CN120683038A

CN120683038A - A bovine ectoderm stem cell line and its establishment method and application

Info

Publication number: CN120683038A
Application number: CN202510339999.0A
Authority: CN
Inventors: 韩建永; 郅明雷; 麻柱
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2024-03-21
Filing date: 2025-03-21
Publication date: 2025-09-23
Also published as: CN120683259A

Abstract

The invention discloses a bovine ectodermal stem cell line, and an establishment method and application thereof. The bovine ectodermal stem cell line has the pluripotency of a bovine intermediate stem cell line and expresses one or more pluripotency markers, is capable of stable passage, retains the multipotent transcriptome characteristics and normal karyotype similar to intermediate (Formative) ectodermal cells after more than 112 passages, and has the potential to differentiate into three germ layers. The bovine intermediate stem cell line has potential for myogenic differentiation, primordial germ cell-like cell differentiation and as Somatic Cell Nuclear Transfer (SCNT) donor cells, which indicates that they have broad application prospects in promoting cell culture meat production and animal breeding.

Description

Cattle ectoderm stem cell line and establishment method and application thereof

The present application claims priority from China patent office, application No. 2024103257603, application name "a bovine intermediate stem cell line, in vitro preparation method and use thereof", filed on 21, 2024, 03, which is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the field of stem cell biology, in particular to a bovine ectodermal stem cell line, and an establishment method and application thereof.

Background

Embryonic ectodermal cells can differentiate into intact fetuses and serve as an important source of pluripotent stem cell lines (pluripotent STEM CELLS, PSCs). PSCs derived from ectoderm at different developmental stages exhibit different pluripotent states, which can be specifically classified as primitive statesIntermediate state (Formative) or originating state (Primed). Human and mouse PSCs have been widely used in the related fields of embryo development, directed differentiation, and disease modeling.

Stable livestock PSCs not only helps to understand embryo development and pluripotency of livestock, but is also the best cell source for animal breeding and cultured meat production. However, the development of stable pluripotent stem cell lines in large animals is slow due to species specificity. Recently, porcine orthointestinal metadermal stem cells. And their potential use in various fields has attracted a great deal of attention. However, studies on bovine embryogenesis and PSCs molecular basis have far fallen behind pigs, mice, humans and non-human primates. Establishing exoembryo-derived pluripotent stem cells from cattle (pluripotent STEM CELLS, PSCs) is challenging because the molecular basis for bovine embryo development and pluripotent stem cell self-renewal is not clear. Therefore, the method for further tracking the bovine embryonic stem cell lines at different development stages has important practical application value.

Disclosure of Invention

The invention aims to solve the technical problem of providing a bovine ectodermal stem cell, and an in-vitro preparation method and application thereof. Stable bovine ectodermal stem cell lines were successfully established with the optimized 3i/LAF culture system. The research provides valuable high-quality seed cells and new ways for researching the pluripotency of the livestock stem cells, and simultaneously advances the research of stem cell related breeding and cell culture meat production.

In a first aspect, the present invention provides a bovine ectodermal stem cell line characterized in that said bovine ectodermal stem cell line has the pluripotency of bovine ectodermal stem cells and expresses one or more pluripotency markers capable of stable passaging

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 or more times.

In certain embodiments, the pluripotency marker is selected from one or more of POU5F1, NANOG, SOX2, CDH1, SSEA1, and SSEA 4;

in certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E7-E14;

In certain embodiments, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10-E12;

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

in certain embodiments, the E7-E14 belongs to the EPI lineage.

In certain embodiments, the EPI lineage highly expresses one or more of TDGF1, POU5F1, and NANOG.

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.

In certain embodiments, the bovine ectodermal stem cell line highly expresses one or more of NANOG, LEFTY2, and NODAL genes compared to bESCs in the original state (Primed);

In certain embodiments, the bovine ectodermal stem cell line expresses one or more of the GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, HES1 genes low compared to bEDSCs in the original state (Primed).

In certain embodiments, 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 compared to bESCs in the original state (Primed);

in certain embodiments, the bovine ectodermal stem cell line has up-regulated gene expression that is involved in stem cell proliferation and intercellular adhesion regulation compared to bEDSCs;

In certain embodiments, the bovine ectodermal stem cell line also expresses one or more of the HAND1, GATA3, TGFB2, ZFP42, VMO1, MEIS2, BMP4, IGF1R, CCND2, and SPRY4 genes low compared to bEDSCs.

In certain embodiments, the stem cell proliferating gene comprises ELL3 and/or FGF4;

In certain embodiments, the genes that modulate intercellular adhesion comprise one or more of NODAL, FOXA2, and EPCAM;

In certain embodiments, the bovine ectodermal stem cell line also highly expresses one or more of DPPA3, LMO1, actrl 1, and WNT3A genes compared to bEDSCs.

In certain embodiments, the bovine ectodermal stem cell line exhibits a higher LIN28A/B and/or NODAL expression level as compared to biPSCs or bEPSCs;

In certain embodiments, the bovine ectodermal stem cell line also highly expresses one or more of the NODAL, LIFR, IL6R, PDGFRA, ACVR1B, ETS1, IL6ST, SALL4 genes compared to biPSCs;

In certain embodiments, the bovine ectodermal stem cell line also low expresses one or more of GDF15, BCL3, CD44, ETV2, FGFR4, KLF15, GDF1, BMP4, COX17, and MYC genes compared to biPSCs. In certain embodiments, the bovine ectodermal stem cell line also highly expresses one or more of LIFR, ESRRB, IL6R, ACVR1B, KLF5, SALL4, CDH1 genes compared to bEPSCs.

In certain embodiments, the bovine ectodermal stem cell line also expresses one or more of MSC, GCK, TGFB, MAPK15, KLF15, GDF1, ID4, FOS, COX17 genes low compared to bEPSCs. In certain embodiments, the bovine ectodermal stem cell line is dependent on activation of FGF/ERK and/or tgfβ/SMADs signaling pathway and inhibition of WNT/β -catenin signaling pathway;

In certain embodiments, the FGF/ERK signaling pathway-related genes include one or more of MAPK1, MAPK14, FGF2, PDGFA, FGFR1, and FGFR 2;

in certain embodiments, the TGF-beta/SMADs signaling pathway-related genes include one or more of INHBA, NODAL, ACVR A/2B, BMP4 and BMPR 1A/1B;

in certain embodiments, the WNT/β -catenin signaling pathway-related genes include one or more of TCF7, APC, WNT11, CTNNB1, FZD2, and WNT 3A.

In another aspect, the invention discloses a culture medium for culturing bovine ectodermal stem cell lines, characterized in that the culture medium comprises a basal medium and an additive component;

In certain embodiments, the basal medium comprises DMEM/F12 medium and/or Neurobasal medium;

in certain embodiments, the DMEM/F12 medium and the Neurobasal medium are in a mass ratio or volume ratio of 1:1;

In certain embodiments, 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 (154 aa), ROCK inhibitor Y-27632, or any combination thereof are also included in the basal medium;

in certain embodiments, the CHIR99021 is used at a concentration of 1 μm;

in certain embodiments, the IWR-1-endo is used at a concentration of 0.5. Mu.M;

in certain embodiments, the WH-4-023 is used at a concentration of 1. Mu.M;

in certain embodiments, the recombinant human LIF is used at a concentration of 10ng/mL;

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

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

In certain embodiments, the ROCK inhibitor Y-27632 is used at a concentration of 5 μm.

Also included in the basal medium is 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 (154 aa) (10 ng/mL, peproTech, 100-18B), ROCK inhibitor Y-27632 (5 μm, SELLECKCHEM, S1049), or any combination thereof.

The added components in the medium included 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％ nonessential amino acids (Thermo FISHER SCIENTIFIC, 11140-050), 0.1mM beta-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).

In another aspect, the present invention discloses a method for the in vitro preparation of bovine ectodermal stem cell lines, characterized in that said method comprises the step of culturing individual embryonic cells isolated from the embryos of bovine E10-E14 in the above-mentioned medium;

in certain embodiments, the E7-E14 belongs to the EPI lineage.

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

In another aspect, the invention discloses the use of a bovine ectodermal stem cell line as defined in any one of the above or a bovine ectodermal stem cell line prepared by the above method for inducing the production of muscle cells or providing a nuclear transfer donor cell.

In another aspect, the invention discloses a method of preparing a muscle cell, the method comprising the step of culturing a bovine ectodermal stem cell line as defined in any one of the above or a bovine ectodermal stem cell line prepared by the above method in myogenic medium to obtain a muscle cell.

In another aspect, the invention discloses a method for preparing bovine primordial germ cell-like cells, which is characterized in that the method comprises the steps of culturing the bovine ectodermal stem cell line as defined in any one of the above or the bovine ectodermal stem cell line prepared by the above method in a PGC induction culture system to obtain bovine primordial germ cell-like cells;

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

In another aspect, the present invention discloses a bovine nuclear transfer method comprising the step of culturing a bovine ectodermal stem cell line as described above or prepared by any one of the methods as a nuclear transfer donor cell or a nuclear transfer donor cell to obtain bovine cells, tissues, organs, whole individuals.

The beneficial effects are that:

This study uses single cell transcriptome sequencing to demonstrate the similarity of bovine and porcine in early embryonic development and multipotent 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 the present application maintains the pluripotent transcriptome characteristics and normal karyotype similar to intermediate (Formative) ectodermal cells after more than 112 passages and has the potential to differentiate into three germ layers. The present application further evaluates bEpiSCs's potential for myogenic differentiation, primordial germ-like cell differentiation, and as a Somatic Cell Nuclear Transfer (SCNT) donor cell. The research provides valuable high-quality seed cells and new ways for researching the pluripotency of the livestock stem cells, and simultaneously advances the research of cell culture meat production and stem cell related breeding.

