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

Abstract

Provided are a method for producing a porcine blastoid from porcine embryonic stem cells (pESC), and a blastoid obtained by the method. Also provided are media for inducing the pESC to form blastula-like embryos and media for producing and culturing the pESC.

Description

Technical Field

[0001] The present invention relates to a method for generating a porcine blastocyst-like structure from porcine embryonic stem cells (pESC), and a blastoid obtained by the method. The present invention also relates to a culture medium for inducing pESC into a blastocyst-like structure and a culture medium for generating pESC. Background Art

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

[0003] Among them, blastocysts can be used to study key events in preimplantation embryogenesis and early embryonic development. In mice, co-culturing mouse embryonic stem cells (ESC) and mouse trophoblast stem cells (TSC) can produce blastocysts (non-patent document 1); mouse expanded pluripotent stem cells (EPSC) and epiblast stem cells (EpiSC) can self-assemble in a 3D culture system to form blastocysts (non-patent documents 2 to 5); mouse ESC, TSC and extraembryonic endoderm stem cells (XEN) can be assembled into embryonic-like structures (ETX) (non-patent documents 6 and 7), and can also form embryonic-like structures with complete gastrula and further develop into organ progenitor cells similar to E8.5 embryos (non-patent documents 8 and 9). In humans, using primitive PSCs ( The use of ESCs or EPSCs can produce blastocysts similar to human blastocysts in morphology, cell lineage composition, and transcriptome characteristics (non-patent literatures 10 to 14); the use of somatic cell reprogramming or intermediate cells in the process of transition from the initial state to the primitive state can also form human blastocysts (non-patent literatures 15 and 16); the use of ESCs and EPSCs can form human embryonic models after implantation (non-patent literatures 17 to 20). In addition, blastocysts from non-human primates (such as cynomolgus monkeys) can be formed using primitive ESCs, which can develop to gastrulation and early pregnancy stages in vitro (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 have shown that ESCs of mice, humans and monkeys can be induced to produce blastocysts. However, ESCs from livestock animals (such as pigs, cattle and sheep) cannot be induced to form blastocysts. So far, only blastocysts of cattle can be formed by assembling EPSCs and TSCs together (non-patent literature 22). At present, there are no reports on the formation of pig blastocysts, and it is still unclear whether pESCs can be induced to form blastocysts.

[0005] Non-patent literature

[0006] [Non-patent document 1] Rivron, NC et al., Blastocyst-like structures generated solely from stem cells. Nature 557, 106-111 (2018).

[0007] [Non-patent document 2] Li, R. et al., Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures. Cell 179, 687-702e618 (2019)

[0008] [Non-patent document 3] Sozen, B. et al., Self-Organization of Mouse Stem Cells into an Extended Potential Blastoid. Dev Cell 51, 698-712 (2019)

[0009] [Non-patent document 4] Liu, K. et al., Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm. Protein Cell 14, 262-278 (2023).

[0010] [Non-patent document 5] Kime, C. et al., Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells. Stem Cell Rep 13, 485-498 (2019).

[0011] [Non-patent document 6] Zhang, S. et al., Implantation initiation of self-assembledembryo-like structures generated using three types of mouse blastocyst-derived stem cells. Nat Commun 10, 496 (2019).

[0012] [Non-patent document 7] Amadei, G. et al., Inducible Stem-Cell-Derived Embryos Capture Mouse Morphogenetic Events In Vitro. Dev Cell 56, 366-382e369 (2021).

[0013] [Non-patent document 8] Tarazi, S. et al., Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs. Cell 185, 3290-3306e3225 (2022).

[0014] [Non-patent document 9] Amadei, G. et al., Embryo model completes gastrulation to neurulation and organogenesis. Nature 610, 143-153 (2022).

[0015] [Non-patent document 10] Yanagida, A. et al., Naive stem cell blastocyst model captures human embryo lineage segregation. Cell Stem Cell 28, 1016-1022e1014 (2021).

[0016] [Non-patent document 11] Yu, L. et al., Blastocyst-like structures generated from human pluripotent stem cells. Nature 591, 620-626 (2021).

[0017] [Non-patent document 12] Kagawa, H. et al., Human blastoids model blastocyst development and implantation. Nature 601, 600-605 (2022).

[0018] [Non-patent document 13] Fan, Y. et al., Generation of human blastocyst-like structures from pluripotent stem cells. Cell Discov 7, 81 (2021).

[0019] [Non-patent document 14] Sozen, B. et al., Reconstructing aspects of human embryogenesis with pluripotent stem cells. Nat Commun 12, 5550 (2021).

[0020] [Non-patent document 15] Liu, X. et al., Modeling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591, 627-632 (2021).

[0021] [Non-patent document 16] Tu, Z. et al., Modeling human pregastrulation development by 3D culture of blastoids generated from primed-to-naive transitioningintermediates. Protein Cell 14, 337-349 (2023).

[0022] [Non-patent document 17] Pedroza, M. et al., Self-patterning of human stem cells into post-implantation lineages. Nature (2023).

[0023] [Non-patent document 18] Weatherbee, BAT et al., A model of the post-implantation human embryo derived from pluripotent stem cells. Nature (2023).

[0024] [Non-patent document 19] Ai, Z. et al., Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res (2023).

[0025] [Non-patent document 20] Liu, L. et al., Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. Cell (2023).

[0026] [Non-patent document 21] Li, J. et al., Cynomolgus monkey embryo model captures gastrulation and early pregnancy. Cell Stem Cell 30, 362-377e367 (2023).

[0027] [Non-patent document 22] Pinzon-Arteaga, CA et al., Bovine blastocyst-like structures derived from stem cell cultures. Cell Stem Cell 30, 611-616e617 (2023).

[0028] [Non-patent document 23] Cao, Z. et al., Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis. Theriogenology 81, 496-508 (2014).

