Culture medium and method for obtaining extraembryonic endoderm stem cells from porcine blastocyst and embryonic stem cells

Abstract

The invention relates to a culture medium and a method for obtaining extraembryonic endoderm stem cells (XEN) from porcine blastocysts and embryonic stem cells (ESCs), and belongs to the technical field of biology. The invention develops a two-step induction method for directly converting pig ESCs into pig XEN without gene modification and two culture mediums 4FY and 2FYSC. The invention provides a high-quality model for exploring separation characteristics and intercellular communication of embryos and extraembryonic pedigree in early embryonic development of large animals, provides a good cell model for researching dynamic development of extraembryonic pedigree, and provides a high-quality material for regenerative medicine and agricultural breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a culture medium and a method for obtaining extraembryonic endoderm stem cells (XEN) from pig blastocysts and embryonic stem cells (ESCs). Background Art

[0002] Pigs are similar to humans in morphology, anatomy, physiological structure, organ size, and nutritional metabolism, and are often considered an ideal animal model for human disease and regeneration research. During early embryonic development in pigs, the fertilized egg undergoes the first cell fate determination to form the inner cell mass and trophoblast; then the second fate determination occurs, and the inner cell mass further develops to form the epiblast (Epiblast / EPI) and hypoblast (Hypoblast / HYPO or primitive endoderm). [1] Currently, stem cells can be isolated from the inner cell mass, EPI or HYPO. For example, porcine embryonic stem cells (ESCs) can be isolated from the inner cell mass or EPI, and porcine extraembryonic endoderm stem cells (XEN) can be isolated from the inner cell mass or HYPO. This provides a good model for studying early embryonic development and high-quality materials for regenerative medicine and agricultural breeding.

[0003] Porcine ESCs can be isolated from porcine blastocysts, inner cell mass or EPI using different culture systems [2-8] It has been reported that porcine XEN can be isolated from porcine blastocysts. [9-11] However, the extraembryonic chimerism ability of some pig XEN is still unclear. [9-10] Some blastocyst derivatives or XEN cannot be digested into single cells for passaging [9-11] , which brings inconvenience to downstream applications such as gene editing. It has been reported that pig embryo-derived stem cells with extended pluripotency can be initially transformed into XEN-like cells, but the induction process is unclear, and it is still unclear whether these XEN-like cells can be stably propagated for a long time and their biological characteristics such as chimerism ability. [9] ; Construction of porcine induced pluripotent stem cells dependent on the exogenous gene OSKM can transform them into XEN, but it is not clear whether the cell karyotype is normal during long-term passage

[12] ; Porcine fetal fibroblasts were transferred into the inducible mouse reprogramming factor OSKM, and the cells were used as donors to construct somatic cell nuclear transfer embryos. The embryos were isolated to establish a stem cell line that depends on doxycycline induction. The stem cells were converted into XEN using a medium containing small molecules.

[10] .

[0004] At present, there has been no report on the isolation and establishment of porcine ESCs from porcine blastocysts that can be stably propagated and are not dependent on exogenous genes, and whether these porcine ESCs can be converted into XEN with biological characteristics such as stable propagation ability, normal karyotype and extraembryonic chimerism ability. Summary of the invention

[0005] The object of the present invention is to provide a culture medium and a method for obtaining extraembryonic endoderm stem cells from pig blastocysts and embryonic stem cells.

[0006] The present invention provides a culture medium and method for directly converting pig blastocysts and ESCs into pig XEN without gene editing. Converting pig ESCs into pig XEN that is stably propagated in vitro can be used to analyze the separation characteristics of embryonic and extraembryonic lineages in early pig embryonic development, and also provide cell resources for exploring pig embryonic and extraembryonic tissue development and cell-to-cell interactions.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect of the present invention, there is provided a culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells, comprising 4FY culture medium and 2FYSC culture medium.

[0009] The 4FY culture medium comprises a basal culture medium and supplementary components added to the basal culture medium, wherein the basal culture medium is a mixture of Neurobasal culture medium and DMEM / F12 culture medium in a 1:1 (v / v) ratio; the supplementary components include: N2 supplement, B27 supplement, NEAA, GlutaMAX, penicillin / streptomycin, FBS, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1 and Y-27632.