Drawings

FIG. 1 shows the generation and characteristics of bEpiSCs.

A, establishing bEpiSCs strategy.

B growth efficiency of bovine embryos and cell lines at different stages in 3i/LAF medium.

Morphology of growths (up) and bEpiSCs (down), white arrows indicate morphology of selected and passaged bEpiSCs colonies. Scale bar, 200 μm.

And D, bEpiSCs population doubling time.

E bEpiSCs monoclonal efficiency.

F bEpiSCs Alkaline Phosphatase (AP) staining test. Scale bar, 100 μm.

And G bEpiSCs, performing karyotyping. For each cell line, 45 metaphase cells were examined.

Immunostaining of the pluripotency markers POU5F1, NANOG and SOX2 in H bEpiSC. DAPI was used for nuclear staining. Scale bar, 100 μm.

Immunostaining of the pluripotent surface markers CDH1, SSEA1 and SSEA4 in bEpiSCs. DAPI was used for nuclear staining. Scale bar, 50 μm.

J, in vitro EB differentiation experiment. Immunostaining of ectodermal nerve-specific marker protein β -III-Tubulin, mesodermal muscle-specific marker protein α -SMA, and endodermal-specific marker protein SOX 17. DAPI was used for nuclear staining. Scale bar, 100 μm.

K: teratoma formation test in vivo. Hematoxylin and eosin (H & E) staining of bEpiSCs teratomas. Scale bar, 50 μm.

For D and E, error bars represent ± SD (n=3 independent experiments), ns. P is more than or equal to 0.05. Similar results were obtained in three independent experiments for (C) and (F-K).

FIG. 2 is that 3i/LAF is critical for long-term subculture of bEpiSCs.

A clone morphology and AP staining were observed with a 3i/LAF group as a control, treated with a cofactor treatment bEpiSCs. Scale bar, 200 μm. And B, analyzing the clone sizes of bEpiSCs culture systems after the factor-reducing treatment. More than 30 clones were counted per treatment group. Real-time quantitative PCR analysis of differences in gene expression in group 3I/LAF and group bEpiSCs I/F.

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

F, comparing the mRNA expression level representing the pluripotency and lineage marker genes in FGF2 deleted group and 3i/LAF group using qRT-PCR. G. mRNA expression levels of representative pluripotency and BMP signaling pathway marker genes in the Activin A deleted group and 3i/LAF group were compared using qRT-PCR method.

MRNA expression levels representing pluripotency and JAK/STAT3 signaling pathway marker genes in LIF deleted and 3i/LAF groups were compared using qRT-PCR.

For (B-H), error bars represent ± SD (n=3 independent experiments), ns. P is greater than or equal to 0.05, P is less than 0.01, P is less than 0.001, and P is less than 0.0001. Similar results were obtained in three separate experiments for (A-H), see FIG. 5.

FIG. 3 is bEpiSCs transcriptomic analysis.

PCA panels show the distribution of scRNA-seq data obtained from bovine embryonic lineage cells and bEpiSCs, based on different embryonic lineages and bEpiSC lines, with color-coded clusters.

Bovine embryonic lineage cells and bEpiSCs were subjected to Spearman correlation analysis. The color gradient from blue to red indicates a gradual increase in correlation from low to high.

Violin plot shows bovine embryo pedigree and in bEpiSCs from different embryo stagesFormative and primed pluripotency represent the expression level of the gene.

PCA plots were generated to compare bEpiSCs to the publicly established Primed bESCs, bEDSCs, bEPSCs, and biPSCs 18, with each data point representing a different cell line.

Differentially expressed genes were identified between bEpiSCs and published bPSCs (DEGs). By comparison, red bars represent genes up-regulated in bEpiSCs and green bars represent genes down-regulated in bEpiSCs, compared to bPSCs as published.

The scatter plot shows bEpiSCs and published comparison of average gene expression levels between bPSCs, orange highlighting up-regulated genes, blue highlighting down-regulated genes. Appropriate annotation of the key genes is shown in FIG. 6.

Fig. 4 is a potential application of bEpiSCs.

And A is bEpiSC application prospect in a model diagram.

BEpiSCs group morphology at different stages of myogenic differentiation. Scale bar, 200 μm.

And C, quantitatively determining mRNA expression of the myogenic differentiation related gene by qRT-PCR.

Immunostaining of muscle cell-associated proteins in myogenic differentiation bEpiSC. Scale bar, 200 μm.

E PGCLCs embryoid body morphology formed by cattle bEpiSCs.

F-PGCLCs Gene expression by cattle bEpiSCs.

G, cloning morphology of GFP-bEpiSCs, scale bar, 50. Mu.m.

GFP-bEpiSCs clone embryo morphology, scale bar, 100. Mu.m.

BEpiSCs Nuclear transfer cloning efficiency statistics.

J, cloning morphology of bovine cloned embryo neonatal GFP-bEpiSCs, scale bar, 200 μm.

For C, F, error bars represent ± SD (n=3 independent experiments), P <0.0001. Similar results were obtained in three independent experiments for (B-H).

FIG. 5 is a culture modification of bEpiSCs, related to FIG. 2.

Cloning morphology and AP staining of bEpiSCs treated with CHIR (C) and IWR (I) at different concentrations, concentration units, μM, scale bar, 200 μM.

B, quantitative expression of pluripotency and lineage marker genes, C (CHIR) and I (IWR), concentration units, μM by qRT-PCR.

The morphology of bEpiSCs clones treated with different concentrations of IWP2 and XAV939, scale bar, 200 μm. mRNA expression levels of bEpiSCs pluripotent and lineage specific marker genes cultured at different concentrations of IWP2 and XAV939 were quantitated. For B and D, error bars represent ± SD (n=3 independent experiments), ns. P is greater than or equal to 0.05, P is less than 0.01, P is less than 0.001, and P is less than 0.0001. Similar results were obtained in three independent experiments for (A-D).

Fig. 6 is a comparative analysis of bEpiSC with published bPSC and pig pgEpiSC, related to fig. 3.

The gene ontology and KEGG pathways were determined by pairwise comparison of bPSCs.

A scatter plot was generated to compare the average gene expression levels between bEpiSCs and pig pgEpiSCs, with orange highlighting up-regulated genes and blue highlighting down-regulated genes. Appropriate annotations were made for the key genes.

Gene ontology and KEGG pathway enrichment of differentially expressed genes in bEpiSCs and pig pgEpiSCs.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions thereof will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, which should not be construed as limiting the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In the description of the present invention, it is to be understood that the terminology used is for the purpose of description only and is not to be interpreted as indicating or implying relative importance.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

All the mouse experiments and dairy cow experimental procedures in the following examples were previously approved by the ethical review Committee of animal experiments, animal welfare and animal experiments, and approval number AW10204202-3-1.

A mouse

(ICR) IGS and BALB/c nude mice were purchased from Beijing VITAL RIVER laboratory animal technologies Inc. for Mouse Embryo Fibroblast (MEFs) isolation and bEpiSCs teratoma formation assays. MEFs were treated with mitomycin C (SELLECKCHEM, S8146) to prepare bEpiSCs feeder cells.

Cattle

Bovine embryo single cell collection and single cell transcriptome analysis were performed using the Holstein bovine embryos at E5, E6, E7, E10, E12 and E14. Embryo day n (E (n)) embryos were obtained n days after mating. For bEpiSCs derivatization, E7, E10, E12 and E14 embryos were used.

Bovine embryo collection and embryo single cell separation

The embryonic cells used in this study were derived entirely from Holstein cow embryos. E5-E7 embryos are obtained by thawing and culturing frozen bovine embryos until the desired developmental stage is reached. The zona pellucida was treated with Pronase (Sigma, 10165921001) for 15-30 seconds and then washed and removed using a solution of dpbs+0.1% bsa. Subsequently, the embryonic cells are mechanically isolated and transferred to a lysate. E10, E12 and E14 embryos were obtained by in vivo transplantation and subsequent rinsing, and individual embryo cells were isolated and collected by a combination of enzymatic treatment and mechanical manipulation.

Bovine ectoderm stem cell culture medium

The composition of the bovine ectodermal stem cell culture system is the same as that of the previously reported 3i/LAF culture system, and the content of each composition component is correspondingly optimized. Specifically, the 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) with the addition of 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), 0.1mM beta-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). To prepare a 3i/LAF culture system, it is also necessary to add to BM the following small molecule inhibitors or cytokines ：CHIR99021(1μM,Selleckchem,S1263)、IWR-1-endo(0.5μM,Selleckchem,S7086)、WH-4-023(1μM,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 (154 aa) (10 ng/mL, peproTech, 100-18B). After optimization, the ROCK inhibitor Y-27632 (5 μm, SELLECKCHEM, S1049) was added. In addition, XAV939 (1. Mu.M, SELLECKCHEM, S1180) may be substituted for IWR-1-endo. bEpiSCs was incubated with mitomycin C treated mouse fibroblasts.

In the present application, E refers to Embryonic DAY, i.e., the number of days of development of fertilized eggs. Typically, the day of fertilization is E0, and so on. Namely, E7, E10, E12 and E14 refer to 7 days, 10 days, 12 days and 14 days, respectively, of fertilized egg development.

Experimental model and study participant detailed information

Material availability

All bEpiSCs generated in this study were available from the primary contact and provided a complete material transfer protocol.

Data and code availability

The scRNA-seq, RNA-seq dataset generated during this study was obtained at Gene Expression Omnibus (GEO) with the accession code GSE256201.

