expression of the mesendoderm-related gene GATA6 is upregulated ( Figure 2 In Figure 5, -FGF2, -Action A, and -LIF represent the subtraction of the corresponding small molecules, while the other concentrations remain unchanged), indicating that LIF supplementation is beneficial. In summary, our study demonstrates that the 3i / LAF culture system effectively maintains the multipotency of bEpiSCs, while emphasizing the positive effects of all factors on their self-renewal capacity.

[0222] Example 3. Transcriptome Analysis of bEpiSCs

[0223] Single-cell transcriptome characterization of bovine embryos can serve as a clear benchmark for assessing the pluripotency state of bovine embryonic stem cells. To further evaluate the gene expression signature and pluripotency state of bEpiSCs:

[0224] We first compared the single-cell transcriptome data of bEpiSCs obtained from bovine embryos at different stages with the single-cell transcriptome data of bovine embryonic lineages (Table 2). PCA showed that the developmental and pluripotent characteristics of bEpiSCs were closely related to those of the E10-E12 epiblast ( Figure 3 Middle A). Spearman correlation analysis showed that E7 and E10bEpiSCs showed a stronger correlation with E10 epiblast, while E12 and E14bEpiSCs showed a higher correlation with E12 epiblast ( Figure 3 (B). We further compared bEpiSCs isolated at different embryonic stages with single cells from bovine embryonic lineages. In terms of pluripotency, we observed downregulation of naive pluripotency-related genes in bEpiSCs. Conversely, genes associated with developing pluripotency were found to be upregulated in bEpiSCs. In addition, the expression levels of initiated pluripotency-related genes were lower in bEpiSCs ( Figure 3 Middle C), indicating that bEpiSCs exhibit characteristics of intermediate (Formative) pluripotency.

[0225] Next, we performed a comparative analysis of the bulk RNA transcriptome between the E10 bEpiSCs established in this study and previously reported bovine PSCs, including primed bESCs15, bEDSCs16, bEPSCs, and biPSCs18. PCA analysis revealed differences in pluripotency at the level of Dim2, indicating that bEpiSCs exhibited a greater degree of similarity to bEDSCs ( Figure 3 Middle D).

[0226] To distinguish bEpiSCs from other bovine PSCs, we performed comparative analyses to identify unique features of bEpiSCs.

[0227] Compared with primed bESCs, E10bEpiSCs expressed higher levels of NANOG, LEFTY2, NODAL, and other stem cell markers associated with pluripotency regulation (FGF4, ZFP42, ETV3L, DPPA3, ACVRL1, TDGF1, and TBX3). Furthermore, they expressed lower levels of GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, and HES1.

[0228] Furthermore, compared with bEDSCs, E10bEpiSCs showed upregulated expression of genes involved in stem cell proliferation (ELL3 and FGF4) and intercellular adhesion regulation (NODAL, FOXA2, and EPCAM). Furthermore, E10bEpiSCs showed higher expression levels of DPPA3, LMO1, ACVRL1, and WNT3A. Furthermore, E10bEpiSCs showed lower expression levels of HAND1, GATA3, TGFB2, ZFP42, VMO1, MEIS2, BMP4, IGF1R, CCND2, and SPRY4.

[0229] In addition, compared with biPSCs, E10bEpiSCs showed higher expression levels of LIN28A / B, NODAL, LIFR, IL6R, PDGFRA, ACVR1B, ETS1, IL6ST, and SALL4 genes. Furthermore, their expression levels of GDF15, BCL3, CD44, ETV2, FGFR4, KLF15, GDF1, BMP4, COX17, and MYC genes were lower.

[0230] In addition, compared with bEPSCs, E10bEpiSCs showed higher expression levels of LIN28A / B, NODAL, LIFR, ESRRB, IL6R, ACVR1B, KLF5, SALL4, and CDH1 genes. Furthermore, their expression levels of MSC, GCK, TGFB2, MAPK15, KLF15, GDF1, ID4, FOS, and COX17 genes were lower.

[0231] In addition, compared with biPSCs or bEPSCs, E10bEpiSCs showed higher expression levels of LIN28A / B and NODAL genes, both of which are known for their inhibitory effects on cell differentiation and the involvement of IL6R in the PI3K-Akt signaling pathway ( Figure 3 middle E and 3 middle F; Figure 6 Middle A).