[0029] [Non-patent document 24] Okae, H. et al., Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22, 50-63e56 (2018).

[0030] [Non-patent document 25] Ramos-Ibeas, P. et al., In vitro culture of ovine embryos upto early gastrulating stages. Development 149 (2022).

[0031] [Non-patent document 26] Zheng, GXY et al., Massively parallel digital transcriptional profiling of single cells. Nat Commun 8 (2017).

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

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

[0035] [Non-patent document 30] Alquicira-Hernandez, J. et al., scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data. Genome Biol 20 (2019). SUMMARY OF THE INVENTION

[0037] The inventors first developed a novel culture system that can reproducibly produce porcine ESCs (pESCs). On the other hand, the inventors further developed a three-dimensional (3D) two-step differentiation system for inducing pESCs to produce porcine blastocyst-like structures (hereinafter referred to as blastocyst-like structures) in vitro. It has been confirmed that the blastocyst-like structures are almost indistinguishable from blastocysts in terms of morphology, cell composition, and single-cell transcriptome. The inventors also provided two different sets of in vitro culture conditions, both of which can maintain porcine blastocyst-like structures in vitro for at least 18 days.

[0038] The use of blastocysts from large animals (e.g., blastocysts derived from pESCs) can achieve in vitro modeling of early embryogenesis, improve the breeding system of livestock animals such as pigs, and accelerate the process of breeding excellent varieties.

[0039] Given that pigs and humans have similar physiological and anatomical structures, pig organs are considered to be suitable substitutes for humanized organs. The present invention provides an excellent in vitro model for exploring human-pig cell interactions and helps advance research progress in producing human organs in pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 shows the origin and characteristics of pESC. Figure 1a, Representative morphology of derivatives and pESCs formed by pESCs isolated from porcine blastocysts. 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 pESC. Scale bar, 50 μm. Figure 1d, Karyotype analysis of pESC. Figure 1e, Representative morphology of embryoid bodies. Scale bar, 50 μm. Figure 1f, Representative immunofluorescence staining results of tubulin, a marker of ectoderm, α-SMA, a marker of mesoderm, and GATA6, a marker of endoderm. Scale bar, 50 μm. Figure 1g, Hematoxylin and eosin staining results of teratoma sections formed by pESCs in mouse subcutaneous tissue. Scale bar, 100 μm.

[0041] Figure 2 shows the generation of porcine blastocysts using a 3D two-step strategy. Figure 2a, a schematic diagram of the formation of porcine blastocysts from pESC using a 3D two-step method. Figure 2b, representative images of cell clusters and blastocysts at designated time points during the formation of porcine blastocysts. Scale bar, 50 μm. Figure 2c, representative images of porcine embryonic day 6 blastocysts (hereinafter referred to as porcine E6 PA blastocysts) and embryonic day 7 blastocysts (hereinafter referred to as porcine E7 PA blastocysts), and blastocysts (day 6) generated by pESC using a 3D two-step method. Scale bar, 200 μm. Figure 2d, the formation efficiency of pESC to generate blastocysts (n=16). Formation efficiency refers to the ratio of blastocysts to all aggregates. Figure 2e, the diameters of porcine blastocysts (n=53), porcine E6 PA blastocysts (n=50), and E7 PA blastocysts (n=33). The diameters were statistically analyzed by ImageJ. The p value was calculated using an unpaired t-test. Figure 2f, X / Y ratios of porcine blastocysts (n=53), porcine E6 PA blastocysts (n=50) and E7 PA blastocysts (n=33). p values ​​were calculated using unpaired t-test. Figure 2g, Representative immunofluorescence staining of SOX2, a marker of epiblast (also referred to herein as EPI), GATA3, a marker of trophectoderm (also referred to herein as TE), and GATA6, a marker of hypoblast (also referred to herein as HYPO), in pESC-derived blastocysts. Scale bar, 50 μm. FIG. 2h, The number of cells of each cell lineage in each porcine blastocyst (n=19), porcine E6 PA blastocyst (n=16), and E7 PA blastocyst (n=10). FIG. 2i, The ratio of each cell lineage in each porcine blastocyst (n=19), porcine E6 PA blastocyst (n=16), and E7 PA blastocyst (n=10). FIG. 2j, Representative immunofluorescence staining of ZO-1, a marker used to indicate tight junctions between cells, in porcine blastocysts and porcine PA blastocysts. Scale bar, 50 μm.

[0042] Figure 3 shows single-cell transcriptional profiles of porcine blastocysts. Figure 3a, UMAP is used to represent the distribution of two samples of porcine blastocysts. Figure 3b, UMAP shows the expression of EPI markers (SOX2, POU5F1), HYPO markers (GATA4, GATA6), and TE markers (GATA2, GATA3). Figure 3c, Cells derived from porcine blastocysts are colored by origin: epiblast-like cells (ELC, yellow), hypoblast-like cells (HLC, purple), and trophoblast-like cells (TLC, red). Figure 3d, Dot plot shows the expression of EPI markers (SOX2, POU5F1), HYPO markers (GATA4, GATA6), and TE markers (GATA2, GATA3). Figure 3e, Heat map shows the expression of cell lineage-specific genes in ELC, HLC, and TLC.

[0043] Figure 4 shows the joint analysis of single-cell sequencing data of porcine blastocysts and embryos. Figure 4a, principal component analysis (PCA) (non-patent document 29) of porcine blastocysts and embryos. EB (early blastocysts, E6), LB (late blastocysts, E7), HB (hatching blastocysts, E8), EBi (early double-layer embryos, E9), LBi (late double-layer embryos, E10), PPS (pre-primitive streak embryos, E11), EPS (early primitive streak embryos, E12), PS (primitive streak embryos, E13) and LPS (late primitive streak embryos, E14). Figure 4b, PCA of LB, HB and EBi. Figure 4c, PCA analysis shows that porcine embryos at E7, E8 and E9 include 5 cell clusters. Figure 4d, similarity scores of EPI (POU5F1 and SOX2), TE (GATA2 and GATA3) and HYPO (GATA4 and GATA6). Figure 4e, cells derived from pig embryos are colored according to their origin: EPI (yellow), HYPO (purple) and TE (red). Figure 4f,UMPA diagram of the combined analysis of porcine blastocyst-derived cells and porcine embryo-derived cells. The pie chart shows the percentages of cells of the three lineages. The percentages of 85.2% / 59.4% / 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 regions. The percentages of 76.4% / 97.7% / 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 regions.

