Method for inducing porcine embryonic stem cells to form porcine blastula and culture medium

Abstract

A method for generating porcine blastocysts from porcine embryonic stem cells (pESCs) is provided, as well as the blastocysts obtained by this method. Culture media for inducing pESCs to form blastocysts and culture media for generating and culturing pESCs are also provided.

Description

Technical Field

[0001] This invention relates to a method for generating porcine blastocyst-like structures from porcine embryonic stem cells (pESCs), and to the blastoids obtained by this method. The invention also relates to a culture medium for inducing pESCs into blastocyst-like structures and a culture medium for generating pESCs. Background Technology

[0002] Pluripotent stem cells have the potential to generate embryonic models that can simulate developmental processes in vitro. Embryonic models include blastocysts, embryoids (EBs), and gastrulae derived from pluripotent stem cells (PSCs), which can provide high-quality models for analyzing the genetic and molecular biological events of embryonic development in vitro.

[0003] Among these, blastocysts can be used to study key events in preimplantation embryogenesis and early embryonic development. In mice, co-culturing mouse embryonic stem cells (ESCs) and mouse trophoblastic stem cells (TSCs) can generate blastocysts (Non-Patent Literature 1); mouse extended pluripotent stem cells (EPSCs) and epiblastic stem cells (EpiSCs) can self-assemble into blastocysts in a 3D culture system (Non-Patent Literature 2 to 5); mouse ESCs, TSCs, and extraembryonic endoderm stem cells (XENs) can assemble into embryo-like structures (ETXs) (Non-Patent Literature 6 and 7), and can also form embryo-like structures with complete gastrula that further develop into organ progenitor cells similar to E8.5 embryos (Non-Patent Literature 8 and 9). In humans, using primitive PSCs ( PSCs or EPSCs can generate blastocysts that resemble human blastocysts in morphology, cell lineage composition, and transcriptome characteristics (Non-Patent Literature 10-14); human blastocysts can also be formed using somatic cell reprogramming or intermediate cells during the transition from the primordial to the primitive state (Non-Patent Literature 15 and 16); and embryonic models of humans can be formed using ESCs and EPSCs after implantation (Non-Patent Literature 17-20). Furthermore, blastocysts derived from non-human primates (e.g., cynomolgus monkeys) can be formed using primitive ESCs, which can develop in vitro to gastrulation and early in vivo pregnancy stages (Non-Patent Literature 21). However, due to ethical restrictions, it is currently impossible to test the in vivo developmental capacity of human blastocysts.

[0004] The above studies demonstrate that ESCs from mice, humans, and monkeys can induce blastocyst formation. However, ESCs from livestock (such as pigs, cattle, and sheep) cannot yet be induced to form blastocysts. To date, only bovine blastocysts have been formed by assembling EPSCs and TSCs together (Non-Patent Literature 22). Currently, there are no reports of blastocyst formation in pigs, and it remains unclear whether pESCs can be induced to form blastocysts.

[0005] Non-patent literature

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[0032] [Non-patent document 27] Zhao, CC, Hu, SE, Huo, X. & Zhang, Y. Dr. seq2: A quality control and analysis pipeline for parallel single cell transcriptome and epigenome data. Plos One 12 (2017).

[0033] [Non-Patent Literature 28] Hao, YH et al., Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 (2021).

[0034] [Non-Patent Literature 29] Zhi, M. et al., Generation and characterization of stable pigpregastrulation epiblast stem cell lines. Cell Res 32, 383-400 (2022).

[0035] [Non-Patent Literature 30] Alquicira-Hernandez, J. et al., scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data. Genome Biol 20 (2019). Invention Overview

[0036] The inventors first developed a novel culture system for the reproducible generation of porcine ESCs (pESCs). Furthermore, they developed a three-dimensional (3D) two-step differentiation system for inducing porcine blastocyst-like structures (hereinafter referred to as blastocysts) from pESCs in vitro. These blastocysts have been shown to be virtually indistinguishable from blastocysts in terms of morphology, cellular composition, and single-cell transcriptome. The inventors also provided two different in vitro culture conditions, both of which enable the porcine blastocysts to be maintained in vitro for at least 18 days.

[0037] Using blastocysts from large animals (e.g., blastocysts derived from pESCs) can enable in vitro modeling of early embryogenesis and improve breeding systems for livestock such as pigs, accelerating the process of developing superior breeds.

[0038] Given that pigs and humans share similar physiological and anatomical structures, pig organs are considered suitable alternatives for humanized organs. This invention provides an excellent in vitro model for exploring human-pig cell interactions and helps advance research progress in generating human-derived organs in pigs. Attached Figure Description

[0039] Figure 1 shows the origin and characteristics of pESCs. Figure 1a, derivatives of pESCs isolated from porcine blastocysts and representative morphology of pESCs. Scale bar, 50 μm. Figure 1b, representative immunofluorescence staining results of pluripotent markers, including POU5F1, SOX2, NANOG, OTX2, and E-cadherin. Scale bar, 20 μm. Figure 1c, alkaline phosphatase (AP) staining of pESCs. Scale bar, 50 μm. Figure 1d, karyotype analysis of pESCs. Figure 1e, representative morphology of embryoid bodies. Scale bar, 50 μm. Figure 1f, representative immunofluorescence staining results of ectoderm-labeled tubulin, mesoderm-labeled α-SMA, and endoderm-labeled GATA6. Scale bar, 50 μm. Figure 1g, hematoxylin and eosin staining results of teratoma sections formed from pESCs in mouse subcutaneous tissue. Scale bar, 100μm.

[0040] Figure 2 shows the generation of porcine blastocysts using a 3D two-step strategy. Figure 2a, Schematic diagram of porcine blastocyst formation from pESC using the 3D two-step method. Figure 2b, Representative images of cell clusters and blastocysts at specified time points during porcine blastocyst formation. Scale bar, 50 μm. Figure 2c, Representative images of porcine blastocysts at day 6 (hereinafter referred to as porcine E6 PA blastocysts) and day 7 (hereinafter referred to as porcine E7 PA blastocysts), as well as blastocysts (day 6) generated by pESC using the 3D two-step method. Scale bar, 200 μm. Figure 2d, Formation efficiency of blastocysts generated by pESC (n = 16). Formation efficiency refers to the ratio of blastocysts to all aggregates. Figure 2e, Diameters of porcine blastocysts (n = 53), porcine E6 PA blastocysts (n = 50), and E7 PA blastocysts (n = 33). Diameters were statistically analyzed using ImageJ. P-values ​​were calculated using an unpaired t-test. Figure 2f shows the X / Y ratios of porcine blastocysts (n=53), porcine E6PA blastocysts (n=50), and E7PA blastocysts (n=33). The p-values ​​were calculated using an unpaired t-test. Figure 2g1. Representative immunofluorescence staining of SOX2 (marker of epiblast, also referred to as EPI in this paper), GATA3 (marker of trophectoderm, also referred to as TE in this paper), and GATA6 (marker of hypoblast, also referred to as HYPO in this paper) in pESC-derived blastocysts. Scale bar, 50 μm. Figure 2h: Cell number of each cell lineage in each porcine blastocyst (n=19), porcine E6 PA blastocyst (n=16), and E7 PA blastocyst (n=10). Figure 2i: Ratio of each cell lineage in each porcine blastocyst (n=19), porcine E6 PA blastocyst (n=16), and E7 PA blastocyst (n=10). Figure 2j: Representative immunofluorescence staining of ZO-1, a marker indicating tight intercellular junctions, in porcine blastocysts and porcine PA blastocysts. Scale bar, 50 μm.