[0232] To investigate the similarities and differences between bEpiSCs and porcine pgEpiSCs, we performed a comparative analysis of gene expression between these two cell types at the bulk RNA level. Analysis of differentially expressed genes (DEGs) revealed no significant differences between bovine and porcine EpiSCs in terms of pluripotency maintenance (e.g., POU5F1, NANOG, LIN28B) or germ layer differentiation (e.g., BMP4, EOMES, NODAL). Figure 6 However, changes were only observed in ion transport (ATOX1 gene, ATP4A gene, and GCK gene) and cell activation (GLI3 gene, IRF1 gene, and EZH2 gene) ( Figure 6 Middle C). These findings suggest that bovine and porcine EpiSCs share similar regulatory networks for maintaining pluripotency in the 3i / LAF system.

[0233] In conclusion, we successfully established bovine formative EpiSCs, which displayed gene expression signatures distinct from those of other published bovine PSCs.

[0234] Table 2

[0235]

[0236] Example 4: Potential Applications of bEpiSCs

[0237] This example also evaluated the potential of bEpiSCs for myogenic differentiation and as donor cells for somatic cell nuclear transfer (SCNT). Figure 4 Middle A).

[0238] Specifically, myogenic differentiation basal medium (MDBM) consisted of DMEM / F12, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR, and 200 μM ascorbic acid.

[0239] In the first stage, E10b EpiSCs were dissociated into small pieces and cultured for 3 days in MDBM supplemented with 1% B27 supplement, 3 μM CHIR99021 and 2 μM SB431542 (Selleckchem, S1067).

[0240] In the second stage, from day 4 to day 6, the culture medium was changed to a medium containing a combination of 3 μM CHIR99021, 2 μM SB431542, 500 nm LDN193189 (Stemgent, 04-0074) and 20 ng / mL recombinant human FGF-basic (154 a.a.).

[0241] For the third stage, the culture medium was replaced with 10 ng / mL HGF (Peprotech, 100-39H), 10 ng / mL IGF-1 (Peprotech, 100-11), 20 ng / mL recombinant human FGF-basic (154 a.a.), and 0.5 μM LDN193189 for 2 days.

[0242] In the fourth stage, bEpiSCs begin to differentiate into muscle precursor cells. bEpiSCs-MPCs were treated with 10 ng / mL IGF-1 for 4 days.

[0243] In the fifth stage, cells were treated with a combination of 10 ng / mL HGF and 10 ng / mL IGF-1 for 20 to 25 days to promote skeletal muscle maturation. For skeletal muscle maturation, cells were treated with N2 medium consisting of DMEM / F12 supplemented with 15% KOSR, 1% N2 supplement, 1% penicillin-streptomycin, and 1% non-essential amino acids to obtain differentiated muscle cells derived from bEpiSCs.

[0244] 1. Cell observation results at each stage are as follows Figure 4 As shown in B (stage 1-5 represent the above stages 1 to 5 respectively), it can be seen that the cells gradually differentiate from stem cell clone morphology to mesoderm and then to muscle cell-like cells.

[0245] 2. Total RNA was extracted from bEpiSCs and bEpiSC-derived differentiated muscle cells (stage 5 bEpiSCs) using the RNA prep Pure Cell / Bacteria kit (TIANGEN, DP430), and then reverse transcribed into cDNA using 5× All-In-OneRT Master Mix (Abm, G490). Subsequently, PCR amplification was performed on the Archimed Real Time System (ROCGENE) using 2× RealStar Green Power Mixture (GenStar, A311-05). Comparison CT (2 -ΔΔCT) method to analyze the data. GAPDH was used as an internal control to calculate the ΔCT value. All experiments were performed with three independent biological replicates. qPCR analysis showed that bEpiSCs-derived muscle cells (stage 5) expressed MYOG, MYMK, MYH3, and MYH11 ( Figure 4 Middle C).

[0246] 3. Further immunostaining experiments on differentiated muscle cells derived from bEpiSCs:

[0247] The cells were washed with DPBS for immunofluorescence (IF) analysis and then fixed in 4% paraformaldehyde (PFA) for 30 minutes at room temperature. Subsequently, the cells were rinsed with DPBS and permeabilized with 0.1% Triton X-100 for 20 minutes. After another round of DPBS washes, the cells were blocked with 3% BSA for 1 hour at room temperature. The primary antibody was incubated overnight at 4°C and then washed three times with a washing solution (DPBS containing 0.1% Triton X-100 and 0.1% Tween 20). The secondary antibody was incubated at room temperature for 1 hour and then washed three times with the same washing solution. Finally, DAPI staining was performed to visualize the cell nuclei for direct observation and photography.