[0044] Figure 5 shows the isolation and establishment of stem cells from porcine blastocysts. Figure 5a, Schematic representation of the establishment of pESCs and pTSCs from blastocysts. Figure 5b, Representative morphology of blastocyst derivatives and pESCs. Scale bar, 50 μm. Figure 5c, Alkaline phosphatase (AP) staining of blastocyst-derived pESCs. 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.

[0045] 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 unpaired t-test. Figure 6d, survival ratio 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 unpaired t-test. Figure 6g, survival ratio of porcine PA blastocysts cultured in vitro.

[0046] Figure 7 The main findings of this study are schematically illustrated.

[0047] Figure 8 shows the generation and optimization of porcine blastocysts. Figure 8a, Representative images of ELCs, TLCs, and HLCs in iBlastoid medium. Scale bar, 50 μm. Figure 8b, Representative results of immunofluorescence staining of GATA3, SOX2, and GATA6, showing the differentiation of pESCs into TE, EPI, and HYPO lineage cells in the indicated medium. Scale bar, 50 μm. Figure 8c, Schematic diagram of the induction of porcine blastocysts from pESCs using the one-step method. Figure 8d, Representative images 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 the one-step method and the two-step method.

[0048] FIG. 9 shows the expression of three lineage markers in porcine blastocysts and blastocysts. Figure 9a , Immunofluorescence staining of EPI markers (SOX2, POU5F1), TE markers (GATA3, CDX2), and HYPO markers (GATA4) 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.

[0049] 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 are scored similarly. Figure 10d, UMAP shows the expression of selected EPI genes (ETV5, DNMT3B), HYPO genes (NID2, COL4A1) and TE genes (SFN, KRT8). Figure 10e, dot plots show gene expression of selected EPI, HYPO and TE lineages.

[0050] 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 based on scPred in porcine blastocyst-derived cells. 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 ELCs, HLCs, TLCs and other cells derived from blastocysts, respectively.

[0051] Figure 12: Immunofluorescence staining of IVC (in vitro culture) 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. DETAILED DESCRIPTION OF THE INVENTION

[0053] A first aspect of the present invention provides a method for generating a porcine embryo-like structure from porcine embryonic stem cells (pESCs), comprising culturing the pESCs in a blastocyst-like induction medium to form a porcine blastoid.

[0054] In some preferred embodiments, the embryo-like structure is a blastocyst.

[0055] In some preferred embodiments, the pESCs are derived from pig blastocysts, embryos or parthenogenetic blastocysts.

[0056] In some embodiments, the blastocyst-like induction medium contains a combination of at least one hypoblast-like cell (HLC) differentiation factor and at least one trophoblast-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.

[0057] In some preferred embodiments, the blastocyst-like induction medium further comprises factors for maintaining pluripotency and / or increasing cell survival, and the factors are optionally selected from LIF, activin A, IGF1, IL-6, sIL-6 receptor α, XAV939, Y-27632 and DZNep.

[0058] In some more preferred embodiments, the blastocyst induction medium contains LIF, BMP4, bFGF, CHIR99021, SB431542, DZNep and TSA, and optionally, the blastocyst induction medium also contains IL-6, IGF1, sIL-6 receptor α, activin A, XAV939 and Y-27632.

[0059] In a further preferred embodiment, the blastocyst induction medium comprises Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, LIF, IGF1, BMP4, bFGF, CHIR99021, XAV939, SB431542, Y-27632, DZNep and TSA mixed in a 1:1 (v / v) ratio.

[0060] In some preferred embodiments, according to the blastocyst induction medium as described above, wherein:

[0061] The concentration of activin A is 5-15 ng / mL, preferably 8-12 ng / mL, more preferably 10 ng / mL;

[0062] The concentration of bFGF is 5-15 ng / mL, preferably 8-12 ng / mL, more preferably 10 ng / mL;

[0063] The concentration of CHIR99021 is 1-2 μM, preferably 1.3-1.7 μM, and more preferably 1.5 μM;

[0064] The concentration of SB431542 is 0.5-1.5 μM, preferably 0.8-1.2 μM, more preferably 1 μM;

[0065] The concentration of TSA is 2-3 nM, preferably 2.3-2.7 nM, more preferably 2.5 nM;

[0066] The concentration of BMP4 is 1-10 ng / mL, preferably 3-7 ng / mL, more preferably 5 ng / mL;

[0067] The concentration of 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;

[0068] 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;

[0069] The concentration of 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;

[0070] 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;

[0071] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0072] The concentration of Y-27632 is 3-15 μM, preferably 5-10 μM, and more preferably 7.5 μM;

[0073] The concentration of DZNep is 2-3 nM, preferably 2.3-2.7 nM, more preferably 2.5 nM;

[0074] The content of FBS is 0.02-0.15%, preferably 0.05-0.1%, and more preferably 0.075%;

[0075] The content of KOSR is 1-10%, preferably 3-7%, more preferably 5%;

[0076] The concentration of 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, more preferably 100 μM;

[0077] and / or,

[0078] The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL, preferably 30-70 μg / mL, more preferably 50 μg / mL; in some preferred embodiments, 2-phospho-L-ascorbic acid sodium salt is preferred.

[0079] In some more preferred embodiments, an example of the blastocyst induction medium is the iBlastoid medium described herein.