[0010] In some embodiments of the present invention, in the 4FY medium, the concentration of activin A is 15-30 ng / mL, preferably 17-23 ng / mL, and more preferably 20 ng / mL;

[0011] In the 4FY medium, 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;

[0012] In the 4FY medium, the concentration of IL-6 is 10-30 ng / mL, preferably 16-24 ng / mL, more preferably 20 ng / mL; in some more preferred embodiments, the IL-6 is human IL-6;

[0013] In the 4FY medium, 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;

[0014] In the 4FY medium, the concentration of Y-27632 is 1-10 μM, preferably 3-7 μM, and more preferably 5 μM;

[0015] In the 4FY medium, the content of KOSR is 1-10%, preferably 3-7%, and more preferably 5%;

[0016] In the 4FY medium, the concentration of 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, and more preferably 100 μM;

[0017] In the 4FY medium, the concentration of 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.

[0018] In the 4FY medium, the content of the N2 supplement is 0.5%;

[0019] In the 4FY medium, the content of the B27 supplement is 1%;

[0020] In the 4FY medium, the content of NEAA is 1%;

[0021] In the 4FY culture medium, the content of GlutaMAX is 1%;

[0022] In the 4FY medium, the content of penicillin / streptomycin is 1%;

[0023] In the 4FY culture medium, the content of FBS is 0.15%.

[0024] The 2FYSC culture medium comprises a basal culture medium and supplementary components added to the basal culture medium, wherein the basal culture medium is a mixture of Neurobasal culture medium and DMEM / F12 culture medium in a 1:1 (v / v) ratio; the supplementary components include: N2 supplement, B27 supplement, NEAA, GlutaMAX, penicillin / streptomycin, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, human EGF, human FGF-basic, Y-27632, SB431542 and CHIR99021.

[0025] In some embodiments, in the 2FYSC medium:

[0026] The concentration of human EGF is 1-100 ng / mL, preferably 25-75 ng / mL, more preferably 50 ng / mL;

[0027] The concentration of human FGF-basic is 1-100 ng / mL, preferably 10-50 ng / mL, more preferably 20 ng / mL;

[0028] The concentration of Y-27632 is 1-20 μM, preferably 5-15 μM, more preferably 10 μM;

[0029] The concentration of SB431542 is 0.1-2 μM, preferably 0.5-1.5 μM, and more preferably 1 μM;

[0030] The concentration of CHIR99021 is 0.1-5 μM, preferably 1.0-4.0 μM, more preferably 2 μM;

[0031] The content of KOSR is 1-10%, preferably 3-7%, more preferably 5%;

[0032] The concentration of 2-mercaptoethanol is 50-200 μM, preferably 75-150 μM, more preferably 100 μM;

[0033] and / or,

[0034] 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.

[0035] In the 2FYSC medium, the content of the N2 supplement is 0.5%;

[0036] In the 2FYSC medium, the content of the B27 supplement is 1%;

[0037] In the 2FYSC culture medium, the content of NEAA is 1%;

[0038] In the 2FYSC culture medium, the content of GlutaMAX is 1%;

[0039] In the 2FYSC culture medium, the concentration of penicillin / streptomycin is 1%;

[0040] Among the culture medium for obtaining XEN from ESCs provided by the present invention, 2FYSC culture medium can also be used as a culture medium for establishing porcine XEN from blastocysts, and can be used for blastocyst attachment when isolating porcine XEN or for the maintenance culture of porcine XEN.

[0041] The second aspect of the present invention provides a culture medium for obtaining extraembryonic endoderm stem cells from porcine blastocysts, which is the above-mentioned 2FYSC culture medium.

[0042] The third aspect of the present invention provides a method for obtaining extraembryonic endoderm stem cells from porcine embryonic stem cells. Porcine embryonic stem cells (ESCs) are first cultured in 4FY medium; the cells are then digested and transferred into 2FYSC medium to obtain porcine extraembryonic endoderm stem cells (XEN).

[0043] In some embodiments of the present invention, porcine ESCs are first cultured in 4FY medium for a period of time, optionally for at least 1, 2, 3, 4, 5 days, or 1 to 3 days, 2 to 5 days, 3 to 5 days, or 4 to 5 days.

[0044] In some embodiments of the present invention, when porcine ESCs are first cultured in 4FY medium, the cell density of porcine ESCs is 1-20×10 3 , preferably 1-10×10 3 , more preferably 3.3-6.6×10 3 .

[0045] In some embodiments of the present invention, when porcine ESCs are first cultured in 4FY medium, the cells are cultured at 38.5° C., 5% O 2 and 5% CO 2 .

[0046] A fourth aspect of the present invention provides a method for obtaining XEN from porcine blastocysts, wherein the porcine blastocysts are cultured in 2FYSC medium to obtain porcine XEN.