[0248] Immunostaining using specific MYOSIN and FACTIN antibodies demonstrated the expression of MYOSIN and FACTIN in differentiated muscle cells derived from bEpiSCs, indicating that initial muscle differentiation was successful ( Figure 4 Middle D).

[0249] 4. As donor cells for genome-edited animal cloning

[0250] Vector construction

[0251] The GFP plasmid was kept in the laboratory. In summary, we modified the PB-CAG-MCS vector (provided by Professor Wu Sen) to generate the PB-CMV-EF1A-GFP-NLS plasmid (disclosed in the article "Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines," and named it PB-CMV-EF1A-GFP-NLS in the article). Specifically, we replaced the chicken β-actin promoter with the human elongation factor 1α (EF1A) promoter and integrated the GFP-NLS downstream of the EF1A promoter.

[0252] bEpiSCs transfection

[0253] Using Lipofectamine TM PB-CMV-EF1A-GFP-NLS plasmid was transfected into E10bEpiSCs using 3000 reagent (Invitrogen, L3000008):

[0254] Transfection was performed 16 hours after passage of E10bEpiSCs in a 24-well plate. First, 25 μL Opti-MEM TM culture medium, and then add 0.75 μL Lipofectamine TM 3000 reagent and mix thoroughly. Then, take another centrifuge tube and add 25μL Opti-MEM TM culture medium, add 0.5 μg PB-CMV-EF1A-GFP-NLS plasmid and 1 μL P3000 TM Then mix the PB-CMV-EF1A-GFP-NLS plasmid mixture with Lipofectamine TM The reagents were mixed at a 1:1 ratio, incubated for 10-15 minutes, and then added to the cells for culture. E10bEpiSCs containing the PB-CMV-EF1A-GFP-NLS plasmid were obtained.

[0255] bGeneration of EpiSCs cloned embryos:

[0256] Ovaries were obtained from cattle farms around Beijing, and oocytes were extracted from 3-8 mm follicles. Oocytes with three layers of cumulus cells were transferred to maturation medium and matured at 38.5°C and 5% CO2 for 18 hours. The maturation medium was based on TCM199 (Gibco, 12340-030) supplemented with 10% FBS (Gibco, 16000-044), 0.01 IU / mL follicle-stimulating hormone (FSH, Sigma, F4021), 0.01 IU / mL luteinizing hormone (LH, Sigma, L6420), and 1 μg / mL estradiol (Sigma, E2257). After 18 hours of oocyte maturation, 0.1% hyaluronidase (Sigma, H4272) was used to remove excess cumulus, and the oocytes with polar bodies were placed in HM medium containing 7.5μg / mL cytochalasin (Sigma, C6762) for 10 minutes and then transferred to HM medium containing 10% FBS for enucleation. The enucleated oocytes were transferred to maturation medium until the injection of the cell nucleus. bEpiSCs were differentiated for more than 1 week in a basal medium containing 10ng / mL BMP4 (PeproTech, 315-27), 5μM SB431542 and 10ng / mL FGF2, and then used as donor cells for nuclear transplantation. Transparent round donor cells were selected and injected into the perivitelline space, so that the cells were as close to the cytoplasm as possible to improve fusion efficiency. The reconstructed embryo was placed between the two electrodes of the fused cells and aligned with the microneedle so that the somatic cells faced one of the two electrodes. Fusion conditions were: double direct current pulses of 2.5 kV / cm, 10 μs, with 1-s intervals; the fusion solution consisted of 0.3 mmol / L mannitol (Sigma, 1375105), 0.15 mmol / L CaCl2 (Sigma, C7902), and 0.15 mmol / L MgCl2 (Sigma, M2393). The fusion rate was assessed under a stereomicroscope. Reconstructed embryos were then transferred to IVC medium containing 5 μM ionomycin (Sigma, 407950) and cultured for 4 minutes, followed by transfer to IVC medium containing 2 mM 6-DMAP (Sigma, D2629) and cultured for 4 hours. Activated embryos were washed three times in IVC medium and transferred to IVC medium for culture.