[0041] Figure 3 shows the single-cell transcription profile of porcine blastocysts. Figure 3a shows the distribution of two porcine blastocyst samples using UMAP. Figure 3b shows the expression of EPI markers (SOX2, POU5F1), HYPO markers (GATA4, GATA6), and TE markers (GATA2, GATA3) using UMAP. Figure 3c shows the cells derived from porcine blastocysts stained by origin: epiblastocyst-like cells (ELC, yellow), hypoblastocyst-like cells (HLC, purple), and trophoblastocyst-like cells (TLC, red). Figure 3d shows the expression of EPI markers (SOX2, POU5F1), HYPO markers (GATA4, GATA6), and TE markers (GATA2, GATA3) using a dot plot. Figure 3e shows the expression of cell lineage-specific genes in ELC, HLC, and TLC using a heatmap.

[0042] Figure 4 shows the combined analysis of single-cell sequencing data from porcine blastocysts and embryos. Figure 4a, Principal component analysis (PCA) of porcine blastocysts and embryos (Non-Patent Literature 29). EB (early blastocyst, E6), LB (late blastocyst, E7), HB (hatched blastocyst, E8), EBi (early double-layered embryo, E9), LBi (late double-layered embryo, E10), PPS (pre-protostella embryo, E11), EPS (early protostella embryo, E12), PS (protostella embryo, E13), and LPS (late protostella embryo, E14). Figure 4b, PCA of LB, HB, and EBi. Figure 4c, PCA analysis shows that porcine embryos at stages E7, E8, and E9 comprise five cell clusters. Figure 4d, Similar scores for EPI (POU5F1 and SOX2), TE (GATA2 and GATA3), and HYPO (GATA4 and GATA6). Figure 4e, cells derived from pig embryos are stained according to their origin: EPI (yellow), HYPO (purple), and TE (red). Figure 4fThe UMPA plot shows the percentages of the three cell lineages in a combined analysis of porcine blastocyst-derived cells and porcine embryo-derived cells. The percentages of 85.2%, 59.4%, and 60.9% were calculated by dividing the number of EPI / HYPO / TE lineage cells in regions I / II / III by the total number of embryo-derived cells in the corresponding region. The percentages of 76.4%, 97.7%, and 97.8% were calculated by dividing the number of ELC / HLC / TLC lineage cells in regions I / II / III by the total number of blastocyst-derived cells in the corresponding region.

[0043] Figure 5 shows the establishment of stem cells from porcine blastocysts. Figure 5a, illustration of pESCs and pTSCs established from blastocysts. Figure 5b, representative morphology of blastocyst derivatives and pESCs. Scale bar, 50 μm. Figure 5c, alkaline phosphatase (AP) staining of pESCs derived from blastocysts. Scale bar, 50 μm. Figure 5d, representative immunofluorescence staining results of pluripotent markers POU5F1, SOX2, NANOG, SSEA4, β-catenin, and E-cadherin in blastocyst-derived pESCs. Scale bar, 50 μm. Figure 5e, representative morphology of blastocyst derivatives and pTSCs. Scale bar, 50 μm. Figure 5f, representative immunofluorescence staining results of GATA3, SOX2, and GATA6 in blastocyst-derived TSCs. Scale bar, 50 μm.

[0044] Figure 6 shows porcine blastocysts cultured in vitro. Figure 6a, representative morphology of blastocysts on day 6 of in vitro culture. Scale bar, 50 μm. Figure 6b, representative morphology of blastocysts on day 18 of in vitro culture. Scale bar, 200 μm. Figure 6c, diameter of blastocysts cultured in vitro. Diameters were statistically analyzed by ImageJ. ***p < 0.001. P-values ​​were calculated using an unpaired t-test. Figure 6d, survival rate of blastocysts cultured in vitro. Figure 6e, representative morphology of porcine PA blastocysts on days 4, 6, and 8 of in vitro culture. Scale bar, 50 μm. Figure 6f, diameter of porcine PA blastocysts cultured in vitro. Diameters were statistically analyzed by ImageJ. *p < 0.05; **p < 0.01. P-values ​​were calculated using an unpaired t-test. Figure 6g, survival rate of porcine PA blastocysts cultured in vitro.

[0045] Figure 7 This illustration illustrates the main findings of this study.

[0046] Figure 8 shows the generation and optimization of porcine blastocysts. Figure 8a, representative images of ELC, TLC, and HLC in iBlastoid medium. Scale bar, 50 μm. Figure 8b, representative results of GATA3, SOX2, and GATA6 immunofluorescence staining, showing pESCs differentiating into TE, EPI, and HYPO lineages in the specified medium. Scale bar, 50 μm. Figure 8c, schematic diagram of induction of porcine blastocysts from pESCs using a one-step method. Figure 8d, representative image of porcine blastocysts generated using the one-step method. Scale bar, 200 μm. Figure 8e, representative results of immunofluorescence staining of EPI (SOX2), TE (CDX2), and HYPO (GATA6) markers in reconstructed blastocysts. Scale bar, 50 μm. Figure 8f, comparison of blastocyst generation using a one-step and a two-step method.

[0047] Figure 9 shows the expression of three lineage markers in porcine blastocysts and blastocysts. Figure 9a Immunofluorescence staining of EPI (SOX2, POU5F1), TE (GATA3, CDX2), and HYPO (GATA4) markers in pESC-derived blastocysts. Scale bar, 50 μm. Figure 9b: Immunofluorescence staining of EPI (SOX2), TE (GATA3), and HYPO (GATA6) markers in pESC-derived blastocysts. Scale bar, 50 μm. Figure 9c: Immunofluorescence staining of EPI (SOX2), TE (GATA3), and HYPO (GATA6) markers in porcine blastocysts. Scale bar, 50 μm.

[0048] Figure 10 shows a single-cell panorama of pESC-derived blastocysts. Figure 10a, UMAP analysis shows that porcine blastocysts contain 13 cell clusters. Figure 10b, cell lineage identification based on lineage markers. Figure 10c, ELC, TLC, and HLC similarity scoring. Figure 10d, UMAP showing the expression of selected EPI genes (ETV5, DNMT3B), HYPO genes (NID2, COL4A1), and TE genes (SFN, KRT8). Figure 10e, dot plot showing gene expression in selected EPI, HYPO, and TE lineages.

[0049] Figure 11: Single-cell transcriptome analysis of blastocysts and blastocysts. Figure 11a: Identification of three cell lineages based on lineage markers. Figure 11b: Pie chart showing the percentage of lineage cells inferred from porcine blastocysts based on scPred. The percentages were calculated by dividing the number of cells with predicted lineage characteristics (EPI, HYPO, TE, undefined, and unassigned cells) by the total number of ELC, HLC, TLC, and other cell types derived from blastocysts.