Animal treatment and ethical statement

All the mouse experiments and dairy cow experiment procedures are approved by the ethical examination committee of animal welfare and animal experiments of Chinese agricultural university in advance, and the approval number AW10204202-3-1.

A mouse

Cattle

Bovine embryo collection and embryo single cell separation

The embryonic cells used in this study were derived entirely from Holstein cow embryos. E5-E7 embryos are obtained by thawing and culturing frozen bovine embryos until the desired developmental stage is reached. The zona pellucida was treated with Pronase (protease Pronase, sigma, 10165921001) for 15-30 seconds, then washed and removed using a solution of dpbs+0.1% bsa. Subsequently, the embryonic cells are mechanically isolated and transferred to a lysate. E10, E12 and E14 embryos were obtained by in vivo transplantation and subsequent rinsing, and individual embryo cells were isolated and collected by a combination of enzymatic treatment and mechanical manipulation.

Bovine ectoderm stem cell culture medium

The composition of the bovine ectodermal stem cell culture system is the same as that of the previously reported 3i/LAF culture system ², and the content of each component is correspondingly optimized. Specifically, the 3i/LAF medium (BM) was a 1:1 mixture of DMEM/F12 medium (Thermo FISHER SCIENTIFIC, 10565-018) and Neurobasal medium (Thermo FISHER SCIENTIFIC, 21103-049) with the addition of 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), 0.1mM beta-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. Mu.g/mL ascorbic acid (Sigma-Aldrich, A4544). To prepare a 3i/LAF culture system, it is also necessary to add to BM the following small molecule inhibitors or cytokines ：CHIR99021(1μM,Selleckchem,S1263)、IWR-1-endo(0.5μM,Selleckchem,S7086)、WH-4-023(1μM,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 (154 aa) (10 ng/mL, peproTech, 100-18B). After optimization, the ROCK inhibitor Y-27632 (5 μm, SELLECKCHEM, S1049) was added. In addition, XAV939 (1. Mu.M, SELLECKCHEM, S1180) may be substituted for IWR-1-endo. bEpiSCs was incubated with mitomycin C treated mouse fibroblasts.

Derivatization of bovine ICMs, ectoderms and ectoderms bEpiSCs

Bovine ICMs, epiblast and ectoderm were isolated by mechanical separation and treated with TrypLE ^TM Express (a non-animal derived recombinase that is used primarily to dissociate various adherent mammalian cells, gibco, 12605010) for 3 minutes and then plated onto feeder cells supplemented with 3i/LAF medium. Cultures were incubated at 37 ℃ at 5% o2 and 5% co 2. Spherical growths were collected by digestion with Acceutase cell dissociation reagent (Gibco, A11105-01) and passaged every 3 days at a 1:4 ratio.

Experimental method

Single cell RNA library preparation and sequencing

Single cell RNA-seq libraries were prepared using the modified Smart-seq2 protocol as previously described in study ^7,8. Briefly, individual embryonic cells were transferred to lysis buffer containing an 8bp barcode. Subsequently, the first strand cDNA was reverse synthesized and amplified in a Reverse Transcription (RT) mixture consisting of 4U RNase inhibitor, 100U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1mM dNTPs (TAKARA, 4019), 60mM MgCl ₂ and 3. Mu.M RT primer with 10. Mu.M TSO primer. After PCR amplification, the product was purified using 0.8XBeckman's AMPure XP beads (A63882). Biotin PCR enrichment was then performed to further improve library quality. Finally, a single cell RNA-seq library was constructed according to the instructions provided by the KAPA PCR library amplification/Illumina series (KAPA KK 8054). High quality libraries were sequenced using Illumina HiSeq Xten platform (Novogene) with paired end reads 150bp in length. The primers used in the experiments are listed in the key resource table.

Cell population doubling time

BEpiSCs was seeded in 12 well plates at a density of 3 x 10 ⁵ and a growth curve of bEpiSCs was drawn. Cells were then digested and counted at 12, 24, 36, 48 and 60 hour intervals using a Luna ^TM automated cell counter. Three replicates were performed for each time point. Doubling time was calculated using the following formula Doubling Time (DT) =12× [ lg 2/(lgN _t-lgN₀) ], where 12 was the cell culture time (hours), N _t was the number of cells cultured at 48 hours, and N ₀ was the number of cells recorded at 36 hours.

Single cell cloning efficiency analysis

Bovine EpiSCs were digested with TrypLE ^TM Express (a non-animal derived recombinase that is used primarily to dissociate various adherent mammalian cells Gibco, 12605010) and filtered through a 40 μm cell filter. Cells were seeded in 6-well plates with 100, 500 and 1000 cells, respectively. After 3 days of culture, the number of clones formed was counted, and the single cell clone formation rate was calculated and averaged.

Nuclear analysis

Prior to karyotyping, 1%KaryoMAX Colcemid solutions (mitotic inhibitors, mainly used in cell culture experiments, capable of arresting cells in the metaphase of mitosis, gibco, 15212012) were added to bEpiSCs medium and incubated for 1 hour. bEpiSC was then dissociated into single cells using TrypLE ^TM Express (a non-animal derived recombinase that is used primarily to dissociate various adherent mammalian cells, gibco, 12605010) and collected by centrifugation. Subsequently bEpiSCs was suspended in a hypotonic solution of 0.075M KCl (Sigma, P5405) and incubated at 37℃for 15 minutes. After this step bEpiSCs was fixed with methanol and acetic acid in a 3:1 ratio, and this procedure was repeated three times. The resulting bEpiSCs 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 examined per cell line.

Alkaline Phosphatase (AP) staining

For details on AP staining (AP is a Pluripotent Stem Cell (PSC) phenotype marker, including undifferentiated Embryonic Stem Cells (ESC), induced Pluripotent Stem Cells (iPSC) and Embryonic Germ Cells (EGC), which have the ability to self-renew and differentiate into all three germ layers (ectodermal, mesodermal and endodermal) procedures and notes, please refer to alkaline phosphatase assay kit (Millipore, SCR 004).

Immunofluorescence analysis

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 min. After another round of DPBS washing, cells were blocked with 3% bsa for 1 hour at room temperature. The primary antibody was incubated overnight at 4℃and then washed three times with a wash 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 liquid. Finally, DAPI (nuclear dye DAPI) staining was performed to visualize the nuclei, thereby achieving direct observation and photography. The antibodies of the present application are shown in Table 4.

TABLE 4 Table 4

Embryoid body differentiation

Following digestion bEpiSCs was seeded at a density of 1X 10 ⁶ cells per well and cultured in 35mm low attachment plate MEF medium, 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) at 70rpm on a horizontal shaker for 5-7 days. Embryoid Bodies (EBs) were then transferred to 12-well plates and incubated in the same medium for one week with medium changes twice daily. Immunofluorescent staining was then performed with adherent cells.

Teratoma formation

1X 10 ⁷ bEpiSCs prepared by centrifugation collection after digestion was resuspended in 50. Mu.L BM and subsequently subcutaneously injected into the neck of BALB/c nude mice. After 4-5 weeks, teratomas were collected and subjected to subsequent H & E analysis.

H & E analysis

Teratomas were washed twice in DPBS and fixed with 4% pfa for 2 days at a temperature of 4 ℃. Subsequently, teratoma tissues were dehydrated with an alcoholic gradient (70%, 80%, 90%, 95%, finally 100% per hour) and then transferred to xylene and embedded in paraffin. The samples were sectioned to 5 μm thickness, then dewaxed in xylene and rehydrated using reduced concentration ethanol. Finally, the samples were stained with hematoxylin (blue staining of chromatin in the nucleus and nucleic acid in the cytoplasm, sigma-Aldrich, MHS 16) and eosin (red staining of components in the cytoplasm and extracellular matrix, sigma-Aldrich, HT 110116) and then observed under a microscope (Leica, DM 5500B).

RT–qPCR

Total RNA was extracted from bEpiSCs using RNA prep Pure Cell/bacterioria kit (TIANGEN, DP 430) and then reverse transcribed into cDNA using 5×all-In-One RT Master Mix (Abm, G490). Subsequently, PCR amplification was performed on ARCHIMED REAL TIME SYSTEM (ROCGENE) using 2X REALSTAR GREEN Power mix (GenStar, A311-05). The data were analyzed using the comparative CT (2 ^-ΔΔCT) method. The Δct value was calculated using GAPDH as an internal reference. All experiments were performed in triplicate with independent biological replicates. Primer sequences for real-time PCR can be found in the key resource table.

BEpiSCs myogenic differentiation

The protocol was identical to pig pgEpiSCs ¹. Briefly, myogenic Differentiation Basal Medium (MDBM) consisted of DMEM/F12, 1% non-essential amino acids, 0.1mM beta-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR and 200. Mu.M ascorbic acid. In the first stage bEpiSCs was separated into small pieces and incubated for 3 days in MDBM with 1% b27 supplement, 3 μm CHIR99021 and 2 μm SB431542 (SELLECKCHEM, S1067). In the second phase, from day 4 to day 6, the medium was changed to a combination containing 3. Mu.M CHIR99021, 2. Mu.M SB431542, 500nm LDN193189 (Stemgent, 04-0074) and 20ng/mL recombinant human FGF-basic (154 a.a.). For the third stage, the broth was incubated with 10ng/mL HGF (Peprotech, 100-39H), 10ng/mL IGF-1 (Peprotech, 100-11), 20ng/mL recombinant human FGF-basic (154 a.) and 0.5. Mu.M LDN193189 for 2 days. In the fourth stage bEpiSCs begins to differentiate into myoprecursor cells. bEpiSCs-MPCs were treated with IGF-1 at a concentration of 10ng/mL for 4 days, and in the fifth stage, a combination of 10ng/mL HGF and 10ng/mL IGF-1 was used for 20 to 25 days to promote skeletal muscle maturation. For skeletal muscle maturation, cells were treated with N2 medium consisting of DMEM/F12, N2 medium consisting of DMEM/F12 supplemented with 15% KOSR, 1% N2 supplement, 1% penicillin-streptomycin and 1% non-essential amino acids.