[0080] In some embodiments, culturing the pESCs in a blastocyst induction medium to form porcine blastocysts comprises suspending the cultured pESCs in a blastocyst induction medium, optionally for at least 3, 4, 5, 6, 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.

[0081] In some embodiments, the method for generating a porcine embryo-like structure (preferably, a porcine blastocyst) from porcine embryonic stem cells (pESC) as described above further comprises:

[0082] Dissociate pESCs into single cells.

[0083] Single cells were cultured into cell clusters using ESC pre-treatment medium.

[0084] Transferring cell clusters directly to blastocyst induction medium without dissociating the cell clusters into single cells, and

[0085] The cell clusters were cultured in blastocyst induction medium to form porcine blastocysts.

[0086] In some preferred embodiments, the ESC pre-treatment medium comprises Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632 mixed in a 1:1 (v / v) ratio.

[0087] In some more preferred embodiments, in the ESC pre-treatment medium as described above:

[0088] The concentration of activin A is 15-30 ng / mL, preferably 17-23 ng / mL, more preferably 20 ng / mL;

[0089] 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;

[0090] 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;

[0091] 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;

[0092] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0093] The concentration of Y-27632 is 1-10 μM, preferably 3-7 μM, and more preferably 5 μM;

[0094] The content of FBS is 0.05-0.3%, preferably 0.1-0.2%, more preferably 0.15%;

[0095] The content of KOSR is 1-10%, preferably 3-7%, more preferably 5%;

[0096] The concentration of 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, more preferably 100 μM;

[0097] and / or,

[0098] The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL, preferably 30-70 μg / mL, more preferably 50 μg / mL; in some preferred embodiments, 2-phospho-L-ascorbic acid sodium salt is preferred.

[0099] In some most preferred embodiments, a preferred example of the ESC pre-treatment medium is the 4FXY medium described herein.

[0100] Therefore, the first aspect of the present invention also provides the ESC pre-treatment medium as described above. In some embodiments, the ESC pre-treatment medium is used to culture single cells of pESC into cell clusters before transferring to blastocyst-like induction medium.

[0101] The second aspect of the present invention provides an embryo-like structure obtained according to the method of the first aspect of the present invention, optionally, the embryo-like structure is a blastocyst.

[0102] The third aspect of the present invention provides a blastocyst induction medium as described above, wherein the medium contains a combination of at least one hypoblast-like cell (HLC) differentiation factor and at least one trophoblast-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.

[0103] The fourth aspect of the present invention provides a culture medium for culturing pESC, the culture medium comprising Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632 mixed in a 1:1 (v / v) ratio. In some embodiments, the above-mentioned culture medium for culturing pESC can be used for blastocyst attachment when isolating pESC or for the maintenance culture of pESC.

[0104] In some embodiments, when the culture medium is used for blastocyst attachment during isolation of pESC, the content of FBS is 4.5-5.5%, preferably 5-5.3%, and more preferably 5.15%.

[0105] In other embodiments, when the culture medium is used for the maintenance culture of pESC, the content of FBS is 0.05-0.3%, preferably 0.1-0.2%, and more preferably 0.15%.

[0106] In some embodiments, the above-mentioned medium for culturing pESC may further contain IWR1; preferably, the concentration of IWRI is 2-3 μM, more preferably 2.5 μM. In some more preferred embodiments, when the above-mentioned medium for culturing pESC is used for the maintenance culture of pESC, the medium further contains IWR1; preferably, the concentration of IWRI is 2-3 μM, more preferably 2.5 μM.

[0107] In some more preferred embodiments, in the culture medium for culturing pESC as described above:

[0108] The concentration of activin A is 15-30 ng / mL, preferably 17-23 ng / mL, more preferably 20 ng / mL;

[0109] 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;

[0110] 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;

[0111] 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;

[0112] The concentration of XAV939 is 2-3 μM, preferably 2.3-2.7 μM, and more preferably 2.5 μM;

[0113] The concentration of Y-27632 is 1-10 μM, preferably 3-7 μM, and more preferably 5 μM;

[0114] The content of KOSR is 1-10%, preferably 3-7%, more preferably 5%;

[0115] The concentration of 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, more preferably 100 μM;

[0116] and / or,

[0117] The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL, preferably 30-70 μg / mL, more preferably 50 μg / mL; in some preferred embodiments, 2-phospho-L-ascorbic acid sodium salt is preferred.

[0118] In some more preferred embodiments, a preferred example of the culture medium for blastocyst attachment when isolating pESCs is the culture medium described herein with 5% FBS additionally added to the 4FIXY or 4FXY medium.

[0119] In some other more preferred embodiments, a preferred example of the culture medium for maintaining pESC culture is 4FIXY described herein.

[0120] Methods and Materials Used in Examples

[0121] Establishment of porcine ESCs

[0122] Porcine oocytes with multiple layers of 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 an activation medium and then incubated in a PZM-3 medium (Non-Patent Document 23). For the establishment of pESCs, the entire PA blastocyst was digested with 0.5% pronase (Sigma-Aldrich, P8811) to remove the zona pellucida, and 5% FBS was additionally added to 4FXY medium or 4FIXY medium, followed by culture on feeder cells in the above medium until the blastocyst adhered. TrypLE TM The derivatives were dissociated by Select and re-seeded on mouse embryonic fibroblast feeder cells treated with mitomycin C in 4FIXY medium. After about three days, the cells formed fine colonies. The cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0123] 4FIXY medium consisted of Neurobasal medium (Gibco, 21103049) and DMEM / F12 (Gibco, 10565018) mixed in a 1:1 (v / v) ratio, 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), 0.15% 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 2-phospho-L-ascorbic acid sodium salt (Sigma, 49752) and 5 μM Y-27632 (TargetMol, T1725). 4FXY medium is based on 4FIXY medium minus IWRI, and the other components and their concentrations are the same.