[0050] Figure 12: Immunofluorescence staining of IVC (in vitro cultured) blastocysts. Figure 12a: Immunofluorescence staining of blastocysts cultured on day 18 in iBlastoid medium. Scale bar, 200 μm. Figure 12b: Immunofluorescence staining of blastocysts cultured on day 18 in N2B27+AY medium. Scale bar, 200 μm. Invention Details

[0051] A first aspect of the present invention provides a method for generating porcine embryo-like structures from porcine embryonic stem cells (pESCs), comprising culturing pESCs in a blastocyst induction medium to form porcine blastoids.

[0052] In some preferred embodiments, the embryo-like structure is a blastocyst.

[0053] In some preferred embodiments, the pESC is derived from a pig blastocyst, embryo, or parthenogenetic blastocyst.

[0054] In some embodiments, the blastocyst induction medium contains a combination of at least one hypoblastocyst-like cell (HLC) differentiation factor and at least one trophoblastocyst-like cell (TLC) differentiation factor. In other preferred embodiments, the HLC differentiation factor is optionally selected from activin A, bFGF, and CHIR99021, and the TLC differentiation factor is optionally selected from CHIR99021, SB431542, TSA, and BMP4.

[0055] In some preferred embodiments, the blastocyst induction medium further contains factors for maintaining pluripotency and / or increasing cell survival, the factors optionally selected from LIF, activin A, IGF1, IL-6, sIL-6 receptor α, XAV939, Y-27632 and DZNep.

[0056] In some preferred embodiments, the blastocyst induction medium contains LIF, BMP4, bFGF, CHIR99021, SB431542, DZNep, and TSA. Optionally, the blastocyst induction medium also contains IL-6, IGF1, sIL-6 receptor α, activin A, XAV939, and Y-27632.

[0057] In a further preferred embodiment, the blastocyst induction medium comprises Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, LIF, IGF1, BMP4, bFGF, CHIR99021, XAV939, SB431542, Y-27632, DZNep and TSA.

[0058] In some preferred embodiments, the blastocyst induction culture medium as described above is used, wherein:

[0059] The concentration of activator A is 5-15 ng / mL, preferably 8-12 ng / mL, and more preferably 10 ng / mL;

[0060] The concentration of bFGF is 5-15 ng / mL, preferably 8-12 ng / mL, and more preferably 10 ng / mL;

[0061] The concentration of CHIR99021 is 1-2 μM, preferably 1.3-1.7 μM, and more preferably 1.5 μM;

[0062] The concentration of SB431542 is 0.5-1.5 μM, preferably 0.8-1.2 μM, and more preferably 1 μM;

[0063] The concentration of the TSA is 2-3 nM, preferably 2.3-2.7 nM, and more preferably 2.5 nM;

[0064] The concentration of BMP4 is 1-10 ng / mL, preferably 3-7 ng / mL, and more preferably 5 ng / mL;

[0065] The concentration of the LIF is 1-10 ng / mL, preferably 3-7 ng / mL, more preferably 5 ng / mL; in some more preferred embodiments, the LIF is human LIF;

[0066] The concentration of IGF1 is 10-100 ng / mL, preferably 40-60 ng / mL, more preferably 50 ng / mL; in some more preferred embodiments, the IGF1 is human IGF1;

[0067] The concentration of the IL-6 is 5-15 ng / mL, preferably 8-12 ng / mL, and more preferably 10 ng / mL; in some more preferred embodiments, the IL-6 is human IL-6;

[0068] The concentration of the sIL-6 receptor α is 5-15 ng / mL, preferably 8-12 ng / mL, and more preferably 10 ng / mL; in some more preferred embodiments, the sIL-6 receptor α is human sIL-6 receptor α.

[0069] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0070] The concentration of Y-27632 is 3-15 μM, preferably 5-10 μM, and more preferably 7.5 μM;

[0071] The concentration of DZNep is 2-3 nM, preferably 2.3-2.7 nM, and more preferably 2.5 nM;

[0072] The FBS content is 0.02-0.15%, preferably 0.05-0.1%, and more preferably 0.075%.

[0073] The content of KOSR is 1-10%, preferably 3-7%, and more preferably 5%;

[0074] The concentration of the 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, and more preferably 100 μM;

[0075] And / or,

[0076] The concentration of the 2-phosphate-L-ascorbate salt is 10-100 μg / mL, preferably 30-70 μg / mL, and more preferably 50 μg / mL; in some preferred embodiments, sodium 2-phosphate-L-ascorbate is preferred.

[0077] In some more preferred embodiments, an example of the blastocyst induction medium is the iBlastoid medium described herein.

[0078] In some embodiments, culturing pESCs in blastocyst induction medium to form porcine blastocysts includes suspending the cultured pESCs in the blastocyst induction medium, optionally for at least 3, 4, 5, 6, or 7 days, or 3 to 7 days, 4 to 7 days, 5 to 7 days, 6 to 7 days, 3 to 4 days, or 3 to 5 days.

[0079] In some embodiments, the method for generating porcine embryo-like structures (preferably porcine blastocysts) from porcine embryonic stem cells (pESCs) as described above further includes:

[0080] pESCs were dissociated into single cells.

[0081] Single cells were cultured into cell clusters using ESC pretreatment medium.

[0082] Cell clusters were directly transferred to blastocyst induction medium without dissociating the cell clusters into individual cells, and...

[0083] Cell clusters were cultured in blastocyst induction medium to form porcine blastocysts.

[0084] In some preferred embodiments, the ESC pretreatment medium comprises Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632.

[0085] In some preferred embodiments, in the ESC pretreatment medium as described above:

[0086] The concentration of activator A is 15-30 ng / mL, preferably 17-23 ng / mL, and more preferably 20 ng / mL;

[0087] The concentration of IGF1 is 10-100 ng / mL, preferably 40-60 ng / mL, more preferably 50 ng / mL; in some more preferred embodiments, the IGF1 is human IGF1;

[0088] The concentration of IL-6 is 10-30 ng / mL, preferably 16-24 ng / mL, and more preferably 20 ng / mL; in some more preferred embodiments, the IL-6 is human IL-6;

[0089] The concentration of the sIL-6 receptor α is 10-30 ng / mL, preferably 16-24 ng / mL, and more preferably 20 ng / mL; in some more preferred embodiments, the sIL-6 receptor α is human sIL-6 receptor α.

[0090] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0091] The concentration of Y-27632 is 1-10 μM, preferably 3-7 μM, and more preferably 5 μM;

[0092] The FBS content is 0.05-0.3%, preferably 0.1-0.2%, and more preferably 0.15%;

[0093] The content of KOSR is 1-10%, preferably 3-7%, and more preferably 5%;

[0094] The concentration of the 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, and more preferably 100 μM;

[0095] And / or,

[0096] The concentration of the 2-phosphate-L-ascorbate salt is 10-100 μg / mL, preferably 30-70 μg / mL, and more preferably 50 μg / mL; in some preferred embodiments, sodium 2-phosphate-L-ascorbate is preferred.

[0097] In some of the most preferred embodiments, a preferred example of the ESC pretreatment medium is the 4FXY medium described herein.

[0098] Therefore, a first aspect of the invention also provides an ESC pretreatment medium as described above. In some embodiments, the ESC pretreatment medium is used to culture single cells of pESCs into cell clusters before transfer to a blastocyst induction medium.