RRNA-seq (rRNA has a high ratio in total RNA and usually does not contain useful transcriptome information), removal of rRNA can improve sequencing efficiency, reduce sequencing cost, and improve data quality

Total RNA was extracted from four bEpiSCs samples using RNEASY MINI kit (Qiagen, 74106), respectively. To construct a strand-specific RNA-seq library, we used the rRNA removal protocol (Global-Zero Gold rRNA Removal Kit, illumina, GZG 1224), bindingUltra^TMDirectional RNA Library Prep Kit for(NEB, E7420S) to operate. All libraries were quantified using the Qubit dsDNA high sensitivity assay kit (Invitrogen, life Technologies, Q32851) and sequenced on Illumina HiSeq 4000 platform.

Vector construction

GFP plasmid was stored in the laboratory. In summary, we generated PB-CMV-EF1A-GFP-NLS plasmid by modifying the PB-CAG-MCS vector (provided by Wu Sen teachings). Specifically, we replaced the chicken β -actin promoter with a human elongation factor 1α (EF 1A) promoter and integrated GFP-NLS downstream of the EF1A promoter.

BEpiSCs transfection

Bovine EpiSCs were transfected with Lipofectamine ^TM reagent (Invitrogen, L3000008). Specifically, transfection was performed after 16 hours of passage of normal cells in 24-well plates. First, 25. Mu.L of Opti-MEM ^TM medium was added to the centrifuge tube, followed by 0.75. Mu.L of Lipofectamine ^TM 3000 reagent and thoroughly mixed. Then, another centrifuge tube was taken, 25. Mu.L of Opti-MEM ^TM medium was added, 0.5. Mu.g of DNA and 1. Mu. L P3000 ^TM reagent were added, and mixed well. The DNA mixture was then mixed with Lipofectamine ^TM reagent in a 1:1 ratio, incubated for 10-15 minutes, and then cells were added for culture.

Production of bEpiSCs cloned embryos

Ovaries were obtained from cattle farms around Beijing, and oocytes in 3-8mm follicles were retrieved. Oocytes with three layers of cumulus cells were transferred into maturation medium and matured for 18 hours at 38.5 ℃ and 5% co ₂. Maturation medium was based on TCM199 (Gibco, 12340-030), 10% FBS (Gibco, 16000-044), 0.01IU/mL follicle stimulating hormone (FSH, sigma, F4021), 0.01IU/mL luteinizing hormone (LH, sigma, L6420), 1. Mu.g/mL estradiol (Sigma, E2257). After 18 hours of oocyte maturation, excess cumulus was removed using 0.1% hyaluronidase (Sigma, H4272), the polar oocyte was placed in HM medium containing 7.5. Mu.g/mL cytochalasin (Sigma, C6762) for 10 minutes, and then transferred to HM medium containing 10% FBS for enucleation. The enucleated oocytes are transferred into maturation medium until the nuclei are injected. bEpiSCs was differentiated in basal medium containing 10ng/mL BMP4 (PeproTech, 315-27), 5. Mu.M SB431542 and 10ng/mL FGF2 for more than 1 week, and then subjected to nuclear transfer as donor cells. Transparent circular donor cells were selected and injected into the perioval space to bring the cells as close as possible to the cytoplasm to increase fusion efficiency. The reconstructed embryo is placed between two electrodes of the fused cell and aligned with the microneedle such that the somatic cell is facing one of the two electrodes. The conditions for the fusion were double DC pulses of 2.5kV/cm,10 μs at 1s intervals, 0.3mmol/L mannitol (Sigma, 1375105), 0.15mmol/LCaCl ₂ (Sigma, C7902) and 0.15mmol/L MgCl ₂ (Sigma, M2393). The fusion rate was examined under a split microscope. The recombinant embryos were then transferred to IVC medium containing 5. Mu. M ionomycin (Sigma, 407950) for 4 min and then transferred to IVC medium containing 2mM 6-DMAP (Sigma, D2629) for 4 h. The activated embryos were washed three times in IVC medium and transferred to IVC medium for culturing.

Quantitative statistical analysis

Single cell RNA-seq Low level processing and filtration

For the STRT-seq dataset, the Raw reads were split by an 8bp cell barcode located on Read 2, allowing 2 mismatches. In addition, the 8bp Unique Molecular Identifier (UMIs) located on Read 2 is switched onto the paired Read 1 identification line. Read 1 is then treated to remove Template Switching Oligonucleotide (TSO) primers, low quality bases, and polyA sequence ⁵¹. The trimmed reads were aligned with the respective reference genomes (cattle: bos_taurus. ARS_UCD.12; pig: sscofa 1.1). Unique Molecular Identifiers (UMI) were counted using kallisto (v-0.46.0) ³.

Identification of differentially expressed genes at embryonic stages of different lineages

According to the differentiation process during bovine embryo development, we divide bovine embryo cells into three major lineages, embryonic lineages, including the anterior ICM (inner cell mass) of E5, the ICM (inner cell mass) of E6 and E7, the ectoderm of E10 and E12 and the ectoderm of E14, TE (trophectoderm) lineages, including the anterior trophectoderm of E5 pre-TE (E5 (morula late)), TE of E6, E7, E10, E12 and E14, and the endoderm lineages, including the endoderm of E10, E12 and the definitive endoderm of E14 (C in FIG. 1).

Construction of expression propensity

In order to track the dynamic changes of DEGs during embryonic development (differential expression genes of the Trophectoderm (TE) lineage (DEGs) show a clear expression trend, the expression patterns of these genes at different stages of development reflect the differentiation and functional changes of the trophectoderm), we constructed the expression trend of DEGs in the ectoderm lineage. We first calculated the average expression level of each gene in each lineage at a specific embryonic development time point, respectively. Average expression levels of embryo lineages were rescaled and analyzed by the k-means clustering method of parameters k=10 and iter.max=100, grouping DEG with similar trends in embryo development into separate clusters.

Cross species comparative analysis

We downloaded a list of homologous genes for four species through BioMart tools in the genome browser 105 (http:// dec2021.Archive. Ensembl. Org/index. Html), keeping 16841 genes, all 1:1 homologous genes. We then retained 1:1 homologous genes in the bovine and porcine embryo lineage dataset. For the comprehensive analysis between porcine and bovine embryo datasets we used Seurat CCA method for anchoring and dataset alignment. The top 2000 features in the dataset that are repeatedly variable are selected and the anchor points are identified using a "FindIntegrationAnchors" function with parameters "reduction=' cca, k, anchor=5, normalization. Method= 'SCT' ". The dataset is then integrated based on the identified anchor points using a "INTEGRATEDATA" function with the parameters "dims =1:30, k.weight=50, normalized. Method= 'SCT' ". The average expression of the genes in the integrated assay was obtained using the "AverageExpression" function and the Spearman correlation coefficient between the different cell types of the two species was calculated using the "cor" function. Using the "FINDMARKERS" function, two species were determined using the Wilcoxon rank sum test, respectivelyFold change in gene expression levels between the state and formative state, formative state and prime state. Only |'avg_ logFC' | >0.25 and 'p_val' <0.05 are considered DEGs.

Pseudo-time analysis

The developmental trajectories of embryonic cells were reconstructed using R-package Monocle (v-1.3.1) ⁴. UMI matrix was used as input and cells were pseudo-time ordered using the variable genes obtained Seurat. Individual cells of both species were further sequenced using destiny (v-2.14.0) R package ⁵ to determine their development sequence according to the Differential Expression Gene (DEGs) between all embryonic day cells of the pig calculated by the "FINDALLMARKERS" function.

RNA-seq data processing and analysis

The expression level of the protein-encoding gene (gene annotation file [ GTF ] from Ensembl-Bos_taurus. ARS_UCD.12) was quantified as a million Transcripts (TPM) using kallisto (v-0.46.0). DEGs between different cell types was identified using DEseq tool (v-1.30.1) ⁶. We used the Benjamini-Hochberg adjusted False Discovery Rate (FDR) <0.05 and absolute log ₂ (fold change) >2 as cut-off values for statistical significance.

BEpiSCs transcriptome correlation analysis with embryonic cells

For the comprehensive analysis between bEpiSC and bovine embryo datasets we used the Seurat RPCA method for anchoring and dataset alignment. The top 2000 features in the dataset that are repeatedly variable are selected and anchor points are identified using a "FindIntegrationAnchors" function with parameters "reduction= 'rpca', k, anchor = 15". The dataset is then integrated based on the identified anchor points using a "INTEGRATEDATA" function with the parameters "dims =1:30, k.weight=50". Scaling and PCA are applied on the combined dataset and the parameters "dims =1:5'. Key labels are visualized by the" FeaturePlot "function, using the integrated PCA coordinates as input to the clustering and t-SNE visualization workflow.

Functional enrichment analysis

Functional enrichment analysis was performed on selected genes using METASCAPE (http:// metacape. Org). Bovine genes are mapped to their human ortholog, human (homo sapiens) being the target species of analysis. Enrichment analysis was performed using all genes in the genome as background set, with the Gene Ontology (GO) -biological process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways as the ontological sources. The term minimum count ≡ 3, adjusted P <0.01 and enrichment factor ≡ 1.5 are considered significant and similar items are clustered. The critical pathways are depicted using a log ₁₀ (P-value) histogram.