[0124] Cultivation of porcine ESCs

[0125] pESCs were maintained on mitomycin C-treated mouse embryonic fibroblast feeder cells and enzymatically passaged every 3-4 days. TM Select (38.5°C, 5 min) to dissociate cells into single cells, centrifuge (250g, 5 min), resuspend, and inoculate in 4FIXY medium at a ratio of 1:3. Cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0126] EB (embryoid body) formation

[0127] Using TrypLE TMSelect (38.5°C, 5 minutes) to separate pESCs before differentiation and plate them in ultra-low attachment multi-well plates in EB formation medium for 7 days. EBs were attached to gelatin-coated plates for 3 days in DMEM (HyClone, SH30022.01B) containing 15% FBS and 5 μM Y-27632, cultured in DMEM containing 15% FBS for 4 days, and collected for analysis. Cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0128] EB formation medium consisted of Neurobasal medium (Gibco) and DMEM / F12 (Gibco) mixed at a ratio of 1:1 (v / v), 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).

[0129] Production of porcine blastocysts

[0130] In a one-step method, pESCs were cultured at 38.5°C with TrypLE TM Select digestion for 5 minutes to form single cells. The cells were collected and resuspended in iBlastoid medium. Approximately 40,000 single cells per well were directly seeded into ultra-low attachment multi-well plates and cultured in iBlastoid medium for 5-7 days. In the two-step induction method, pESCs were dissociated with TrypLETM Select for 5 minutes at 38.5°C to form single cells. The cells were collected and resuspended in 4FXY medium. Approximately 40,000 single cells per well were seeded into ultra-low attachment multi-well plates and cultured in 4FXY medium for 1-2 days. The cell clusters formed by pESCs were then cultured in iBlastoid medium for 3-5 days. All porcine blastocysts produced were manually picked using a mouth pipette under a stereomicroscope for downstream experiments. Blastocysts were cultured at 38.5°C, 20% O2 and 5% CO2.

[0131] iBlastoid medium consisted of Neurobasal (Gibco) medium and DMEM / F12 (Gibco) mixed in a 1:1 (v / v) ratio and contained 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 2-phospho-L-ascorbic acid sodium salt (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), 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).

[0132] Isolation of stem cell lines from porcine blastocysts

[0133] Single porcine blastocysts were transferred onto mitomycin C-treated mouse embryonic fibroblast feeder cells in 4-well plates and cultured in 4FIXY medium (for pESCs) and TSC medium (for pTSCs). TM Derivatives observed within 7 days were selected and dissociated and passaged onto mouse embryonic fibroblast feeder cells treated with mitomycin C in the corresponding medium. 5% FBS was added to the medium until blastocysts attached. Cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0134] TSC culture medium was modified according to a previous report (non-patent document 24), which consisted of Neurobasal medium and DMEM / F12 mixed in a 1:1 (v / v) ratio, and N2 and B27 supplements, NEAA, GlutaMAX, penicillin / streptomycin, 100 μM 2-mercaptoethanol, 50 μg / mL 2-phospho-L-ascorbic acid sodium salt, 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) were added.

[0135] Long-term culture of porcine blastocysts in vitro

[0136] Porcine blastocysts were manually picked using a mouth pipette and transferred to new ultra-low attachment multi-well plates containing iBlastoid medium or N2B27+AY medium. Blastocysts were cultured at 38.5°C, 20% O2 and 5% CO2.

[0137] N2B27+AY medium is a modified form of bovine and sheep embryo IVC medium (non-patent literature 22, 25). N2B27+AY medium consists of Neurobasal medium and DMEM / F12 mixed at a ratio of 1:1 (v / v), and N2 and B27 supplements, NEAA, GlutaMAX, penicillin / streptomycin, 5% KOSR, 100 μM 2-mercaptoethanol, 20 ng / mL activin A, and 10 μM Y-27632 are added.

[0138] Immunofluorescence staining

[0139] Cells, blastoids, and blastocysts were fixed with 4% paraformaldehyde (PFA) for approximately 30 minutes at room temperature, washed in DPBS for 5 minutes, and permeabilized with 0.2% Triton X-100 in DPBS for 30 minutes. For surface marker staining, samples were not permeabilized. Samples were then blocked with blocking buffer for 40 minutes at room temperature. Primary antibodies were diluted in primary antibody dilution buffer. Blastocysts / blastocysts or cells in 96-wells were incubated in primary antibodies overnight at 4°C. Samples were washed 3 times with DPBS for 15 minutes each and incubated with fluorescent dye-conjugated secondary antibodies diluted in secondary antibody dilution buffer for 1 hour at room temperature. Samples were washed 3 times with DPBS. Finally, samples were counterstained with DAPI for 3 minutes at room temperature. Blastocysts were imaged on 8-well μ-slides (ibidi). Blastocysts were imaged after being pressed with a coverslip.

[0140] Karyotype analysis

[0141] Before karyotyping, pESCs were treated with medium containing 4 μg / mL colchicine for 2 hours. The cells were digested, centrifuged, resuspended in 0.075M KCl hypotonic solution, and incubated at 37°C for 15 minutes. pESCs were fixed with a 3:1 (v / v) mixture of pre-cooled methanol and acetic acid, and this step was repeated 3 times. The resuspended pESCs were dropped onto a pre-cooled slide, dried and stained with Giemsa. Karyotype was analyzed under a 100X microscope.

[0142] Teratoma formation

[0143] Digestion of about 0.5-1×10 7 pESCs were isolated and centrifuged (250 g, 5 min) and injected subcutaneously into the dorsal subcutaneous tissue of NCG mice. Approximately 10 weeks later, teratomas were collected and sliced.

[0144] Hematoxylin and eosin staining

[0145] Teratomas were collected and fixed with 4% PFA for at least 24 h. Teratoma tissues were dehydrated with graded ethanol, transferred to xylene, embedded in paraffin, and cut into 5 μm thick serial cross-sectional sections. Cross-sectional sections were dewaxed in xylene, rehydrated with graded ethanol, and stained with hematoxylin and eosin.