[0099] A second aspect of the invention provides an embryo-like structure obtained by the method according to the first aspect of the invention, wherein the embryo-like structure is a blastocyst.

[0100] A third aspect of the invention provides a blastocyst induction culture medium as described above, wherein the culture medium contains a combination of at least one hypoblastocyst-like cell (HLC) differentiation factor and at least one trophoblastocyst-like cell (TLC) differentiation factor, wherein the HLC differentiation factor is optionally selected from activin A, bFGF and CHIR99021, and the TLC differentiation factor is optionally selected from CHIR99021, SB431542, TSA and BMP4.

[0101] A fourth aspect of the invention provides a culture medium for culturing pESCs, the medium comprising Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939, and Y-27632. In some embodiments, the above-described culture medium for culturing pESCs can be used for blastocyst adhesion during pESC isolation or for the maintenance culture of pESCs.

[0102] In some embodiments, when the culture medium is used for blastocyst adhesion during pESC isolation, the FBS content is 4.5-5.5%, preferably 5-5.3%, and more preferably 5.15%.

[0103] In other embodiments, when the culture medium is used for the maintenance culture of pESCs, the FBS content is 0.05-0.3%, preferably 0.1-0.2%, and more preferably 0.15%.

[0104] In some embodiments, the culture medium for culturing pESCs described above may further contain IWR1; preferably, the concentration of IWR1 is 2-3 μM, more preferably 2.5 μM. In some more preferred embodiments, when the culture medium for culturing pESCs described above is used for the maintenance culture of pESCs, the culture medium further contains IWR1; preferably, the concentration of IWR1 is 2-3 μM, more preferably 2.5 μM.

[0105] In some preferred embodiments, in the culture medium for culturing pESCs as described above:

[0106] The concentration of activator A is 15-30 ng / mL, preferably 17-23 ng / mL, and more preferably 20 ng / mL;

[0107] The concentration of IGF1 is 10-100 ng / mL, preferably 40-60 ng / mL, more preferably 50 ng / mL; in some more preferred embodiments, the IGF1 is human IGF1;

[0108] The concentration of IL-6 is 10-30 ng / mL, preferably 16-24 ng / mL, and more preferably 20 ng / mL; in some more preferred embodiments, the IL-6 is human IL-6;

[0109] The concentration of the sIL-6 receptor α is 10-30 ng / mL, preferably 16-24 ng / mL, and more preferably 20 ng / mL; in some more preferred embodiments, the sIL-6 receptor α is human sIL-6 receptor α.

[0110] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0111] The concentration of Y-27632 is 1-10 μM, preferably 3-7 μM, and more preferably 5 μM;

[0112] The content of KOSR is 1-10%, preferably 3-7%, and more preferably 5%;

[0113] The concentration of the 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, and more preferably 100 μM;

[0114] And / or,

[0115] The concentration of the 2-phosphate-L-ascorbate salt is 10-100 μg / mL, preferably 30-70 μg / mL, and more preferably 50 μg / mL; in some preferred embodiments, sodium 2-phosphate-L-ascorbate is preferred.

[0116] In some more preferred embodiments, a preferred example of the culture medium for blastocyst adhesion during pESC isolation is the culture medium described herein, which is a medium with an additional 5% FBS added to a 4FIXY or 4FXY medium.

[0117] In some other, more preferred embodiments, a preferred example of the culture medium used for pESC maintenance culture is 4FIXY as described herein.

[0118] Methods and materials used in the embodiments

[0119] The establishment of pig ESC

[0120] Porcine oocytes with multilayered cumulus cells were collected from follicles, matured in vitro for 42-46 hours, and treated with hyaluronidase to remove cumulus cells. The oocytes were then exposed to a 60V pulse for 30 μs in activation medium, followed by incubation in PZM-3 medium (Non-Patent Literature 23). For pESC establishment, the entire PA blastocyst was digested with 0.5% streptomycin (Sigma-Aldrich, P8811) to remove the zona pellucida, and then cultured in 4FXY or 4FIXY medium with an additional 5% FBS until the blastocyst adhered to the culture vessel. Using TrypLE... TM Select cells dissociated the derivative and re-seeded them onto mitomycin C-treated mouse embryonic fibroblast feeder cells in 4-FIXY medium. After approximately three days, the cells formed dense colonies. The cells were cultured at 38.5°C, 5% O2, and 5% CO2.

[0121] 4. FIXY medium consisted of Neurobasal medium (Gibco, 21103049) and DMEM / F12 (Gibco, 10565018) mixed 1:1 (v / v), supplemented with N2 (Gibco, 17502048) and B27 (Gibco, 17504044) supplements, NEAA (Gibco, 11140050), GlutaMAX (Gibco, 35050061), penicillin / streptomycin (Gibco, 15140122), 5% KOSR, 100 μM 2-mercaptoethanol (Sigma, M3148), and 0.15% hydroxychloroquine. The medium consisted of FBS (Gibco, 10099141C), 20 ng / mL human IL-6 (Peprotech, AF-200-06), 20 ng / mL human sIL-6 receptor α (Peprotech, 200-06RC), 20 ng / mL activin A (Peprotech, 120-14-1000), 50 ng / mL human IGF1 (MCE, HY-P7018), 2.5 μM XAV939 (Selleck, S1108), 2.5 μM IWR1 (Selleck, S7086), 50 μg / mL sodium 2-phosphate-L-ascorbate (Sigma, 49752), and 5 μM Y-27632 (TargetMol, T1725). 4FXY medium was prepared by subtracting IWRI from 4FIXY medium, with all other components and concentrations identical.

[0122] Cultured pigs ESC

[0123] pESCs were maintained on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts and passaged every 3-4 days via enzymatic digestion. They were then treated with TrypLE. TM Cells were dissociated into single cells using the Select method (38.5℃, 5 min), centrifuged (250 g, 5 min), resuspended, and seeded at a 1:3 ratio in 4-FIXY medium. Cells were cultured at 38.5℃, 5% O2, and 5% CO2.

[0124] EB (embryomorph) formation

[0125] Using TrypLE TMPredifferentiated pESCs were isolated using Select (38.5℃, 5 min) and plated in EB-forming medium into ultra-low adhesion multi-well plates for 7 days. EB was then attached to gelatin-coated plates in DMEM (HyClone, SH30022.01B) containing 15% FBS and 5 μM Y-27632 for 3 days, followed by 4 days of culture in DMEM containing 15% FBS. Cells were then collected for analysis. Cells were cultured at 38.5℃, 5% O2, and 5% CO2.

[0126] The EB formation medium consisted of Neurobasal medium (Gibco) and DMEM / F12 (Gibco) mixed at a 1:1 (v / v) ratio, supplemented with N2 (Gibco) and B27 (Gibco) supplements, NEAA (Gibco), GlutaMAX (Gibco), penicillin / streptomycin (Gibco), 5% KOSR, 100 μM 2-mercaptoethanol (Sigma) and 5 μM Y-27632 (TargetMol).