Statistical analysis

Two-factor analysis of variance was performed on the RT-qPCR data of FIGS. 2C-H, 5B, and 5D, and the data of FIGS. 2B, 1D, and 1E were analyzed. Multiple comparison tests with Dunnett were used for B in FIG. 2, D in FIG. 2, E in FIG. 2 and B in FIG. 6. The H in F-2 in FIGS. 2C, 2 was tested by multiple comparisons with Bonferroni. The cell population doubling time and single cell cloning efficiency were compared using Tukey multiple comparison assays for E in fig. 2D and 2.

TABLE 3 Key resources Table

Selecting a cloning step:

In the stem cell line establishment process, the adherent growth characteristic of embryonic cells is one of key links. However, in addition to normal anchorage growth of epiblast, anchorage of other germ cells such as epiblast and trophectoderm cells may occur, which adversely affects the purity and quality of stem cell clones. Thus, selection of clones with a typical dome-like morphology is a key step to ensure successful stem cell line establishment. To this end, we established the following clone selection criteria:

1. clones must exhibit a steric dome-like structure, which is a hallmark of typical embryonic stem cell clones.

2. Clones should have clear and well-defined boundaries to facilitate differentiation and selection.

3. During the growth process, the clone center position should not be depressed, and the whole should not be in a monolayer adherence shape, so as to ensure that the three-dimensional structure of the clone is maintained.

4. For flat and non-monolayer clones, whole digestion followed by subculture can be performed to see if dome-shaped clones can form in the secondary cells. Typically, after 1-2 rounds of screening, the clones will exhibit a uniform dome-like morphology and the growth rate and size of the clones will also tend to be consistent. At this time, it can be considered that stem cell line construction has been successful. 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.

Optimizing embryo establishment methods in different periods:

For the E6-8 day embryo, taking the E7 embryo as an example, a full embryo inoculation mode is adopted, and during inoculation, a glass needle is required to divide the embryo, and an inner cell mass is exposed on feeder cells, so that the adherent growth of epiblast stem cells is facilitated.

For E9-E14 embryos, the blastoderm of the embryo is isolated prior to inoculation, and then the blastoderm is digested into a cell mass by enzymatic digestion, and the cell mass is inoculated onto feeder cells.

Example 1 establishment and characterization of bovine ectodermal stem cells (Bovine epiblast STEM CELLS, bEpiSCs)

1. Ectoderm (Epiblast isolation) was excised from bovine embryos (including E7, E10, E12, and E14) at different stages of development by mechanical isolation, treated with TrypLE ^TM Express (Gibco, 12605010) for 3min, and then plated onto feeder cells (mouse fetal fibroblasts) supplemented with 3i/LAF medium, as shown in FIG. 1A. Cultures were incubated at 37 ℃ in an environment containing 5% O ₂ and 5% CO ₂. Spherical growths were harvested by digestion with Acceutase cell dissociation reagent (Gibco, A11105-01) and passaged every 3 days in a 1:4 ratio to give the initial spherical growths (also called outgrowth, the primary clone formed after attachment of embryonic cells), which were passaged successfully to obtain the E7bEpiSCs cell line (also called E7bEpiSCs line or E7bEpiSCs in the present application), the E10bEpiSCs cell line (also called E10bEpiSCs line or E10bEpiSCs in the present application), the E12bEpiSCs cell line (also called E12bEpiSCs line or E12bEpiSCs in the present application) and the E14bEpiSCs cell line (also called E14bEpiSCs line or E14bEpiSCs in the present application) (cell line refers to a cell line capable of passaging and having a certain number and stability of bovine ectodermal stem cells) respectively.

2. The embryo at different developmental stages was subjected to statistics of the efficiency of the establishment of lines, and the statistical results are shown in the following table (B in fig. 2).

TABLE 1

Stage of bovine embryo development	Embryo quantity	Ratio 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

3. Selecting and subculturing bovine ectodermal stem cells (bEpiSCs):

The bEpiSCs (E7 bEpiSCs, E10bEpiSCs, E12bEpiSCs, E14 bEpiSCs) were cultured on mitomycin C (SELLECKCHEM, S8146) treated Mouse Embryonic Fibroblasts (MEF) feeder cells (5X 10 ⁴ cells/cm ²), respectively. Fresh 3i/LAF medium was changed every 12 h. To maintain bEpiSCs in an undifferentiated state, it is important that (a) the freshly prepared 3i/LAF medium should be kept at 4℃for no more than one week and should not be frozen, (b) the passage density must be appropriate-bEpiSCs seeding density is about 3-5X 10 ⁴cells/cm², and (C) fresh feeder cells and appropriate densities (3-4X 10 ⁴cells/cm²) must be ensured. The cells were passaged bEpiSCs with Acceutase cell separation reagent (Gibco, A11105-01) at a passaging ratio of 1:3-1:5 every 2-3 days. The specific number of days and proportion of traffic should be adjusted according to the actual situation.

After subculturing to the passage time of the lower half of fig. 1C, the cells were observed under a microscope, and the observation results were shown in fig. 1C (outgrowth is the primary clone formed after the embryonic cells were attached, and 19/14/11/15 in bEpiSCs-p19/p14/p11/P15 refers to the passage number (p=passages)), and it can be seen that all bEpiSCs lines exhibited dome morphology.

4. The cell proliferation capacity and single cell colony formation efficiency of bEpiSCs of embryo origin at different developmental stages were analyzed:

doubleing time (cell doubling time):

The above bEpiSCs (E7 bEpiSCs, E10bEpiSCs, E12bEpiSCs, E14 bEpiSCs) were each seeded on 12-well plates at a cell density of 3X 10 ⁵, and a bEpiSCs growth curve was drawn. Cells were then digested and counted every 12 hours, 24 hours, 36 hours, 48 hours and 60 hours using a Luna ^TM automated cytometer. 3 replicates were performed at each time point. The doubling time was calculated as (DT) =12× [ lg 2/(lgNt-lgN 0) ], 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 FIG. 1D.

Efficiency of single cell colony formation:

The bEpiSCs was digested with TrypLE ^TM Express (Gibco, 12,605,010) and filtered through a 40 μm cell filter, respectively. Cells were seeded in 6-well plates of 100, 500 and 1000 cells, respectively. After 3d 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 FIG. 1E.

As a result, as shown in FIGS. 1D and E (X in bEpiSCs-PX indicates the number of passages (P=passages)), it can be seen that there was no significant difference in the proliferation capacity of bEpiSCs cells derived from different embryonic ectoderms, the doubling time was 12 hours, and the single cell colony formation efficiency was about 23%, indicating that the cell lines derived from different embryonic ectoderms had good consistency.

5. Whereas E10EPI is a key stage in the formation of pluripotency, E10bEpiSCs was selected for pluripotency determination, specifically E10bEpiSCs was stained using alkaline phosphatase detection kit (Millipore, SCR 004), and the staining results are shown as F in FIG. 1, alkaline phosphatase staining was observed positively in colonies, and positive results demonstrated pluripotency.

Karyotyping of E10 bEpiSCs:

1%KaryoMAX Colcemid solution (Gibco, 15212012) was added to bEpiSCs medium and incubated for 1 hour. bEpiSCs was then dissociated into single cells using TrypLE ^TM Express (Gibco, 12605010) and collected by centrifugation. Subsequently bEpiSCs was suspended in a hypotonic solution of 0.075M KCl (Sigma, P5405) and incubated at 37℃for 15 minutes. After this step bEpiSCs was fixed with methanol and acetic acid in a 3:1 ratio, and this procedure was repeated three times. The resulting bEpiSCs 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 examined per cell line. As a result, as shown in FIG. 1G, karyotyping showed bEpiSCs to have a chromosome count of normally 60, indicating that it has a bovine species normal chromosome karyotype.

6. 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 min. After another round of DPBS washing, cells were blocked with 3% bsa for 1 hour at room temperature. The primary antibody was incubated overnight at 4℃and then washed three times with a wash 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 liquid. Finally, DAPI staining was performed to visualize the nuclei, allowing direct observation and photography (as in panels H-I). As a result, as shown by H-I in FIG. 1, it can be seen that the pluripotency analysis demonstrated high level expression of POU5F1, NANOG and SOX 2. In addition, pluripotency-related surface markers such as CDH1, SSEA1, and SSEA4 are also highly expressed.

After digestion of E10bEpiSCs, cells were seeded at a density of 1X 10 ⁶ cells per well and cultured in MEF medium on 35mm low attachment plates for 5-7 days in DMEM (Gibco, 11960-044), 10% FBS (Gibco, 16000-044), 1% penicillin-streptomycin (Thermo FISHER SCIENTIFIC, 15140-122) and 1% GlutaMAX (Thermo FISHER SCIENTIFIC, 35050-061) were added and cultured on a horizontal shaker at 70 rpm. Subsequently, embryoid Bodies (EB) were transferred to 12-well plates and incubated in the same medium for one week, the medium was changed twice daily, followed by immunofluorescent staining with adherent cells, and as a result, in vitro differentiation assay demonstrated that E10bEpiSCs had the ability to form embryoid bodies and differentiate into three germ layers, as shown in J in fig. 1.

1X 10 ⁷ E10bEpiSCs prepared by centrifugation collection after digestion was resuspended in 50. Mu.L BM and subsequently subcutaneously injected into the neck of BALB/c nude mice. After 4-5 weeks, teratomas were collected and subjected to subsequent H & E analysis. The results are shown in figure 1, K, and the in vivo teratoma experiments demonstrate that these cells are capable of producing teratomas with organized structures representing all three germ layers. In summary, E10bEpiSCs has the basic PSCs features.