[0146] Bulk RNA sequencing and analysis

[0147] A total of 1 μg 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. Library preparations were sequenced on an Illumina NovaSeq platform (Illumina, USA) and 150 bp paired-end reads were generated. RNA-seq reads were trimmed using Trim Galore (v0.6.4) and then mapped to the susScr11 reference genome using HISAT2 (v2.2.1), and the transcript levels of each gene in each sample were quantified using StringTie (v2.2.1). Samples were converted to FPKM (fragments per kilobase of exon per million mapped reads).

[0148] Single-cell sequencing

[0149] Porcine blastocysts with EPI-like, TE-like compartments and cavities were manually picked with a mouth 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 samples were centrifuged at 500g for 5 minutes. The cell pellet was resuspended in DMEM / F12 and filtered through a 40μm cell strainer. Single cell suspension was performed using the 10xGenomics Chromium Single Cell Kit for single cell sequencing.

[0150] Single-cell sequencing data collection

[0151] The pig data set includes CRA003960 (Non-Patent Document 29).

[0152] Single-cell sequencing data analysis

[0153] Quality control was performed using Dr.seq2 (v2.2.1) (non-patent document 27), and scRNA-seq reads were mapped to the mm10 reference genome using 10x Genomics CellRanger (v7.1.0) (non-patent document 26). Subsequently, Seurat (v4.3.0) (non-patent document 28) was used to integrate the samples using the transcription level of each gene in each sample as input. After standardization, the Seurat functions RunUMAP() and FindCluster() were used for dimensionality reduction and clustering. For each cell type, the FindAllMarkers() function was used to find genes with specific expression with an average logarithmic fold change of >0.8 and a differential expression percentage of >30%. Based on the E7-E9 embryonic data set (non-patent document 29), scPred (non-patent document 30) was used to classify cells derived from pig embryos into cell types, including EPI, HYPO, and TE, and other cells with multiple characteristics were defined as "undefined". This prediction model was applied to single-cell sequencing data of porcine blastocysts, and blastocyst-derived cells were divided into four cell categories (EPI lineage, HYPO lineage, TE lineage, and undefined).

[0154] Data analysis

[0155] p values ​​were calculated using unpaired t-test and are shown in the relevant figures. p values ​​< 0.05 were considered to be significantly different. Specific embodiments

[0156] Example 1 Establishment of pESC using 4FIXY medium

[0157] Prior to generating porcine blastocysts, the inventors used a novel culture medium to establish pESCs from parthenogenetic (PA) blastocysts. Based on transcriptome data of early porcine embryonic development and published 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 WNT signaling inhibitors XAV939 and IWR1, and ROCK inhibitor Y-27632, for culturing pESCs. The resulting pESCs can be passaged as single cells by enzymatic hydrolysis at a ratio of 1:3 every 3-4 days and form dense colonies with smooth and clear edges (Figure 1a). Immunofluorescence staining showed that pESCs expressed pluripotent markers, including POU5F1, SOX2, NANOG, OTX2, and E-cadherin (Figure 1b). During long-term culture, pESCs were alkaline phosphatase (AP) positive and had a normal karyotype (Figure 1c, d). In addition, pESC can form embryoid bodies (EBs) and differentiate into three germ layers in vitro (Figure 1e, f). In immunodeficient mice, pESC can form teratomas containing endoderm, mesoderm, and ectoderm (Figure 1g). These results show that the inventors have successfully established pESC under 4FIXY culture conditions.

[0158] Example 2 Efficient generation of blastocyst-like cells using pESC

[0159] In order to test whether pESC can be assembled into blastocyst-like cells in vitro, the inventors first detected their ability to differentiate into extraembryonic lineages, including trophectoderm (trophectoderm, also referred to as TE herein) and hypoblast, also referred to as HYPO herein). For this reason, the inventors established new culture conditions (therefore referred to as blastocyst-like induction medium, iBlastoid medium herein is an example thereof) to culture pESC, and co-cultured for 3 days. The inventors found that epiblast-like cells (also referred to as ELC herein), trophoblast-like cells (also referred to as TLC herein) and hypoblast-like cells (also referred to as HLC herein) gradually appeared (Fig. 8a). Immunofluorescence staining showed that GATA3 and GATA6 positive cells representing TLC and HLC respectively could be identified (Fig. 8b). Both GATA3 and GATA6 positive cells can be observed in the same view with SOX2 positive cells (ELC) (Fig. 8b). These results show that pESC has the potential to differentiate into extraembryonic lineages in vitro, which is a prerequisite for producing blastocyst-like cells.

[0160] Then, the inventors tried to rebuild pig blastocysts (Fig. 8c) under 3D culture conditions. First, the inventors prepared blastocyst induction culture medium (iBlastoid in this article is an example) based on the expression data set of pig early embryonic development, including cytokine LIF, activin A, IGF1, IL-6 and sIL-6 receptor α. The inventors also used chemical reagents CHIR99021, SB431542, TSA and BMP4 to support the formation of TLC. In addition, activin A, bFGF and CHIR99021 were also added to the culture medium because they contribute to the differentiation of HLC. In a one-step method, pESC is digested into single cells and directly inoculated into ultra-low attachment multi-well plates with a specified number of cells in iBlastoid culture medium. The inventors show that pig blastocysts containing blastocyst-like cavities, EPI-like compartments and TE-like outer layers gradually appear on days 5-7 (Fig. 8d). Immunofluorescence staining showed 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).