[0047] In some embodiments of the present invention, porcine oocytes with multiple layers of cumulus cells are collected from ovarian follicles, matured in vitro for 42-46 hours, and treated with hyaluronidase to remove cumulus cells; then, the oocytes are exposed to a 60V pulse for 30 μs in an activation medium and subsequently incubated in PZM-3 medium; the whole parthenogenetic blastocyst is digested with 0.5% pronase to remove the zona pellucida, and then transferred to a mouse embryonic fibroblast feeder layer cell treated with mitomycin C and cultured in 2FYSC medium until the blastocyst adheres to the wall; TrypLE TM Derivatives observed within 7-10 days were express dissociated and passaged onto mitomycin C-treated mouse embryonic fibroblast feeder cells in 2FYSC-containing medium.

[0048] In some embodiments of the present invention, when the cells are passaged onto a mouse embryonic fibroblast feeder layer treated with mitomycin C in a 2FYSC medium, the cells are cultured at 38.5° C., 5% O 2 and 5% CO 2 .

[0049] In some embodiments of the present invention, after obtaining porcine XEN, the porcine XEN is maintained on a mouse embryonic fibroblast feeder layer treated with mitomycin C and passaged every 3-4 days by enzymatic digestion. TM Express cells were dissociated into single cells, centrifuged, resuspended, and inoculated into 2FYSC medium at a ratio of 1:3. The cells were cultured at 38.5°C, 5% O2, and 5% CO2.

[0050] Compared with the prior art, the advantages and beneficial effects of the present application are as follows:

[0051] The present invention has developed a two-step induction method and two culture media 4FY and 2FYSC for directly converting pig ESCs into pig XEN without genetic modification. In the process of converting ESCs into XEN, pig ESCs are first cultured in 4FY culture medium for 3 days. 4FY culture medium is an ESCs culture medium (see reference 6 for details) without Wnt inhibitors XAV939 and IWR1, in which Y-27632 helps cell proliferation and survival; the combination of the remaining components can promote pig ESCs to exit pluripotency and form XEN precursor cells. On this basis, the cells are cultured in 2FYSC culture medium to form a stable XEN line. 2FYSC mainly contains cytokines EGF and FGF-basic, and small molecule inhibitors Y27632, SB41542 and CHIR99021, which help the formation and maintenance of XEN. The XEN obtained by this method can be stably propagated for a long time and is suitable for single-cell propagation. It can maintain a normal karyotype, has transcriptional properties similar to HYPO in pigs, can form embryoid bodies, and has the characteristics of being chimeric to mouse extraembryonic tissues.

[0052] The present invention provides a high-quality model for exploring the separation characteristics of embryonic and extraembryonic lineages and intercellular communication in early embryonic development of large animals, provides a good cell model for studying the dynamic development of extraembryonic lineages, and provides high-quality materials for regenerative medicine and agricultural breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows the transformation of porcine ESCs into porcine XEN and its biological characteristics.

[0054] Figure 1 includes Figure 1A-1I ,in, Figure 1A , Figure 2. Pig ESCs. Figure 1B, Figure 2. The first stage of porcine ESCs conversion into XEN cells, with cells cultured in 4FY medium for different days. Figure 1C , The morphology and alkaline phosphatase staining of pig XEN cultured in 2FYSC medium during the second stage of pig ESCs conversion into XEN. Figure 1D , Statistical chart of induction status of different cell lines and cell numbers. Figure 1E , Expression diagram of XEN markers and pluripotency markers in pig XEN and ESCs. Figure 1F , Immunofluorescence staining of SOX17, GATA4, GATA6, and SOX2 in pig XEN. DAPI was used to stain the cell nucleus. Figure 1G , XEN karyotype diagram of porcine ESCs. Figure 1H , Image of embryoid bodies formed by XEN derived from porcine ESCs. Fig. 1I , Expression of XEN, VE, and ParE markers. EB, embryoid body. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0055] FIG. 2 is a diagram showing the isolation of porcine XEN from porcine blastocysts and its biological characteristics.

[0056] Figure 2 includes Figure 2A-2G ,in, Figure 2A , images of pig blastocysts and isolated XEN as well as images of XEN at different generations. Figure 2B , Pig XEN alkaline phosphatase staining. Figure 2C , XEN and XEN marker expression in ESCs. Where <P20 and P50 refer to XEN less than 20 and 50 passages, respectively. Figure 2D , Immunofluorescence staining of SOX17, GATA4, GATA6, SOX2, GATA3, and CDX2 in pig XEN. DAPI was used to stain the cell nucleus. Figure 2E , pig XEN karyotype diagram. Figure 2F , Diagram of embryoid bodies formed by XEN derived from porcine blastocysts. Figure 2G , Expression of XEN, VE, and ParE markers. EB, embryoid body. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0057] FIG. 3 is a diagram showing the analysis of XEN transcriptional characteristics derived from pig blastocysts and ESCs.