[0127] Production of porcine blastocysts

[0128] In the one-step method, pESC is used at 38.5℃ with TrypLE. TM Select digestion for 5 minutes yields single cells. Cells are collected and resuspended in iBlastoid medium. Approximately 40,000 single cells per well are directly seeded into ultra-low adhesion multi-well plates and cultured in iBlastoid medium for 5–7 days. In a two-step induction method, pESCs are dissociated at 38.5°C using TrypLE™ Select for 5 minutes to form single cells. Cells are collected and resuspended in 4FXY medium. Approximately 40,000 single cells per well are seeded into ultra-low adhesion multi-well plates and cultured in 4FXY medium for 1–2 days. Cell clusters formed from pESCs are then cultured in iBlastoid medium for 3–5 days. All resulting porcine blastocysts are manually picked using a pipette under a stereomicroscope for downstream experiments. Blastocysts are cultured at 38.5°C, 20% O2, and 5% CO2.

[0129] iBlastoid medium consists of Neurobasal (Gibco) medium and DMEM / F12 (Gibco) mixed at a 1:1 (v / v) ratio, and contains N2 (Gibco) and B27 (Gibco) supplements, NEAA (Gibco), GlutaMAX (Gibco), penicillin / streptomycin (Gibco), 0.075% FBS, 5% KOSR, 100 μM 2-mercaptoethanol (Sigma), 50 μg / mL sodium L-phosphate ascorbate (Sigma), 10 ng / mL human IL-6 (Peprotech), 10 ng / mL human sIL-6 receptor α (Peprotech), 10 ng / mL activin A (Peprotech), 5 ng / mL human LIF (Peprotech, 300-025), 50 ng / mL human IGF1, 5 ng / mL BMP4 (R&D, 314-BP), and 10 ng / mL bFGF (Peprotech, 100-18B), 1.5μM CHIR99021 (Tocris, 4423), 2.5μM XAV939 (Selleck), 1μM SB431542 (Selleck, S1067), 7.5μM Y-27632 (TargetMol), 2.5nM DZNep (Selleck, S7120) and 2.5nM TSA (Solarbio, IT1250).

[0130] Stem cell lines were isolated from porcine blastocysts.

[0131] Single porcine blastocysts were transferred to mitomycin C-treated mouse embryonic fibroblast feeder cells in 4-well plates and cultured in 4FIXY medium (for pESC) and TSC medium (for pTSC). Using TrypLE... TM Select derivatives observed within 7 days were dissociated and passaged into mitomycin C-treated mouse embryonic fibroblast feeder cells in the appropriate culture medium. 5% FBS was added to the medium until the blastocysts adhered. Cells were cultured at 38.5°C, 5% O2, and 5% CO2.

[0132] Based on a previous report (Non-Patent Document 24), the TSC medium was modified to consist of Neurobasal medium and DMEM / F12 mixed in a 1:1 (v / v) ratio, with the addition of N2 and B27 supplements, NEAA, GlutaMAX, penicillin / streptomycin, 100 μM 2-mercaptoethanol, 50 μg / mL sodium L-ascorbate 2-phosphate, 50 ng / mL human EGF (Peprotech, AF-100-15), 1 μM SB431542, 2 μM CHIR99021, 0.8 mM VPA (Solarbio, IV0010), 10 μM Y-27632 (TargetMol), and 0.5 μM A83-01 (STEMCELL, 72022).

[0133] Long-term in vitro culture of porcine blastocysts

[0134] Porcine blastocysts were manually picked up using a pipette and transferred to new ultra-low adhesion multiwell plates containing iBlastoid medium or N2B27+AY medium. The blastocysts were cultured at 38.5°C, 20% O2, and 5% CO2.

[0135] The N2B27+AY medium is a modified version of bovine and ovine embryo IVC medium (Non-Patent Literature 22, 25). The N2B27+AY medium consists of Neurobasal medium and DMEM / F12 mixed at a 1:1 (v / v) ratio, with the addition of N2 and B27 supplements, NEAA, GlutaMAX, penicillin / streptomycin, 5% KOSR, 100 μM 2-mercaptoethanol, 20 ng / mL activator A and 10 μM Y-27632.

[0136] Immunofluorescence staining

[0137] Cells, blastocysts, and blastocysts were fixed with 4% paraformaldehyde (PFA) at room temperature for approximately 30 minutes, washed in DPBS for 5 minutes, and permeabilized with 0.2% Triton X-100 in DPBS for 30 minutes. For surface label staining, samples were not permeabilized. Samples were then blocked with blocking buffer at room temperature for 40 minutes. Primary antibody was diluted in primary antibody dilution buffer. 96-well blastocysts / blastocysts or cells were incubated overnight at 4°C with primary antibody in the primary antibody solution. Samples were washed three times with DPBS for 15 minutes each time and incubated with secondary antibody (conjugated to a fluorescent dye diluted in secondary antibody dilution buffer) at room temperature for 1 hour. Samples were washed three times with DPBS. Finally, samples were counterstained with DAPI at room temperature for 3 minutes. blastocysts were imaged on 8-well μ-slides (ibidi). Blastocysts were imaged after being pressed with coverslips.

[0138] Karyotype analysis

[0139] Prior to karyotype analysis, pESCs were treated with medium containing 4 μg / mL colchicine for 2 hours. Cells were digested, centrifuged, resuspended in 0.075 M KCl hypotonic solution, and incubated at 37°C for 15 minutes. pESCs were fixed with a pre-chilled 3:1 (v / v) mixture of methanol and acetic acid, and this step was repeated three times. The resuspended pESCs were placed on pre-chilled slides, dried, and stained with Giemsa stain. Karyotype analysis was performed under a 100X microscope.

[0140] Teratoma formation

[0141] Digestion is approximately 0.5-1 × 10⁻⁶. 7 One pESC was collected and centrifuged (250g, 5 minutes) to collect the teratoma, which was then subcutaneously injected into the dorsal subcutaneous tissue of NCG mice. Approximately 10 weeks later, the teratomas were collected and sectioned.

[0142] Hematoxylin and eosin staining

[0143] Teratomas were collected and fixed with 4% PFA for at least 24 hours. The teratoma tissue was dehydrated with graded ethanol, transferred to xylene, embedded in paraffin, and cut into continuous 5 μm thick cross-sectional sections. The cross-sectional sections were dewaxed in xylene, rehydrated with graded ethanol, and stained with hematoxylin and eosin.

[0144] Batch RNA sequencing and analysis

[0145] A total of 1 μg of RNA was used for subsequent library construction, sequencing, and analysis. UltraTM RNA LibraryPrep Kit for Sequencing libraries were generated. Library quality was assessed on an Agilent Bioanalyzer 2100 system. The library preparations were sequenced on an Illumina NovaSeq platform (Illumina, USA), generating 150 bp paired-end reads. RNA-seq reads were corrected using Trim Galore (v0.6.4), then mapped to the susScr11 reference genome using HISAT2 (v2.2.1), and the transcriptional level of each gene in each sample was quantified using StringTie (v2.2.1). Samples were converted to FPKM (kilobase fragments per million mapped reads).

[0146] Single-cell sequencing

[0147] Porcine blastocysts with EPI-like, TE-like compartments and cavities were manually selected using a pipette and dissociated with 0.25% trypsin-EDTA at 37°C for 10–20 minutes to form single cells. Dissociation was terminated with 15% FBS, and the sample was centrifuged at 500g for 5 minutes. The cell pellet was resuspended in DMEM / F12 and filtered through a 40μm cell filter. Single-cell suspension was performed using the 10xGenomics Chromium Single Cell Kit for single-cell sequencing.