[0161] In order to improve the efficiency of porcine blastocyst production, the inventors improved and developed a more robust 3D two-step method (Figure 2a and Figure 8f). In the two-step induction method, pESCs dissociated into single cells were inoculated into ultra-low attachment multi-well plates containing 4FXY medium (4FIXY without IWR1), and cell clusters were formed on days 1-2; the cell clusters were then maintained in iBlastoid medium for 3-5 days (Figure 2a). The proportion of SOX2-positive blastocysts was 25.9% to 55% (Figure 8f). In iBlastoid medium, cell clusters gradually formed blastocyst-like cavities, EPI-like cells, and TE-like cell layers (Figure 2b). The blastocysts formed in vitro on day 6 were morphologically similar to porcine PA blastocysts on embryonic days 6 (E6) and E7 (Figure 2c). The efficiency of porcine blastocyst formation was 4.95% to 29.68% (Figure 2d). In addition, the diameter of the porcine blastocyst (average 183 μm) was similar to that of the porcine E6 PA blastocyst (average 192 μm), and both were smaller than the E7 PA blastocyst (average 224 μm) (Figure 2e). The X / Y ratio of the porcine blastocyst was approximately equal to 1, indicating that the blastocyst was round, similar to the porcine PA blastocyst at E6 and E7 (Figure 2f). In summary, the inventors showed that porcine blastocysts can be efficiently generated from pESCs in a 3D system.

[0162] Example 3: Porcine blastocyst exhibits characteristics of blastocyst

[0163] To explore whether the reconstructed porcine blastocysts contain the three cell lineages in the blastocyst, the inventors performed immunofluorescence staining to detect markers of EPI (SOX2), HYPO (GATA6), and TE (GATA3), and showed that the porcine blastocysts contain these cells ( Figure 2g The inventors also confirmed other markers of each lineage in porcine blastocysts, such as EPI (POU5F1), HYPO (GATA4), and TE (CDX2) ( Figure 9a , b). Then, the inventors calculated the number of cells and the proportion of the three cell lineages in porcine blastocysts. Blastocysts contain more cells than E6 and E7 PA blastocysts. Compared with E6 PA blastocysts, blastocysts contain more ELCs and TLCs, and the ratio of ELCs and TLCs is comparable to that of E6 PA blastocysts (Figure 2h, i). In addition, the number of HLCs in porcine blastocysts and E6 PA blastocysts is similar, while the proportion of HLCs in porcine blastocysts is lower than that in porcine E6 PA blastocysts (Figure 2h, i). Compared with E7 PA blastocysts, the number and proportion of ELCs are both higher. The number of HLCs and TLCs is similar, but the proportion is lower than that of E7 PA blastocysts (Figure 2h, i). The tight intercellular junctions between blastocyst-like TLCs can be detected by immunofluorescence staining of ZO-1 (Figure 2j). In summary, these data indicate that blastocysts induced by pESCs exhibit characteristics similar to those of porcine blastocysts.

[0164] Example 4 Single-cell transcriptome of porcine blastocyst

[0165] In order 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 can be divided into 13 clusters (Figure 10a). The inventors identified ELC, HLC and TLC using known characteristic markers, including SOX2 and POU5F1 for identifying ELC, GATA6 and GATA4 for identifying HLC, and GATA3 and GATA2 for identifying TLC (Figure 3b, c and Figure 10b). Cell cluster 5 expresses SOX2 and POU5F1, representing ELC; cell clusters 7 and 10 express GATA6 and GATA4, representing HLC; seven clusters (0, 1, 2, 3, 4, 6, 12) express GATA3 and GATA2, marked as TLC (Figure 3b and Figure 10a). For clusters 8, 9, and 11, the percentage of cells expressing marker genes was less than 50%, and the corrected average expression levels of all marker genes were less than 0.1, making it difficult to determine their cell types. Therefore, these cell clusters were excluded. Each lineage cell of the porcine blastocyst showed a unique gene expression pattern, similar to its corresponding lineage cell in the pig embryo (Figure 3d, e and Figure 10c). For example, EPI genes (DNMT3B, ETV5, ZIC2) are unique to the ELC cluster. The HYPO gene NID2 is unique to the HLC cluster; another HYPO gene COL4A1 is highly expressed in the HLC cluster. TE genes (KRT8, SFN) are most highly expressed in the TLC cluster (Figure 10d, e). These results confirm the presence of EPI, HYPO, and TE-like lineage cells in porcine blastocysts.

[0166] Next, the inventors studied whether pESC-derived porcine blastocysts are similar to porcine blastocysts at the transcriptome level. The inventors 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 blastocyst-derived cells were closer to blastocyst cells (Figure 4a). In order to further evaluate the similarity between lineage cells from blastocysts and blastocysts, two comparative methods were used for integrated analysis to perform unbiased and quantitative analysis of blastocyst-derived cells. In early porcine embryonic development, the first lineage separation begins at E5; the second lineage separation begins at E6 and ends at E7; subsequently, EPI begins to transform into ectoderm from E10, indicating that three lineage cells, EPI, TE and HYPO, can be separated at E7, E8 and E9. Therefore, in order to study the association between the three lineage cells derived from blastocyst-like and blastocyst-like, namely, ELC and EPI, TLC and TE, HLC and HYPO, single-cell transcriptome data of porcine E7, E8 and E9 embryos were selected for integrated analysis (Figure 4b). EPI markers SOX2 and POU5F1, HYPO markers GATA6 and GATA4, and TE markers GATA3 and GATA2 were used to identify EPI, HYPO and TE (Figure 4c-e and Figure 11a). Subsequently, UMAP analysis was performed, and the results showed that ELC, TLC and HLC in blastocyst-like overlapped with the corresponding parts of EPI, TE and HYPO in blastocyst in the same region ( Figure 4f ). In addition, 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 corresponding lineage groups had EPI, HYPO, and TE lineage features, respectively (Figure 11b).

[0167] Overall, these results indicate that pESC-induced blastocysts have three characteristic lineages and therefore have very similar transcriptome profiles to blastocysts.