[0257] Specific steps for embryo transfer and embryo flushing:

[0258] 1. Recipient cattle transplantation operation steps

[0259] 1.1 The recipient cow was given epidural anesthesia between the 1st and 2nd caudal vertebrae and the vulva was wiped.

[0260] 1.2 Re-load the embryos into a 0.25 mL plastic straw, and place the straw into the transplant gun. Place the transplant gun with the straw in a hard jacket, secure it with a plastic ring, and then replace it with a soft jacket.

[0261] 1.3 Transplant the embryo into the upper 1 / 3 to 1 / 2 of the uterine horn on the side with the corpus luteum of the recipient.

[0262] 2. Non-surgical embryo flushing

[0263] 2.1 Main equipment required for non-surgical embryo flushing

[0264] Stereoscopic microscopes: 1-2, embryonic tube (two-way type): 20# embryonic tube for multiparous cattle, 18# embryonic tube for growing cattle, embryonic tube inner core: 64cm in length, cervical dilator, 50mL syringes: 9-10, 20mL syringes: 2, 10mL syringes: 2, 5mL syringes: 2, egg collection funnel: 1, Φ90mm culture dishes: 2 / head, draw 1cm2 squares on the outer bottom of each culture dish, shears.

[0265] 2.2 Drugs required for non-surgical embryo flushing

[0266] Dulbecco's phosphate buffered saline (PBS), alcohol, iodine tincture, chlorhexidine, normal saline, 2% lidocaine, 0.9% normal saline, oxytetracycline.

[0267] 2.3 Non-surgical embryo flushing technical steps

[0268] The donor cattle were restrained in a six-post pen and anesthetized with 2% lidocaine at the junction of the sacral vertebrae and the first or second caudal vertebrae until the tail lost sensation. The dosage of lidocaine was approximately 5-10 mL per head.

[0269] Use a dilator to dilate the cervix, then slowly insert the embryo-forming tube with the inner core into the uterine horn. When the embryo-forming tube reaches the curvature of the uterine horn, withdraw the inner core about 5 cm and then push the embryo-forming tube toward the front of the uterine horn. When the inner core reaches the curvature of the uterine horn again, withdraw it another 5-10 cm until the embryo-forming tube reaches the front of the uterine horn.

[0270] Inflate the balloon of the embryo flushing tube to about 8-12 mL, remove the inner core of the embryo flushing tube, and connect the interface between the embryo flushing tube and the 50 mL syringe.

[0271] Number 8 50mL syringes 1 to 8, draw 50mL of embryo flushing solution into each syringe, and inject one tube of embryo flushing solution into the uterine horn from the embryo flushing tube each time. Then draw all the recovered solution back into the original syringe. Repeat this process for each uterine horn 4 times, using 200mL of embryo flushing solution.

[0272] Use a 50mL syringe to draw 50mL of embryo flushing solution. Pour the recovered solution from the eight 50mL syringes used for embryo flushing into an oocyte collection funnel at room temperature (18°C-22°C) and filter. When approximately 20mL of recovered solution remains in the oocyte collection funnel, shake the funnel and pour it into a 90mm Φ culture dish. Rinse the walls and bottom of the oocyte collection funnel with PBS until no mucus remains. Prepare the culture dish for microscopic examination.

[0273] We transformed the PB-CMV-EF1A-GFP-NLS plasmid into bEpiSCs cells to obtain GFP-bEpiSCs cells.

[0274] Recipient cattle transplantation steps:

[0275] The recipient cows were given epidural anesthesia between the 1st and 2nd caudal vertebrae, and the vulva was wiped.

[0276] GFP-bEpiSCs cells were loaded into a 0.25 mL plastic straw, which was then placed in a transplant gun. The transplant gun, containing the straw, was covered with a hard jacket, secured with a plastic ring, and then covered with a soft jacket.

[0277] The embryo is transferred to the upper 1 / 3-1 / 2 of the uterine horn on the side with the corpus luteum of the recipient.