[0148] Single-cell sequencing data collection

[0149] The dataset for pigs includes CRA003960 (non-patent document 29).

[0150] Single-cell sequencing data analysis

[0151] Quality control was performed using Dr.seq2 (v2.2.1) (Non-Patent Literature 27), and scRNA-seq reads were mapped to the mm10 reference genome using 10x Genomics CellRanger (v7.1.0) (Non-Patent Literature 26). Subsequently, Seurat (v4.3.0) (Non-Patent Literature 28) was used to integrate samples with the transcriptional level of each gene in each sample as input. After standardization, dimensionality reduction and clustering were performed using the Seurat functions RunUMAP() and FindCluster(). For each cell type, the FindAllMarkers() function was used to find genes with a mean logarithmic fold change >0.8 and a differential expression percentage >30%. Based on the E7-E9 embryo dataset (Non-Patent Literature 29), cell types derived from porcine embryos were classified using scPred (Non-Patent Literature 30), including EPI, HYPO, and TE; other cells with multiple characteristics were designated as "undefined". This predictive model was applied to single-cell sequencing data of porcine blastocysts, classifying cells derived from blastocysts into four cell categories (EPI lineage, HYPO lineage, TE lineage, and undefined).

[0152] Data Analysis

[0153] The p-values ​​were calculated using an unpaired t-test and are shown in the relevant figures. A p-value < 0.05 was considered statistically significant. Specific Implementation

[0154] Example 1 uses 4FIXY medium to establish pESC

[0155] Prior to generating porcine blastocysts, the inventors established pESCs from parthenogenetic (PA) blastocysts using a novel culture medium. Based on transcriptomic data from early porcine embryonic development and publicly available culture media, the inventors tested four cytokines—activin A, IGF1, IL-6, and sIL-6 receptor α—as well as small molecule chemical inhibitors, including the WNT signaling inhibitors XAV939 and IWR1, and the ROCK inhibitor Y-27632, for culturing pESCs. The resulting pESCs could be passaged as single cells every 3–4 days at a 1:3 ratio via enzymatic digestion, forming dense, well-defined colonies (Fig. 1a). Immunofluorescence staining showed that pESCs expressed pluripotent markers, including POU5F1, SOX2, NANOG, OTX2, and E-cadherin (Fig. 1b). During long-term culture, pESCs were alkaline phosphatase (AP) positive and exhibited a normal karyotype (Fig. 1c, d). Furthermore, pESCs can form embryoid bodies (EBs) and differentiate into three germ layers in vitro (Fig. 1e, f). In immunodeficient mice, pESCs can form teratomas containing endoderm, mesoderm, and ectoderm (Fig. 1g). These results demonstrate that the inventors have successfully established pESCs under 4FIXY culture conditions.

[0156] Example 2: Effective generation of blastocysts using pESC

[0157] To test whether pESCs could assemble into blastocysts in vitro, the inventors first examined their ability to differentiate into extraembryonic lineages, including trophectoderm (also referred to as TE) and hypoblast (also referred to as HYPO). For this purpose, the inventors established new culture conditions (hence the name blastocyst induction medium, with iBlastoid medium as an example) to culture pESCs for 3 days. The inventors observed the gradual emergence of epiblast-like cells (also referred to as ELC), trophectoderm-like cells (also referred to as TLC), and hypoblast-like cells (also referred to as HLC) (Fig. 8a). Immunofluorescence staining revealed the identification of GATA3 and GATA6 positive cells, representing TLC and HLC, respectively (Fig. 8b). Both GATA3 and GATA6 positive cells were observed in the same view as SOX2 positive cells (ELC) (Fig. 8b). These results demonstrate that pESCs possess the potential to differentiate into extraembryonic lineages in vitro, a prerequisite for blastocyst generation.

[0158] The inventors then attempted to reconstruct porcine blastocysts under 3D culture conditions (Fig. 8c). First, they prepared a blastocyst induction medium (iBlastoid in this paper is an example) based on an expression dataset of early porcine embryonic development, including the cytokines LIF, activin A, IGF1, IL-6, and sIL-6 receptor α. The inventors also used the chemical reagents CHIR99021, SB431542, TSA, and BMP4 to support TLC formation. Furthermore, activin A, bFGF, and CHIR99021 were added to the medium as they contribute to HLC differentiation. In a one-step process, pESCs were digested into single cells and directly seeded into ultra-low attachment multiwell plates in iBlastoid medium at specified cell numbers. The inventors demonstrated that porcine blastocysts, containing blastocyst-like cavities, EPI-like compartments, and TE-like outer layers, gradually emerged on days 5–7 (Fig. 8d). Immunofluorescence staining revealed the expression of three major lineage markers in these blastocysts, including the EPI marker SOX2, the HYPO marker GATA6, and the TE marker CDX2 (Fig. 8e). The proportion of SOX2-positive blastocysts ranged from 18.4% to 34% (Fig. 8f).

[0159] To improve the efficiency of porcine blastocyst formation, the inventors modified and developed a more robust 3D two-step method (Fig. 2a and Fig. 8f). In the two-step induction method, pESCs dissociated into single cells were seeded into ultra-low adhesion multiwell plates containing 4FXY medium (4FIXY without IWR1), forming cell clusters on days 1-2; the cell clusters were then maintained in iBlastoid medium for 3-5 days (Fig. 2a). The proportion of SOX2-positive blastocysts was 25.9% to 55% (Fig. 8f). In iBlastoid medium, the cell clusters gradually formed blastocyst-like cavities, EPI-like cells, and TE-like cell layers (Fig. 2b). The blastocysts formed in vitro on day 6 were morphologically similar to porcine PA blastocysts at embryonic days 6 (E6) and E7 (Fig. 2c). The porcine blastocyst formation efficiency was 4.95% to 29.68% (Fig. 2d). Furthermore, the diameter of the porcine blastocysts (average 183 μm) is similar to that of porcine E6 PA blastocysts (average 192 μm), both smaller than that of E7 PA blastocysts (average 224 μm) (Fig. 2e). The X / Y ratio of the porcine blastocysts is approximately 1, indicating that the blastocysts are round, similar to porcine PA blastocysts at E6 and E7 (Fig. 2f). In summary, the inventors have demonstrated that porcine blastocysts can be efficiently generated from pESC in a 3D system.

[0160] Example 3: Porcine blastocysts exhibit blastocyst characteristics.