[0168] Example 5 Obtaining stem cells from porcine blastocysts

[0169] Then, the inventors studied whether stem cells can be obtained from these blastocysts, as in blastocysts (Figure 1a). To this end, the inventors showed that porcine blastocysts can be isolated from scratch to obtain ESCs and TSCs in a specified culture medium under the same culture conditions (Figure 5a). Derivatives of blastocyst-like origin can be formed, and pESCs with dense colonies can be produced (Figure 5b), which are positive for AP staining and express pluripotent markers, including POU5F1, E-cadherin, SOX2, NANOG, SSEA4 and β-catenin (Figure 5c, d). Using TSC culture medium, porcine TSCs (pTSCs) can also be established, which express the TE marker GATA3 (Figure 5e, f). In summary, these results show that these blastocysts, like blastocysts, can be isolated and obtained from stem cells.

[0170] Example 6 Long-term in vitro culture of porcine blastocysts

[0171] Then, the inventor explored the possibility of these blastocysts growing and developing in vitro. As far as we know, the IVC (in vitro culture) method of porcine embryos has not yet been established. According to the IVC system of cattle and sheep embryos, the inventors improved these culture conditions and developed N2B27+AY culture medium for blastocyst culture. iBlastoid culture medium is also used for long-term culture in vitro. The porcine blastocysts with EPI-like cells and obvious cavities at the 6th day (expressed as porcine blastocysts IVC day 0) are selected for long-term culture in vitro. When the IVC of porcine blastocysts lasts to the 18th day, it can be seen that the blastocysts continue to grow and expand in the cavity, and the size of the blastocysts increases (Fig. 6a, b). At the 18th day, the diameter range of the porcine blastocysts in N2B27+AY culture medium was 234μm to 1707μm (average 722μm), and in iBlastoid culture medium, it was 171μm to 1234μm (average 384μm) (Fig. 6c). On day 6, the survival rates of blastocysts in N2B27+AY and iBlastoid medium were 27.31% and 49.07%, respectively, and decreased to 1.72% and 2.96%, respectively, on day 18 (Fig. 6d). As a control, porcine PA blastocysts grew for about 6 and 8 days in N2B27+AY and iBlastoid medium, and then became smaller with increasing IVC days (Fig. 6e-g).

[0172] Fluorescent staining of three lineage markers in IVC blastocysts: EPI (SOX2), HYPO (GATA6), and TE (GATA3) showed that IVC blastocysts at day 18 contained SOX2-, GATA3-, and GATA6-positive cells ( FIG. 12 ).

[0173] Overall, these results indicate that pESC-derived blastocysts have the potential to grow in long-term in vitro culture conditions.

Claims

1. A method for generating a porcine embryo-like structure from porcine embryonic stem cells (pESCs), comprising culturing pESCs in a blastocyst-like induction medium to form a porcine blastocyst-like structure, wherein the blastocyst-like induction medium contains a combination of at least one hypoblast-like cell (HLC) differentiation factor and at least one trophoblast-like cell (TLC) differentiation factor, wherein the HLC differentiation factor is optionally selected from activin A, bFGF and CHIR99021, The TLC differentiation factor is optionally selected from CHIR99021, SB431542, TSA and BMP4.

2. The method according to claim 1, wherein the blastocyst-like induction medium further comprises factors for maintaining pluripotency and / or increasing cell survival, the factors being optionally selected from LIF, activin A, IGF1, IL-6, sIL-6 receptor α, XAV939, Y-27632 and DZNep.

3. The method according to claim 1 or 2, wherein the blastocyst-like induction medium contains LIF, BMP4, bFGF, CHIR99021, SB431542, DZNep and TSA, Optionally, the blastocyst induction medium further contains IL-6, IGF1, sIL-6 receptor α, activin A, XAV939 and Y-27632.

4. The method according to any one of claims 1 to 3, wherein the blastocyst induction medium comprises Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, LIF, IGF1, BMP4, bFGF, CHIR99021, XAV939, SB431542, Y-27632, DZNep and TSA mixed in a 1:1 (v / v) ratio.

5. The method according to any one of claims 1 to 4, wherein the cultured pESCs are suspended in blastocyst induction medium, optionally for at least 3, 4, 5, 6, 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.

6. The method according to any one of claims 1 to 5, wherein: The method further comprises: Dissociate pESCs into single cells. Single cells were cultured into cell clusters using ESC pre-treatment medium. Transferring cell clusters directly to blastocyst induction medium without dissociating the cell clusters into single cells, and The cell clusters were cultured in blastocyst induction medium to form porcine blastocysts.

7. The method according to any one of claims 1 to 6, wherein the ESC pre-treatment medium comprises Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632 mixed in a 1:1 (v / v) ratio.

8. The method according to any one of claims 1 to 7, wherein the embryo-like structure is a blastocyst.

9. The embryo-like structure obtained according to the method described in any one of claims 1 to 7, optionally, the embryo-like structure is a blastocyst-like structure.

10. A blastocyst-like induction medium for inducing porcine embryonic stem cells (pESCs) to form porcine embryo-like structures, wherein the medium contains a combination of at least one hypoblast-like cell (HLC) differentiation factor and at least one trophoblast-like cell (TLC) differentiation factor, in, The HLC differentiation factor is optionally selected from activin A, bFGF and CHIR99021, The TLC differentiation factor is optionally selected from CHIR99021, SB431542, TSA and BMP4, Optionally, the embryo-like structure is a blastocyst.

11. An ESC pre-treatment culture medium, comprising Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632 mixed in a 1:1 (v / v) ratio; preferably, the culture medium is used to culture single cells of pESC into cell clusters before transferring to blastocyst induction medium.

12. A culture medium for culturing porcine embryonic stem cells (pESCs), wherein the culture medium comprises Neurobasal medium and DMEM / F12, N2 and B27 supplements, NEAA, GlutaMAX, penicillin and streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1, XAV939 and Y-27632 mixed in a 1:1 (v / v) ratio; optionally, the culture medium further includes IWR1.

Images