[0058] Figure 3 includes Figure 3A-3E ,in, Figure 3A , principal component analysis plot. Figure 3B , cell correlation analysis diagram at different stages. Figure 3C , Analysis of the correlation between pig XEN and early embryo. Figure 3D, heatmap and bubble chart showing pluripotency gene expression. Figure 3E , heatmap and bubble chart showing XEN gene expression. LB (late blastocyst), Sph (spherical embryo), EPI (epiblast); HYPO (hypoblast). XEN B : Blastocyst-derived XEN. XEN E : XEN from porcine ESCs. Day 1 and Day 3 refer to the first and third days of culture of porcine ESCs in the 4FY culture system.

[0059] FIG. 4 is a diagram showing the chromatin accessibility analysis of XEN derived from pig blastocysts and ESCs.

[0060] Figure 4 includes Figure 4A-4G ,in, Figure 4A , principal component analysis plot. Figure 4B , ATAC-seq peak map within 5kb range. Figure 4C , CO, OC, and PO diagrams of cells at each stage. Figure 4D , sample similarity heatmap.

[0061] Figure 4E , Peak number statistics chart. Figure 4F , Representative loci maps of CO, OC, and PO.

[0062] Figure 4G , Expression level diagram of representative genes in RNA-seq. XEN B : Blastocyst-derived XEN. XEN E : XEN from porcine ESCs. Day 1 and Day 3 refer to the first and third days of culture of porcine ESCs in the 4FY culture system.

[0063] Figure 5 shows the chimera of XEN derived from pig blastocysts and ESCs into mouse extraembryonic tissues.

[0064] Figure 5 includes Figure 5A , Figure 5B , Figure 5C ,in, Figure 5A , Figure 3 of E4.5 chimeric embryo. Figure 5B , Immunofluorescence staining of E4.5 chimeric embryos. Figure 5C , E6.5 mosaic embryos with porcine XEN mosaicism to VE and ParE extraembryonic tissues. B : Blastocyst-derived XEN. XEN E : XEN derived from porcine ESCs.

[0065] Figure 6 , Flowchart for obtaining XEN from porcine blastocysts and ESCs. DETAILED DESCRIPTION

[0066] The following examples illustrate exemplary embodiments of the present invention to help those skilled in the art understand other objects, features, advantages and aspects of the present application. It should be understood that, although preferred embodiments of the present application are shown, the following description and specific examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is determined according to the appended claims. Unless otherwise stated, the specific experiments in the following examples are performed according to conventional methods and conditions in the art, or in accordance with the product specifications.

[0067] Methods and Materials Used in Examples

[0068] Culture and maintenance of porcine ESCs

[0069] Porcine ESCs were maintained on mitomycin C-treated mouse embryonic fibroblast feeder cells and enzymatically passaged every 3-4 days. TM The cells were dissociated into single cells by centrifugation (250 g, 5 min) and resuspended, and inoculated into 4FIXY medium at a ratio of 1:3. The cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0070] 4FIXY medium consisted of Neurobasal medium (Gibco, 21103049) and DMEM / F12 (Gibco, 10565018) mixed in a 1:1 (v / v) ratio, supplemented with 0.5% N2 (Gibco, 17502048) and 1% B27 (Gibco, 17504044) supplements, 1% NEAA (Gibco, 11140050), 1% GlutaMAX (Gibco, 35050061), 1% 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). The composition of 4FIXY medium is as in reference 6.

[0071] Method for converting porcine ESCs into porcine XEN

[0072] Phase 1: Pig ESCs were cultured at a rate of 3.3-6.6×10 per square centimeter. 3 The cells were inoculated at a density of 100 cells on a mitomycin C-treated mouse embryonic fibroblast feeder layer and cultured in 4FIXY medium for 1 day. The next day, the medium was replaced with 4FY medium and the cells were cultured for another 3 days. TM The cells were dissociated into single cells by Select (38.5°C, 5 minutes), centrifuged (250g, 5 minutes), resuspended, and inoculated in 2FYSC medium at a ratio of 1:2. The cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0073] 4FY medium consisted of Neurobasal medium (Gibco, 21103049) and DMEM / F12 (Gibco, 10565018) mixed in a 1:1 (v / v) ratio and supplemented with 0.5% N2 (Gibco, 17502048) and 1% B27 (Gibco, 17504044) supplements, 1% NEAA (Gibco, 11140050), 1% GlutaMAX (Gibco, 35050061), 1% 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), 50 μg / mL 2-phospho-L-ascorbic acid sodium salt (Sigma, 49752) and 5 μM Y-27632 (TargetMol, T1725).