[0161] To investigate whether the reconstructed porcine blastocysts contained the three cell lineages of blastocysts, the inventors performed immunofluorescence staining to detect markers of EPI (SOX2), HYPO (GATA6), and TE (GATA3), and showed that porcine blastocysts contained these cell lines. Figure 2g The inventors also identified other markers for various lineages in porcine blastocysts, such as EPI (POU5F1), HYPO (GATA4), and TE (CDX2). Figure 9a (b) The inventors then calculated the cell number and the proportions of the three cell lineages in porcine blastocysts. The blastocysts contained more cells than the E6 and E7 PA blastocysts. Compared to the E6 PA blastocysts, the blastocysts contained more ELCs and TLCs, while the proportions of ELCs and TLCs were comparable to those in the E6 PA blastocysts (Fig. 2h, i). Furthermore, the number of HLCs was similar in porcine blastocysts and E6 PA blastocysts, but the proportion of HLCs in porcine blastocysts was lower than that in porcine E6 PA blastocysts (Fig. 2h, i). Compared to the E7 PA blastocysts, both the number and proportion of ELCs were higher. The number of HLCs and TLCs were similar, but the proportions were lower in porcine blastocysts (Fig. 2h, i). Tight junctions between TLCs in the blastocysts could be detected by immunofluorescence staining with ZO-1 (Fig. 2j). In summary, these data indicate that pESC-induced blastocysts exhibit characteristics similar to porcine blastocysts.

[0162] Example 4: Single-cell transcriptome of porcine blastocysts

[0163] To determine the transcriptional profile of porcine blastocyst-derived cells, the inventors performed single-cell RNA sequencing (scRNA-seq) on two samples (Figure 3a). UMAP analysis showed that porcine blastocyst-derived cells could be divided into 13 clusters (Figure 10a). The inventors identified ELC, HLC, and TLC using known characteristic markers, including SOX2 and POU5F1 for ELC, GATA6 and GATA4 for HLC, and GATA3 and GATA2 for TLC (Figures 3b, c, and 10b). Cell cluster 5 expressed SOX2 and POU5F1, representing ELC; cell clusters 7 and 10 expressed GATA6 and GATA4, representing HLC; and seven clusters (0, 1, 2, 3, 4, 6, 12) expressed GATA3 and GATA2, labeled as TLC (Figures 3b and 10a). For clusters 8, 9, and 11, the percentage of cells expressing marker genes was less than 50%, and the corrected mean expression level of all marker genes was less than 0.1, making it difficult to determine their cell type. Therefore, these cell clusters were excluded. Each lineage cell in porcine blastocysts exhibited a unique gene expression pattern, similar to its corresponding lineage cell in porcine embryos (Fig. 3d, e, and Fig. 10c). For example, EPI genes (DNMT3B, ETV5, ZIC2) were specific to the ELC cluster. The HYPO gene NID2 was specific to the HLC cluster; another HYPO gene, COL4A1, was highly expressed in the HLC cluster. TE genes (KRT8, SFN) were most highly expressed in the TLC cluster (Fig. 10d, e). These results confirm the presence of EPI, HYPO, and TE-like lineage cells in porcine blastocysts.

[0164] Next, the inventors investigated whether porcine blastocysts derived from pESCs were similar to porcine blastocysts at the transcriptomic level. They performed an integrated analysis of Smart-seq2 single-cell transcriptomes obtained from porcine oocytes and embryos at different stages. Principal component analysis (PCA) showed that cells derived from blastocysts were more similar to blastocyst cells (Figure 4a). To further assess the similarity between lineage cells derived from blastocysts and blastocysts, an integrated analysis using two comparative methods was conducted to perform unbiased and quantitative analysis of cells derived from blastocysts. In early porcine embryonic development, the first lineage separation begins at E5; the second lineage separation begins at E6 and ends at E7; subsequently, the EPI transforms into the ectoderm starting at E10, indicating that the three lineage cells—EPI, TE, and HYPO—can be separated at E7, E8, and E9. Therefore, to investigate the associations among the three cell lineages derived from blastocysts and blastocysts—namely, ELC and EPI, TLC and TE, and HLC and HYPO—single-cell transcriptome data from porcine E7, E8, and E9 embryos were integrated for analysis (Figure 4b). EPI, HYPO, and TE were identified using EPI markers SOX2 and POU5F1, HYPO markers GATA6 and GATA4, and TE markers GATA3 and GATA2 (Figures 4c-e and 11a). Subsequent UMAP analysis revealed that ELC, TLC, and HLC in blastocysts overlapped with corresponding regions of EPI, TE, and HYPO in blastocysts, respectively. Figure 4f Furthermore, scPred (an accurate supervised cell type classification method) was used to classify blastocyst-derived cell types based on the E7-E9 embryo dataset. Blastocyst-derived cells were mapped onto the E7-E9 embryo dataset, where lineages were divided into EPI, HYPO, TE, and undefined clusters. scPred predicted that 91%, 85%, and 54% of blastocyst-derived cells corresponding to the respective lineage groups possessed EPI, HYPO, and TE lineage characteristics, respectively (Figure 11b).

[0165] Overall, these results indicate that pESC-induced blastocysts have three characteristic lineages, and are therefore very similar to the transcriptomic features of blastocysts.

[0166] Example 5: Obtaining stem cells from porcine blastocysts

[0167] The inventors then investigated whether stem cells could be obtained from these blastocysts in the same manner as from blastocysts (Fig. 1a). To this end, the inventors demonstrated that ESCs and TSCs could be de novo isolated from porcine blastocysts under the same culture conditions in a specified medium (Fig. 5a). Derivatives derived from blastocysts could be formed, producing pESCs with dense colonies (Fig. 5b) that were positive for AP staining and expressed pluripotent markers, including POU5F1, E-cadherin, SOX2, NANOG, SSEA4, and β-catenin (Fig. 5c, d). Using TSC medium, porcine TSCs (pTSCs) expressing the TE marker GATA3 could also be established (Fig. 5e, f). In summary, these results demonstrate that stem cells can be isolated from these blastocysts, just like from blastocysts.

[0168] Example 6: Long-term in vitro culture of porcine blastocysts

[0169] The inventors then explored the possibility of growing and developing these blastocysts in vitro. To the best of our knowledge, in vitro culture (IVC) methods for porcine embryos have not been established. Based on IVC systems for bovine and ovine embryos, the inventors modified these culture conditions and developed the N2B27+AY medium for blastocyst culture. iBlastoid medium was also used for long-term in vitro culture. Porcine blastocysts with EPI-like cells and a distinct cavity at day 6 (denoted as porcine blastocyst IVC day 0) were selected for long-term in vitro culture. When IVC of the porcine blastocysts continued until day 18, the cavity of the blastocysts continued to grow and expand, and the blastocyst size increased (Fig. 6a, b). At day 18, the diameter of porcine blastocysts in N2B27+AY medium ranged from 234 μm to 1707 μm (mean 722 μm), while in iBlastoid medium it ranged from 171 μm to 1234 μm (mean 384 μm) (Fig. 6c). On day 6, the survival rates of blastocysts in N2B27+AY and iBlastoid media were 27.31% and 49.07%, respectively, decreasing to 1.72% and 2.96% on day 18 (Fig. 6d). As a control, porcine PA blastocysts grew for approximately 6 and 8 days in N2B27+AY and iBlastoid media, respectively, and then decreased in size with increasing IVC days (Fig. 6e-g).

[0170] Fluorescent staining was performed on three lineage markers in IVC blastocysts: EPI (SOX2), HYPO (GATA6), and TE (GATA3). The results showed that IVC blastocysts at day 18 contained SOX2, GATA3, and GATA6 positive cells (Figure 12).

[0171] Overall, these results indicate that pESC-derived blastocysts have the potential to grow under long-term in vitro culture conditions.