Examples 2, 3i/LAF factors are critical for long term maintenance of bEpiSCs

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 (154 aa) (10 ng/mL, peproTech, 100-18B), IWR-1-endo (0.5 μm, SELLECKCHEM, S7086).

To investigate the effect 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 a single factor reduction to assess the function of these molecules. Using the 3i/LAF cultured E10bEpiSCs as a control, we replaced the reduced factor medium 12 hours after the E10bEpiSCs passage experiment of example 1. The cell morphology and AP staining were observed in the medium without factor for 48 hours, and the results were shown in FIG. 2, in which A-B (-CHIR 99021, -WH-4-023, -IWR-1-endo, -LIF (10 ng/mL, peproTech, 300-05), -Activity A and-FGF-basic, respectively, were devoid of corresponding factors), and it was seen that the clone was observed to be significantly flattened, with unclear boundaries and differentiated in E10bEpiSCs from the IWR-1-endo (IWR) no factor group. In contrast, bEpiSCs clones from the FGF2 factor-free group showed significantly slower proliferation rates, smaller clone volumes and more severe apoptosis. The key role of IWR and FGF2 in E10bEpiSCs self-renewal is evident, confirming the previous report on bovine origin (Primed) PSCs.

By comparing the gene expression profiles of E10bEpiSCs cultured with 3I/LAF and IWR/FGF2 (I/F) (with the addition of IWR and FGF2 only), we found that the use of I/F alone resulted in down-regulation of the pluripotency-related gene and up-regulation of the development-related gene (C in FIG. 2). Thus, the combination of IWR and FGF2 may be effective in maintaining the pluripotency of the originating state (Primed) bESCs, but such a combination is insufficient to maintain the pluripotency of E10 bEpiSCs.

To investigate the function of three small molecule inhibitors associated with the WNT/β -catenin signaling pathway in bEpiSCs pluripotency and self-renewal, we collected the analysis of differentially expressed genes (DIFFERENTIALLY EXPRESSED GENE, DEG) using the third generation E10bEpiSCs, with CHIR99021 (CHIR)/IWR/WH 4023 (WH) (representing the removal of CHIR99021, IWR-1-endo and WH-4-023 factors, respectively). Analysis showed that removal of any WNT-related small molecules resulted in abnormalities in the expression of the core pluripotency genes POU5F1, SOX2 and NANOG in bEpiSCs, and a significant decrease in the expression of ZFP42, UTF1 and TDGF1 (D in FIG. 2). In addition, genes involved in mesodermal differentiation and embryonic gastrulation are upregulated, such as BMP4 gene, CDH2 gene, GATA6 gene, WNT5A gene, LEF1 gene, and CTNNB1 gene (E in fig. 2), indicating that the differentiation process of E10bEpiSCs is triggered directly or indirectly upon removal of WNT-related small molecules.

The combination of CHIR and IWR proved to be able to maintain self-renewal of mouse EpiSCs and human ESCs. We studied the effect of different ratios of CHIR to IWR concentrations on E10bEpiSCs pluripotency at the concentration of A in FIG. 5. The concentration of IWR and CHIR factors in the 3I/LAF medium was respectively configured and grouped according to the concentration shown in FIG. 5A (where C is CHIR, I is IWR; in xC/yI, x is CHIR, in. Mu.M, y is IWR, in. Mu.M), and we replaced the reduced factor medium 12 hours after the E10bEpiSCs passage experiment of example 1. The presence of low concentration IWR (> 0.5 μm) counteracts the promotion of EpiSCs differentiation by high CHIR concentration (< 5 μm) as shown in fig. 5 a and B, as shown by culturing for 48 hours in factor-free medium and subjecting it to clone morphology, AP staining and gene expression analysis.

Using the above method, we further investigated by concentration in D in FIGS. 5C and 7 whether there is an IWP2 or XAV939 concentration range that can replace IWP in 3i/LAF medium, studied the effect of IWP2 and XAV939 (replacement factors for IWP) on maintenance of E10bEpiSCs morphology and pluripotency, and found that a concentration range of 1-2.5 μ MXAV939 can be used to effectively replace IWP (D in FIGS. 5C and 7, D in FIG. 5, 3i/LAF in 3i/LAF medium, and x and y in xIWP2 and yXAV939 represent concentrations in μM, respectively).

The concentrations of FGF2, activin a and LIF factors in 3i/LAF medium were respectively configured and grouped as indicated by F, G and H in fig. 2, we replaced the reduced factor medium after 12 hours of passage E10bEpiSCs of example 1, cultured in medium without factor for 48 hours and examined the effect of FGF2, activin a and LIF on bEpiSCs pluripotency, and found that depletion of FGF2 resulted in deregulation of the core pluripotency regulating network and bEpiSCs multilineage differentiation (F, -FGF2, -Action a and LIF in fig. 2 represent respectively minus the corresponding small molecules, the remaining concentrations being unchanged). The removal of activin A resulted in a significant decrease in POU5F1 and NANOG expression, while the expression of mesoendoderm-related genes (e.g., BMP2 gene, BMP4 gene, and IDs gene) was significantly increased (G in FIG. 2), -FGF2, -Action A, and-LIF represent respectively minus the corresponding small molecules, with the remaining concentrations unchanged. Activation of the JAK/STAT3 signaling pathway is critical for maintaining stem cell pluripotency 38. However, the expression of the core JAK/STAT3 signaling gene STAT3 was down-regulated, and the mesoendodermal related gene GATA6 was up-regulated (H, -FGF2, -Action a and-LIF in fig. 2 represent respectively minus the corresponding small molecules, the remaining concentrations unchanged), indicating that LIF supplementation is beneficial. In summary, our study showed that the 3i/LAF culture system effectively maintained bEpiSCs pluripotency while emphasizing the positive impact of all factors on self-renewing capacity.

Transcriptome analysis of examples 3, bEpiSCs

The single cell transcriptome characteristics of bovine embryos can be used as a definitive basis for assessing the pluripotent state of bovine embryonic stem cells. To further evaluate bEpiSCs gene expression signatures and pluripotency states:

We first compared single cell transcriptome data of bEpiSCs obtained from different stages of bovine embryo with single cell transcriptome data of bovine embryo lineage (table 2). PCA showed bEpiSCs development and multipotent features closely related to those of E10-E12 ectoderm (A in FIG. 3). Spearman correlation analysis showed that E7 and E10bEpiSCs showed a stronger correlation with E10 ectoderm, while E12 and E14bEpiSCs showed a higher correlation with E12 ectoderm (B in fig. 3). We further compared bEpiSCs isolated from different embryo stages with single cells of bovine embryo lineage. In terms of pluripotency, we observed down-regulation of the naive pluripotency-related gene in bEpiSCs. In contrast, the formation of the pluripotency-related gene was found to be up-regulated in bEpiSCs. In addition, the expression level of the pluripotency-related gene induced in bEpiSCs was low (C in fig. 3), indicating that bEpiSCs exhibited the characteristics of intermediate (Formative) pluripotency.

Next, we performed comparative analysis of a number of RNA transcriptomes between E10bEpiSCs established in this study and the previously reported bovine PSCs, including bESCs, bEDSCs, bEPSCs and biPSCs18 in the original state (Primed). PCA analysis revealed a pluripotency difference in Dim2 levels, indicating that bEpiSCs and bEDSCs exhibited a greater degree of similarity (D in FIG. 3).

To distinguish bEpiSCS from other bovine PSCs, we performed a comparative analysis to determine the unique features of bEpiSCs.

The NANOG, LEFTY2, NODAL genes of E10bEpiSCs were expressed at higher levels than bESCs in the original state (Primed) than other stem cell markers associated with pluripotency modulation (FGF 4 gene, ZFP42 gene, ETV3L gene, DPPA3 gene, actrl 1 gene, TDGF1 gene, and TBX3 gene). Further, the GATA3 gene, PAX6 gene, TGFB2 gene, PAX2 gene, FGFR4 gene, LMO1 gene, MEIS2 gene, ID2 gene and HES1 gene are expressed at lower levels.

In addition, E10bEpiSCs was involved in up-regulation of gene expression of stem cell proliferation (ELL 3 gene and FGF4 gene) and intercellular adhesion regulation (NODAL gene, FOXA2 gene and EPCAM gene) and DPPA3 gene, LMO1 gene, ACVRL1 gene and WNT3A gene expression levels were higher than bEDSCs. Further, their expression levels of the HAND1 gene, GATA3 gene, TGFB2 gene, ZFP42 gene, VMO1 gene, MEIS2 gene, BMP4 gene, IGF1R gene, CCND2 gene and SPRY4 gene were lower.

In addition, E10bEpiSCs exhibited higher levels of expression of the LIN28A/B gene, NODAL gene, LIFR gene, IL6R gene, PDGFRA gene, ACVR1B gene, ETS1 gene, IL6ST gene, SALL4 gene, as compared to biPSCs. Further, the expression level of GDF15 gene, BCL3 gene, CD44 gene, ETV2 gene, FGFR4 gene, KLF15 gene, GDF1 gene, BMP4 gene, COX17 gene, MYC gene is lower.

In addition, E10bEpiSCs exhibited higher levels of expression of the LIN28A/B gene, NODAL gene, LIFR gene, ESRRB gene, IL6R gene, ACVR1B gene, KLF5 gene, SALL4 gene, and CDH1 gene as compared to bEPSCs. Further, the MSC gene, GCK gene, TGFB2 gene, MAPK15 gene, KLF15 gene, GDF1 gene, ID4 gene, FOS gene, COX17 gene expression level was lower.

Furthermore, E10bEpiSCs exhibited higher levels of LIN28A/B gene and NODAL gene expression compared to biPSCs or bEPSCs, both of which are known for their inhibition of cell differentiation and the involvement of IL6R in the PI3K-Akt signaling pathway (F in FIGS. 3E and 3; A in FIG. 6).