[0278] The results showed that the embryoid body morphology of PGCLCs formed by bovine bEpiSCs was Figure 4 As shown in Figure E, the gene expression of PGCLCs formed by bovine bEpiSCs. As shown in Figure 4F, the expression of TFAP2C and PRDM1 genes in PGCLCs was significantly increased compared with bEpiSCs. We generated GFP-bEpiSCs ( Figure 4 G), which was used as the donor cell for embryo cloning, and blastocysts of GFP-bEpiSCs clones were obtained ( Figure 4 bThe blastocyst formation efficiency of EpiSCss was comparable to that of fibroblast clones, both reaching approximately 30% ( Figure 4 bEpiSCs can be efficiently established de novo using GFP-bEpiSCs cloned embryos ( Figure 4 These findings suggest that bEpiSCs have potential applications in cell-cultured meat production, gene editing, and animal breeding.

[0279] In this study, we used single-cell transcriptome sequencing to comprehensively analyze gene expression patterns during key developmental stages of the bovine early embryo. Our results revealed a transition from a naive, intermediate, and finally a primed state during early epiblast development in the bovine embryo. Specifically, we observed that genes involved in LIF / STAT3 signaling were downregulated during the initial epiblast (EPI) stage, whereas genes involved in WNT / β-catenin signaling were upregulated during the transition from the intermediate to primed EPI stage. These results further highlight the evolutionary conservation of embryonic development and the regulation of pluripotency in artiodactyls (between cattle and pigs). Using an optimized 3i / LAF culture system, we successfully established bovine epiblast stem cells (bEpiSCss), based on the conserved early embryonic development observed in pigs and cattle. These cells demonstrated long-term stability, maintained a normal karyotype, displayed characteristics of the formative epiblast, expressed pluripotent marker genes, possessed the ability to develop all three germ layers in vitro and in vivo, and displayed typical stem cell pluripotency.

[0280] In this study, bEpiSCs cultured under 3i / LAF conditions exhibited characteristics of forming pluripotency. From the perspective of culture systems, both FGF2 and WNT inhibitors are widely used in the culture of bPSCs other than bEPSCs (where FGF2 is not required for the self-renewal of bEPSCs). The 3i / LAF culture system used in this study showed effective use for the pluripotency and self-renewal of bovine PSCs. Our results indicate that the removal of any small molecules or cytokines is not conducive to the maintenance of bEpiSCs pluripotency, highlighting the applicability of 3i / LAF for preserving the pluripotency of bEpiSCs. Interestingly, bEpiSCs can be derived from a wider range of embryonic stages (E7-E14), resolving the inherent heterogeneity of embryonic discs at different developmental stages through cell colony selection. In addition, the transcript level of NODAL expression in bEpiSCs was relatively higher than that observed in bPSCs previously reported, and it can serve as a marker of the pre-gastrulation ectoderm and play a key role in the formation of mesoderm and endoderm, indicating the similarity between bEpiSCs and intermediate (Formative) ectoderm cells.

[0281] Notably, bovine bEpiSCs and porcine pgEpiSCs displayed remarkably similar clonal morphology, pluripotency characteristics, and transcriptomic profiles. This comprehensive study enhances our understanding of the commonalities and differences in the dynamics of embryonic pluripotency across livestock species. Furthermore, bEpiSCs exhibited robust myogenic differentiation potential, providing a new avenue for advancing cell-based meat production. Furthermore, bEpiSCs can serve as donor cells for genome-edited cloned embryos, offering innovative perspectives for stem cell breeding and the subsequent development of genome-editing technologies.

[0282] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

[0283] References:

[0284] 1. Zhu, G., Gao, D., Li, L., Yao, Y., Wang, Y., Zhi, M., Zhang, J., Chen, X., Zhu, Q., Gao, J., et al. (2023). Generation of three-dimensional meat-like tissue from stable pig epiblast stem cells.Nat.Commun.14.10.1038 / s41467-023-44001-8.

[0285] 2. Zhi, ML, Zhang, JY, Tang, QZ, Yu, DW, Gao, S., Gao, DF, Liu, PL, Guo, JX, Hai, T., Gao, J., et al. (2022). Generation and characterization of stable pigpregastrulation epiblast stem cell lines. Cell Res.32,383-400.10.1038 / s41422-021-00592-9.