Claims

1. A method for generating porcine embryo-like structures from porcine embryonic stem cells, comprising culturing porcine embryonic stem cells in a blastocyst induction medium to form porcine blastocysts, wherein the blastocyst induction medium contains a combination of hypoblastocyst-like cell differentiation factors and trophoblastocyst-like cell differentiation factors. The hypodermal-like cell differentiation factors include activin A, bFGF, and CHIR99021. Trophoblastic cell differentiation factors include CHIR99021, SB431542, TSA, and BMP4; The concentration of activator A is 5-15 ng / mL; The concentration of bFGF is 5-15 ng / mL; The concentration of CHIR99021 is 1.3-1.7 μM; The concentration of SB431542 is 0.5-1.5 μM; The concentration of the TSA is 2-3 nM; The concentration of BMP4 is 1-10 ng / mL; The blastocyst induction medium also contains LIF, IGF1, IL-6, sIL-6 receptor α, XAV939, Y-27632 and DZNep.

2. The method according to claim 1, wherein the blastocyst induction medium comprises Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, LIF, IGF1, BMP4, bFGF, CHIR99021, XAV939, SB431542, Y-27632, DZNep and TSA.

3. The method according to claim 1 or 2, wherein the concentration of activator A is 8-12 ng / mL.

4. The method according to claim 1 or 2, wherein the concentration of kinetin A is 10 ng / mL.

5. The method according to claim 1 or 2, wherein the concentration of bFGF is 8-12 ng / mL.

6. The method according to claim 1 or 2, wherein the concentration of bFGF is 10 ng / mL.

7. The method according to claim 1 or 2, wherein the concentration of CHIR99021 is 1.5 μM.

8. The method according to claim 1 or 2, wherein the concentration of SB431542 is 0.8-1.2 μM.

9. The method according to claim 1 or 2, wherein the concentration of SB431542 is 1 μM.

10. The method according to claim 1 or 2, wherein the concentration of the TSA is 2.3-2.7 nM.

11. The method according to claim 1 or 2, wherein the concentration of the TSA is 2.5 nM.

12. The method according to claim 1 or 2, wherein the concentration of BMP4 is 3-7 ng / mL.

13. The method according to claim 1 or 2, wherein the concentration of BMP4 is 5 ng / mL.

14. The method according to claim 1 or 2, wherein the concentration of LIF is 1-10 ng / mL.

15. The method according to claim 1 or 2, wherein the concentration of IGF1 is 10-100 ng / mL.

16. The method according to claim 1 or 2, wherein the concentration of IL-6 is 5-15 ng / mL.

17. The method according to claim 1 or 2, wherein the concentration of sIL-6 receptor α is 5-15 ng / mL.

18. The method according to claim 1 or 2, wherein the concentration of XAV939 is 2-3 μM.

19. The method according to claim 1 or 2, wherein the concentration of Y-27632 is 3-15 μM.

20. The method according to claim 1 or 2, wherein the concentration of DZNep is 2-3 nM.

21. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for at least 3 days.

22. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for at least 4 days.

23. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for at least 5 days.

24. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for at least 6 days.

25. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for at least 7 days.

26. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 3 to 7 days.

27. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 4 to 7 days.

28. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 5 to 7 days.

29. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 6 to 7 days.

30. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 3 to 4 days.

31. The method according to claim 1 or 2, wherein the cultured porcine embryonic stem cells are suspended in blastocyst induction medium for 3 to 5 days.

32. The method according to claim 1 or 2, wherein, The method further includes: Pig embryonic stem cells were dissociated into single cells. Single cells were cultured into cell clusters using ESC pretreatment medium. Cell clusters were directly transferred to blastocyst induction medium without dissociating the cell clusters into individual cells, and... Cell clusters were cultured in blastocyst induction medium to form porcine blastocysts.

33. The method of claim 32, wherein the ESC pretreatment medium comprises Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632.

34. The method according to claim 1 or 2, wherein the embryo-like structure is a blastocyst.

35. The embryo-like structure obtained by the method according to any one of claims 1 to 34, wherein the embryo-like structure is a blastocyst.

36. A blastocyst induction culture medium for inducing porcine embryonic stem cells to form porcine embryo-like structures, wherein the culture medium contains a combination of hypoblastocyst-like cell differentiation factors and trophoblast-like cell differentiation factors. in, The hypodermal-like cell differentiation factors include activin A, bFGF, and CHIR99021. The trophoblast-like cell differentiation factors include CHIR99021, SB431542, TSA, and BMP4. The embryo-like structure is a blastocyst-like structure; The concentration of activator A is 5-15 ng / mL; The concentration of bFGF is 5-15 ng / mL; The concentration of CHIR99021 is 1.3-1.7 μM; The concentration of SB431542 is 0.5-1.5 μM; The concentration of the TSA is 2-3 nM; The concentration of BMP4 is 1-10 ng / mL; The blastocyst induction medium also contains LIF, IGF1, IL-6, sIL-6 receptor α, XAV939, Y-27632 and DZNep.

37. The culture medium according to claim 36, wherein the blastocyst induction medium comprises Neurobasal medium mixed in a 1:1 (v / v) ratio with DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, L-ascorbate 2-phosphate, IL-6, sIL-6 receptor α, activin A, LIF, IGF1, BMP4, bFGF, CHIR99021, XAV939, SB431542, Y-27632, DZNep and TSA.

38. The culture medium according to claim 36 or 37, wherein the concentration of activator A is 8-12 ng / mL.

39. The culture medium according to claim 36 or 37, wherein the concentration of kinetin A is 10 ng / mL.

40. The culture medium according to claim 36 or 37, wherein the concentration of bFGF is 8-12 ng / mL.

41. The culture medium according to claim 36 or 37, wherein the concentration of bFGF is 10 ng / mL.

42. The culture medium according to claim 36 or 37, wherein the concentration of CHIR99021 is 1.5 μM.

43. The culture medium according to claim 36 or 37, wherein the concentration of SB431542 is 0.8-1.2 μM.

44. The culture medium according to claim 36 or 37, wherein the concentration of SB431542 is 1 μM.

45. The culture medium according to claim 36 or 37, wherein the concentration of TSA is 2.3-2.7 nM.

46. ​​The culture medium according to claim 36 or 37, wherein the concentration of TSA is 2.5 nM.

47. The culture medium according to claim 36 or 37, wherein the concentration of BMP4 is 3-7 ng / mL.

48. The culture medium according to claim 36 or 37, wherein the concentration of BMP4 is 5 ng / mL.

49. The culture medium according to claim 36 or 37, wherein the concentration of LIF is 1-10 ng / mL.

50. The culture medium according to claim 36 or 37, wherein the concentration of IGF1 is 10-100 ng / mL.

51. The culture medium according to claim 36 or 37, wherein the concentration of IL-6 is 5-15 ng / mL.

52. The culture medium according to claim 36 or 37, wherein the concentration of sIL-6 receptor α is 5-15 ng / mL.

53. The culture medium according to claim 36 or 37, wherein the concentration of XAV939 is 2-3 μM.

54. The culture medium according to claim 36 or 37, wherein the concentration of Y-27632 is 3-15 μM.

55. The culture medium according to claim 36 or 37, wherein the concentration of DZNep is 2-3 nM.

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