To investigate the dissimilarity between bEpiSCs and pig pgEpiSCs, we performed comparative analysis of gene expression at the level of the bulk RNA for both cell types. Analysis of the Differentially Expressed Genes (DEGs) showed that bovine and porcine EpiSCs were not significantly different in terms of multipotent maintenance (e.g. POU5F1 gene, NANOG gene, LIN28B gene) or germ layer differentiation (e.g. BMP4 gene, EOMES gene, NODAL gene) (fig. 6B). However, changes were observed only in ion transport (ATOX 1 gene, ATP4A gene and GCK gene) and cell activation (GLI 3 gene, IRF1 gene and EZH2 gene) (C in fig. 6). These findings indicate that bovine and porcine EpiSCs have a similar regulatory network in maintaining the multipotent properties in the 3i/LAF system.

In summary, we have successfully established bovine intermediate (Formative) EpiSCs, which exhibit different gene expression profiles than other published bovine PSCs.

TABLE 2

Potential applications of examples 4, bEpiSCs

This example also evaluates bEpiSCs's potential for myogenic differentiation and as a Somatic Cell Nuclear Transfer (SCNT) donor cell (a in fig. 4).

Specifically, myogenic Differentiation Basal Medium (MDBM) consisted of DMEM/F12, 1% non-essential amino acids, 0.1mM beta-mercaptoethanol, 1% penicillin-streptomycin, 15% KOSR and 200. Mu.M ascorbic acid.

In the first stage, E10bEpiSCs was separated into small pieces and incubated for 3 days in MDBM with 1% B27 supplement, 3. Mu.M CHIR99021 and 2. Mu.M SB431542 (SELLECKCHEM, S1067).

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

For the third stage, the broth was incubated with 10ng/mL HGF (Peprotech, 100-39H), 10ng/mL IGF-1 (Peprotech, 100-11), 20ng/mL recombinant human FGF-basic (154 a.) and 0.5. Mu.M LDN193189 for 2 days.

In the fourth stage bEpiSCs begins to differentiate into myoprecursor cells. bEpiSCs-MPCs were treated with IGF-1 at a concentration of 10ng/mL for 4 days,

In the fifth stage, the skeletal muscle is then accelerated by treatment with a composition of 10ng/mL HGF and 10ng/mL IGF-1 for 20 to 25 days. For skeletal muscle maturation, cells were treated with N2 medium consisting of DMEM/F12, N2 medium consisting of DMEM/F12 supplemented with 15% KOSR, 1% N2 supplement, 1% penicillin-streptomycin and 1% non-essential amino acids, resulting in bEpiSCs-derived differentiated muscle cells.

1. Cell observations at each stage are shown in FIG. 4B (stage 1-5 represents the above-described one to five stages, respectively), and it can be seen that cells gradually differentiate from stem cell clone morphology into mesoderm and then into muscle cell-like cells.

2. Total RNA was extracted from differentiated muscle cells derived from bEpiSCs and bEpiSCs (five-stage bEpiSCs) using RNA prep Pure Cell/Bacteria kit (TIANGEN, DP 430), respectively, and then reverse transcribed into cDNA using 5×all-In-One RT Master Mix (Abm, G490). Subsequently, PCR amplification was performed on ARCHIMED REAL TIME SYSTEM (ROCGENE) using 2X REALSTAR GREEN Power mix (GenStar, A311-05). The data were analyzed using the comparative CT (2 ^-ΔΔCT) method. The Δct value was calculated using GAPDH as an internal reference. All experiments were performed in triplicate with independent biological replicates. qPCR analysis showed bEpiSCs-derived muscle cells (stage 5) to express MYOG, MYMK, MYH and MYH11 (C in fig. 4).

3. Experiments with further immunostaining bEpiSCs-derived differentiated muscle cells:

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 min. After another round of DPBS washing, cells were blocked with 3% bsa for 1 hour at room temperature. The primary antibody was incubated overnight at 4℃and then washed three times with a wash 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 liquid. Finally, DAPI staining was performed to visualize the nuclei, enabling direct observation and photography.

Immunostaining with specific MYOSIN and FACTIN antibodies demonstrated MYOSIN and FACTIN expression in bEpiSCs-derived differentiated muscle cells, indicating successful initial muscle differentiation (D in fig. 4).

4. Donor cells as genome editing animal clones

Vector construction

GFP plasmid was stored in the laboratory. In summary, we generated the PB-CMV-EF1A-GFP-NLS plasmid (disclosed in ："Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines", entitled PB-CMV-EF1A-GFP-NLS in the literature) by modifying the PB-CAG-MCS vector (provided by Wu Sen teachings). Specifically, we replaced the chicken β -actin promoter with a human elongation factor 1α (EF 1A) promoter and integrated GFP-NLS downstream of the EF1A promoter.

BEpiSCs transfection

The PB-CMV-EF1A-GFP-NLS plasmid was transfected with E10bEpiSCs using Lipofectamine ^TM reagent (Invitrogen, L3000008):

Transfection was performed 16 hours after passage of E10bEpiSCs in 24 well plates, first 25. Mu.L of Opti-MEM ^TM medium was added to the centrifuge tube, followed by 0.75. Mu.L of Lipofectamine ^TM reagent and thoroughly mixed. Then, another centrifuge tube was taken, 25. Mu.L of Opti-MEM ^TM medium was added, and 0.5. Mu.g of PB-CMV-EF1A-GFP-NLS plasmid and 1. Mu. L P3000 ^TM reagent were added and mixed well. The PB-CMV-EF1A-GFP-NLS plasmid mixture was then mixed with Lipofectamine ^TM reagent in a 1:1 ratio, incubated for 10-15 min, and then cells were added for culture. E10bEpiSCs of PB-CMV-EF1A-GFP-NLS plasmid was obtained.

Production of bEpiSCs clone embryos:

Embryo transplantation and embryo punching specifically comprises the following steps:

1 recipient bovine transplantation procedure

1.1 Recipient cattle were subjected to epidural anesthesia between the 1 st and 2 nd coccyx and the pudendum was swabbed.

1.2 The embryos are reloaded into 0.25mL plastic tubules, which are loaded into a transplantation gun. The transplantation gun sleeve with the tubule is covered with a hard coat, clamped by a plastic ring, and then covered with a soft coat.

1.3 Transplanting the embryo to the position 1/3-1/2 of the uterine horn on the luteal side of the receptor.

2 Non-operative embryo punching

2.1 Main equipment for non-operative embryo punching

1-2 Solid microscopes and embryo flushing pipes (two paths), namely, through a 20# embryo flushing pipe for producing cattle, a 18# embryo flushing pipe for breeding cattle, an inner core of the embryo flushing pipe, wherein the length of the embryo flushing pipe is 64cm, the cervical dilatation rod is 50mL, 9-10 syringes are 2 syringes, 10mL, 2 syringes are 5mL, 2 syringes are 20mL, egg collecting funnels are 1, phi 90mm culture dishes are 2/head, and the outer bottom of each culture dish is divided into 1cm2 square grids and shearing scissors.

2.2 Non-operative embryo-punching medicine

Du's Phosphate Buffer (PBS), alcohol, iodine tincture, benzalkonium chloride, physiological saline, 2% lidocaine, 0.9% physiological saline, oxytetracycline.

2.3 Non-operative embryo punching technical step

The donor cattle were fixed in six columns and extracocsal anesthetics were performed with 2% lidocaine at the junction of the referral and the first coccyx or the second coccyx until the tail was unconscious. The amount of lidocaine is about 5-10 mL/head.

The cervix is expanded by a uterine dilating rod, then the embryo-punching tube with the inner core is slowly inserted into the uterine horn, when the embryo-punching tube reaches the bending position of the uterine horn, the inner core is pulled out by about 5cm, and then the embryo-punching tube is sent to the front end of the uterine horn. When the inner core reaches the bent part of the uterine horn again, the inner core is pulled out outwards for 5-10 cm until the embryo punching tube reaches the front end of the uterine horn.

And (3) inflating the embryo tube air bag by about 8-12 mL, drawing out the inner core of the embryo tube, and connecting the interface between the embryo tube and a 50mL syringe.

850 ML syringes are numbered 1-8, each syringe absorbs 50mL of embryo flushing liquid, one tube of embryo flushing liquid is injected into uterine horns from the embryo flushing tube each time, then all recovery liquid is absorbed back to the original syringe, each uterine horn is repeated for 4 times, and 200mL of embryo flushing liquid is used.

The 50mL embryo flushing liquid is sucked by a 50mL injector, the recovery liquid in the 8 embryo flushing 50mL injectors is injected into an egg collecting funnel for filtration at room temperature (18 ℃ to 22 ℃), and when about 20mL recovery liquid remains in the egg collecting funnel, the egg collecting funnel is shaken to pour the recovery liquid into a phi 90mm culture dish. The egg collection funnel wall and bottom were rinsed with PBS until there was no mucus. And (5) carrying out microscopic examination on the culture dish.

We transferred PB-CMV-EF1A-GFP-NLS plasmid into bEpiSCs cells to obtain GFP-bEpiSCs cells.

The operation steps of transplanting the recipient cattle are as follows:

the recipient cattle are subjected to epidural anesthesia between the 1 st and the 2 nd coccyx and the external pudendum is wiped.

GFP-bEpiSCs cells were loaded into 0.25mL plastic tubules, which were loaded into a transplantation gun. The transplantation gun sleeve with the tubule is covered with a hard coat, clamped by a plastic ring, and then covered with a soft coat.