[0286] 3.Bray,N.L.,Pimentel,H.,Melsted,P.,and Pachter,L.(2016).Near-optimalprobabilistic RNA-seq quantification.Nat.Biotechnol.34,525-527.10.1038 / nbt.3519

[0287] 4.Cao,J.,Spielmann,M.,Qiu,X.,Huang,X.,Ibrahim,D.M.,Hill,A.J.,Zhang,F.,Mundlos,S.,Christiansen,L.,Steemers,F.J.,et al.(2019).The single-celltranscriptional landscape of mammalian organogenesis.Nature 566,496-502.10.1038 / s41586-019-0969-x.

[0288] 5.Angerer,P.,Haghverdi,L.,Büttner,M.,Theis,F.J.,Marr,C.,and Buettner,F.(2016).diffusion maps for large-scale single cell data inR.Bioinformatics32,1241-1243.10.1093 / bioinformatics / btv715.

[0289] 6.Love,M.I.,Huber,W.,and Anders,S.(2014).Moderated estimation offoldchange and dispersion for RNA-seq data with DESeq2.Genome Biol.15,550.ARTN 550.10.1186 / s13059-014-0550-8.

[0290] 7.Gao,S.,Yan,L.,Wang,R.,Li,J.,Yong,J.,Zhou,X.,Wei,Y.,Wu,X.,Wang,X.,Fan,X.,et al.(2018).Tracing the temporal-spatial transcriptomelandscapes ofthe human fetal digestive tract using single-cell RNA-sequencing.Nat.CellBiol.20,721-734.10.1038 / s41556-018-0105-4.

[0291] 8.Wang,M.,Liu,X.X.,Chang,G.,Chen,Y.D.,An,G.,Yan,L.Y.,Gao,S.,Xu,Y.W.,Cui,Y.L.,Dong,J.,et al.(2018).Single-Cell RNA Sequencing AnalysisRevealsSequential Cell Fate Transition during Human Spermatogenesis.Cell StemCell23,599-614.e4.10.1016 / j.stem.2018.08.007.

Claims

1. A bovine ectodermal stem cell line, characterized in that: The bovine ectodermal stem cell line has the pluripotency of bovine ectodermal stem cells and expresses one or more pluripotency markers, wherein the pluripotency markers are selected from one or more of POU5F1, NANOG, SOX2, CDH1, SSEA1 and SSEA4.

2. The bovine ectodermal stem cell line according to claim 1, characterized in that The bovine ectodermal stem cells have intermediate pluripotency and are capable of stably inheriting at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or more times; Preferably, the bovine ectoderm stem cell line is derived from the ectoderm of bovine embryos at E7-E14; Preferably, the bovine ectoderm stem cell line is derived from the ectoderm of bovine embryos at E10-E12; Preferably, the bovine ectoderm stem cell line is derived from the ectoderm of bovine embryo E10; Preferably, the bovine embryos E7-E14 belong to the EPI lineage.

3. The bovine ectodermal stem cell line according to claim 1, characterized in that Compared with primed bESCs, the bovine ectodermal stem cell line highly expresses one or more of NANOG, LEFTY2 and NODAL genes; Preferably, compared with primed bEDSCs, the bovine ectodermal stem cell line underexpresses one or more of GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, and HES1 genes; Preferably, compared with primed bESCs, the bovine ectodermal stem cell line highly expresses one or more of the FGF4 gene, ZFP42 gene, ETV3L gene, DPPA3 gene, ACVRL1 gene, TDGF1 gene and TBX3 gene; Preferably, compared with primed bEDSCs, the bovine ectodermal stem cell line underexpresses one or more of GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, and HES1 genes.

4. The bovine ectodermal stem cell line according to claim 1, characterized in that Compared with bEDSCs, the bovine ectodermal stem cell line has upregulated expression of genes involved in stem cell proliferation and cell-cell adhesion regulation; Preferably, the stem cell proliferation genes include ELL3 and / or FGF4; Preferably, the cell-cell adhesion regulating genes include one or more of NODAL, FOXA2 and EPCAM; Preferably, compared with bEDSCs, the bovine ectodermal stem cell line also highly expresses one or more of DPPA3, LMO1, ACVRL1 and WNT3A genes; Preferably, compared with bEDSCs, the bovine ectodermal stem cell line also underexpresses one or more of the genes HAND1, GATA3, TGFB2, ZFP42, VMO1, MEIS2, BMP4, IGF1R, CCND2 and SPRY4.

5. The bovine ectodermal stem cell line according to claim 1, characterized in that The bovine ectodermal stem cell line exhibits a higher expression level of LIN28A / B and / or NODAL compared to biPSCs or bEPSCs; Preferably, compared with biPSCs, the bovine ectodermal stem cell line also highly expresses one or more of the following genes: NODAL, LIFR, IL6R, PDGFRA, ACVR1B, ETS1, IL6ST, and SALL4; Preferably, compared to biPSCs, the bovine ectodermal stem cell line also underexpresses one or more of the following genes: GDF15, BCL3, CD44, ETV2, FGFR4, KLF15, GDF1, BMP4, COX17, and MYC; Preferably, compared with bEPSCs, the bovine ectodermal stem cell line also highly expresses one or more of LIFR, ESRRB, IL6R, ACVR1B, KLF5, SALL4, and CDH1 genes; Preferably, compared with bEPSCs, the bovine ectodermal stem cell line also underexpresses one or more of MSC, GCK, TGFB2, MAPK15, KLF15, GDF1, ID4, FOS, and COX17 genes.

6. The bovine ectodermal stem cell line according to claim 1, characterized in that The bovine ectodermal stem cell line relies on the activation of the FGF / ERK and / or TGFβ / SMADs signaling pathway and the inhibition of the WNT / β-catenin signaling pathway; Preferably, the FGF / ERK signaling pathway related genes include one or more of MAPK1, MAPK14, FGF2, PDGFA, FGFR1 and FGFR2; Preferably, the TGFβ / SMADs signaling pathway-related genes include one or more of INHBA, NODAL, ACVR2A / 2B, BMP4 and BMPR1A / 1B; Preferably, the WNT / β-catenin signaling pathway related genes include one or more of TCF7, APC, WNT11, CTNNB1, FZD2 and WNT3A.

7. A culture medium for culturing bovine ectodermal stem cell lines, characterized in that: The culture medium includes a basal culture medium and additional components; Preferably, the basal culture medium includes DMEM / F12 culture medium and / or Neurobasal culture medium; More preferably, the mass ratio or volume ratio of the DMEM / F12 medium to the Neurobasal medium is 1:1; Preferably, the basic culture medium further comprises one or more small molecule inhibitors or cytokines selected from the group consisting of: CHIR99021, IWR-1-endo, WH-4-023, recombinant human LIF, recombinant human Activin A and recombinant human FGF-basic (154aa), ROCK inhibitor Y-27632, or any combination thereof.

8. A method for preparing a bovine ectodermal stem cell line in vitro, characterized in that: The method comprises the steps of culturing one or more ectoderm cells isolated from bovine embryos E10-E14 in the culture medium of claim 7; Preferably, the bovine embryos E10-E14 belong to the EPI lineage; Preferably, the bovine ectoderm stem cell line is derived from the ectoderm of bovine embryos at E10-E12; Preferably, the bovine ectoderm stem cell line is derived from the ectoderm of bovine embryo E10.

9. Use of the bovine ectodermal stem cell line according to any one of claims 1 to 6 or the bovine ectodermal stem cell line prepared by the method according to claim 8 in inducing the generation of muscle cells or providing donor cells or cell nuclei for nuclear transplantation.

10. A method for preparing muscle cells, characterized in that: The method comprises the step of culturing the bovine ectoderm stem cell line according to any one of claims 1 to 6 or the bovine ectoderm stem cell line prepared by the method according to claim 8 in a myogenic culture medium to obtain muscle cells.

11. A method for preparing bovine primordial germ cell-like cells, characterized in that: The method comprises the steps of culturing the bovine ectoderm stem cell line according to any one of claims 1 to 6 or the bovine ectoderm stem cell line prepared by the method according to claim 8 in a PGC induction culture system to obtain bovine primordial germ cell-like cells; Preferably, compared with the bovine ectoderm stem cell line, the bovine primordial germ cell-like cells highly express the TFAP2C gene and the PRDM1 gene.

12. A bovine nuclear transplantation method, characterized in that: The method comprises the steps of culturing the bovine ectoderm stem cell line described in any one of claims 1 to 6 or the bovine ectoderm stem cell line prepared by the method of claim 8 as a nuclear transplant donor cell nucleus or nuclear transplant donor cell to obtain bovine cells, tissues, organs, or complete individuals.

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