Embryo transfer to the upper 1/3-1/2 of the uterine horn on the luteal side of the recipient.

The results showed that the PGCLCs embryoid body morphology formed by bovine bEpiSCs is shown as E in FIG. 4, the PGCLCs gene expression profile 4F formed by bovine bEpiSCs is shown as F, and the TFAP2C gene and PRDM1 gene expression levels of PGCLCs are significantly increased compared to those of bEpiSCs. We produced GFP-bEpiSCs (G in FIG. 4) by transfection, which was used as donor cells for embryo cloning, and obtained GFP-bEpiSCs cloned blastocysts (H in FIG. 4). bEpiSCss were comparable to fibroblast clones, and reached about 30% in both blasts (I in FIG. 4). Cloning embryos using GFP-bEpiSCs can be used to efficiently establish bEpiSCs de novo (J in fig. 4). These findings indicate that bEpiSCs has potential applications in cell culture meat production, gene editing, and animal breeding.

In this study, we used single cell transcriptome sequencing to fully analyze gene expression patterns at key developmental stages of early bovine embryos. Our findings reveal that a transition from the primitive (native) to the intermediate (Formative) to the Primed (Primed) state occurs during early ectodermal development of bovine embryos. Specifically, we observed that LIF/STAT3 signaling-related genes were down-regulated during the initial EPI phase, while WNT/β -catenin signaling-related genes were up-regulated during the transition from intermediate (Formative) to originating (Primed) EPI. These results further underscore the modulation of evolutionary conservation and pluripotency of embryo development in artiodactyls (between cattle and pigs). Using an optimized 3i/LAF culture system, we successfully established bovine ectodermal stem cells based on the conserved early embryo development observed in pigs and cattle (bEpiSCss). These cells exhibit long-term stability, maintain normal karyotype, exhibit the characteristics of forming ectoderm, express pluripotent marker genes, have the ability to develop three germ layers in vitro and in vivo, and exhibit typical stem cell pluripotency.

BEpiSCs cultured under 3i/LAF conditions in this study showed the characteristic of forming pluripotency. From the standpoint of the culture system, FGF2 and WNT inhibitors are widely used in cultures of bPSCs other than bEPSCs (where FGF2 is not necessary for self-renewal of bEPSCs). The 3i/LAF culture system used in this study showed an effective use for pluripotency and self-renewal of bovine development PSCs. Our findings indicate that the removal of any small molecule or cytokine is detrimental to the maintenance of bEpiSCs pluripotency, highlighting the applicability of 3i/LAF to the preservation of bEpiSCs pluripotency. Interestingly bEpiSCs can be derived from a broader embryo stage (E7-E14), by cell colony selection to address the inherent heterogeneity of embryo discs at different developmental stages. Furthermore, transcript levels of NODAL expression in bEpiSCs were relatively higher than those observed in bPSCs reported previously, could serve as markers for prointestinal embryogenesis of the anterior ectoderm and play a key role in mesoderm and endoderm formation, suggesting a similarity between bEpiSCs and intermediate (Formative) ectoderm cells.

Notably, bovine bEpiSCs and porcine pgEpiSCs exhibited very similar cloning morphology, pluripotency characteristics, and transcriptome characteristics. This comprehensive study enhances our understanding of the commonality and distinctions of embryo pluripotency dynamics in livestock species. In addition, bEpiSCs shows strong myogenic differentiation potential, thereby providing a new way for promoting the production of cell culture meat. In addition, bEpiSCs can be used as donor cells of genome editing cloned embryos, and brings innovation prospect for the subsequent development of stem cell breeding and genome editing technology.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.

Reference is made to:

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.

2.Zhi,M.L.,Zhang,J.Y.,Tang,Q.Z.,Yu,D.W.,Gao,S.,Gao,D.F.,Liu,P.L.,Guo,J.X.,Hai,T.,Gao,J.,et al.(2022).Generation and characterization of stablepig pregastrulation epiblast stem cell lines.Cell Res.32,383-400.10.1038/s41422-021-00592-9.

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

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.

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

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

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 of the human fetal digestive tract using single-cell RNA-sequencing.Nat.Cell Biol.20,721-734.10.1038/s41556-018-0105-4.

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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 SSEA 4.

2. The bovine ectoembryonic stem cell line of claim 1, wherein said bovine ectodermal stem cell has intermediate multipotency and is capable of stable inheritance at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or more times;

Preferably, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E7-E14;

preferably, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10-E12;

Preferably, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10;

Preferably, the bovine embryo E7-E14 belongs to the EPI lineage.

3. The bovine ectodermal stem cell line of claim 1, wherein said bovine ectodermal stem cell line highly expresses one or more of NANOG, LEFTY2 and NODAL genes compared to bESCs in the original state (Primed);

Preferably, the bovine ectodermal stem cell line low expresses one or more of the GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, HES1 genes compared to bEDSCs in the original state (Primed);

preferably, the bovine ectodermal stem cell line highly expresses one or more of FGF4 gene, ZFP42 gene, ETV3L gene, DPPA3 gene, ACVRL1 gene, TDGF1 gene, and TBX3 gene compared to bESCs in the original state (Primed);

preferably, the bovine ectodermal stem cell line expresses one or more of the GATA3, PAX6, TGFB2, PAX2, FGFR4, LMO1, MEIS2, ID2, HES1 genes low compared to bEDSCs in the original state (Primed).

4. The bovine ectodermal stem cell line according to claim 1, characterized in that the bovine ectodermal stem cell line has up-regulated gene expression involved in stem cell proliferation and intercellular adhesion regulation compared to bEDSCs;

preferably, the stem cell proliferation gene comprises ELL3 and/or FGF4;

Preferably, the genes that regulate intercellular adhesion include one or more of NODAL, FOXA2, and EPCAM;

preferably, the bovine ectodermal stem cell line also highly expresses one or more of DPPA3, LMO1, ACVRL1 and WNT3A genes compared to bEDSCs;

preferably, the bovine ectodermal stem cell line also expresses one or more of the HAND1, GATA3, TGFB2, ZFP42, VMO1, MEIS2, BMP4, IGF1R, CCND2, and SPRY4 genes low compared to bEDSCs.

5. The bovine ectodermal stem cell line of claim 1, wherein the bovine ectodermal stem cell line exhibits a higher LIN28A/B and/or NODAL expression level compared to biPSCs or bEPSCs;

preferably, the bovine ectodermal stem cell line also highly expresses one or more of NODAL, LIFR, IL6R, PDGFRA, ACVR1B, ETS1, IL6ST, SALL4 genes compared to biPSCs;

Preferably, the bovine ectodermal stem cell line also expresses one or more of GDF15, BCL3, CD44, ETV2, FGFR4, KLF15, GDF1, BMP4, COX17, and MYC genes low compared to biPSCs;

Preferably, the bovine ectodermal stem cell line also highly expresses one or more of LIFR, ESRRB, IL6R, ACVR1B, KLF5, SALL4, CDH1 genes compared to bEPSCs;

Preferably, the bovine ectodermal stem cell line also expresses one or more of MSC, GCK, TGFB2, MAPK15, KLF15, GDF1, ID4, FOS, COX17 genes in low compared to bEPSCs.

6. The bovine ectodermal stem cell line according to claim 1, characterized in that it relies on the activation of FGF/ERK and/or tgfβ/SMADs signaling pathway and the inhibition of WNT/β -catenin signaling pathway;

Preferably, the FGF/ERK signaling pathway-related genes include one or more of MAPK1, MAPK14, FGF2, PDGFA, FGFR1, and FGFR 2;

Preferably, the TGF-beta/SMADs signaling pathway-related genes include one or more of INHBA, NODAL, ACVR A/2B, BMP4 and BMPR 1A/1B;

preferably, the WNT/β -catenin signaling pathway-related gene includes one or more of TCF7, APC, WNT11, CTNNB1, FZD2, and WNT 3A.

7. A culture medium for culturing a bovine ectodermal stem cell line, the culture medium comprising a basal medium and an additive component;

preferably, the basal medium comprises DMEM/F12 medium and/or Neurobasal medium;

more preferably, the mass ratio or volume ratio of the DMEM/F12 medium to the Neurobasal medium is 1:1;

Preferably, 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 (154 aa), ROCK inhibitor Y-27632, or any combination thereof are also included in the basal medium.

8. An in vitro method of preparing a bovine ectodermal stem cell line, said method comprising the step of culturing one or more ectodermal cells isolated from bovine embryos E10-E14 in the medium of claim 7;

preferably, the bovine embryo E10-E14 belongs to the EPI lineage;

preferably, the bovine ectodermal stem cell line is derived from the ectoderm of bovine embryo E10.

9. Use of a bovine ectodermal stem cell line according to any one of claims 1-6 or prepared by the method of claim 8 for inducing the production of muscle cells or providing a nuclear transfer donor cell or nucleus.

10. A method for preparing muscle cells, comprising the step of culturing 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 a myogenic medium to obtain muscle cells.

11. A method for preparing bovine primordial germ cell-like cells, comprising the step of culturing the bovine ectodermal stem cell line of any one of claims 1 to 6 or the bovine ectodermal stem cell line prepared by the method of claim 8 in a PGC-induced culture system to obtain bovine primordial germ cell-like cells;

preferably, bovine primordial germ-like cells highly express TFAP2C gene and PRDM1 gene compared to the bovine ectodermal stem cell line.

12. A bovine nuclear transfer method comprising the step of culturing the bovine ectodermal stem cell line of any one of claims 1 to 6 or the bovine ectodermal stem cell line prepared by the method of claim 8 as a nuclear transfer donor cell or a nuclear transfer donor cell to obtain bovine cells, tissues, organs, and whole individuals.