[0074] 2FYSC culture medium was composed of a 1:1 (v / v) mixture of Neurobasal medium (Gibco, 21103049) and DMEM / F12 (Gibco, 10565018), with the addition of 0.5% N2 (Gibco, 17502048) and 1% B27 (Gibco, 17504044) supplements, 1% NEAA (Gibco, 11140050), 1% GlutaMAX (Gibco, 35050061), 1% penicillin / streptomycin (Gibco, 15140122), 5% KOSR, 100 μM 2-mercaptoethanol (Sigma, M3148), 50 ng / mL human EGF (Peprotech, AF-100-15), 20 ng / mL human FGF-basic (Peprotech, 100-18B), 50 μg / mL 2-phospho-L-ascorbic acid sodium salt (Sigma, 49752), 10 μM Y-27632 (Target Mol, T1725), 1 μM SB431542 (Selleck, S1067), 2 μM CHIR99021 (Tocris, 4423).

[0075] Isolation of porcine XEN from porcine blastocysts

[0076] 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 subsequently incubated in PZM-3 medium, as described in reference 6. The entire parthenogenetic blastocyst was digested with 0.5% pronase (Sigma-Aldrich, P8811) to remove the zona pellucida, and then transferred to a mouse embryonic fibroblast feeder layer treated with mitomycin C and cultured in 2FYSC medium. 5% FBS was added to the culture medium until the blastocyst adhered. TrypLE TM Derivatives observed within 7-10 days were express dissociated and passaged onto mitomycin C-treated mouse embryonic fibroblast feeder cells in 2FYSC-containing medium. Cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0077] Maintenance and culture of porcine XEN

[0078] Porcine XEN were maintained on mitomycin C-treated mouse embryonic fibroblast feeder cells and enzymatically passaged every 3-4 days. TM The cells were dissociated into single cells by PCR (38.5°C, 4-5 minutes), centrifuged (250g, 5 minutes), resuspended, and inoculated in 2FYSC medium at a ratio of 1:3. The cells were cultured at 38.5°C, 5% O2 and 5% CO2.

[0079] Pig XEN embryoid body formation

[0080] Using TrypLE TM Express (38.5°C, 5 minutes) to isolate porcine XEN, and the cells were plated in ultra-low attachment multi-well plates and cultured for 7 days in embryoid body medium. After 7 days of culture, the cells were collected for subsequent detection. Embryoid body medium consisted of Neurobasal medium (Gibco) and DMEM / F12 (Gibco) mixed in a 1:1 (v / v) ratio, and 0.5% N2 (Gibco) and 1% B27 (Gibco) supplements, 1% NEAA (Gibco), 1% GlutaMAX (Gibco), 1% penicillin / streptomycin (Gibco), 5% KOSR, 100 μM 2-mercaptoethanol (Sigma), and 10 μM Y-27632 (TargetMol) were added.

[0081] Immunofluorescence staining

[0082] Cells or embryos were fixed with 4% paraformaldehyde for about 50 minutes at room temperature, washed in DPBS for 5 minutes, and permeabilized with 0.2% Triton X-100 in DPBS for 20 minutes. The samples were blocked with blocking buffer for 40 minutes at room temperature. Primary antibodies were diluted in primary antibody dilution buffer, including anti-GATA6 (5851, Cell Signaling Technology), anti-SOX17 (AF1924, R&D), anti-GATA4 (sc-25310, Santa Cruz Biotechnology), anti-SOX2 (MAB2018, R&D), anti-GATA3 (5852, Cell Signaling Technology), and anti-CDX2 (ab76541, Abcam). Cells were incubated in primary antibodies overnight at 4°C. The samples were washed 3 times with DPBS for 5 minutes each time and incubated with fluorescent dye-conjugated secondary antibodies diluted in secondary antibody dilution buffer for 1 hour at room temperature. The secondary antibodies included Alexa 488goatanti-rabbit (4412, Cell Signaling Technology), Alexa 594donkey anti-goat (ab150132, Abcam), Alexa 488goat anti-mouse (4408, Cell Signaling Technology), and Alexa 594goat anti-rabbit (8889, Cell Signaling Technology). The samples were washed 3 times with DPBS. Finally, the samples were counterstained with DAPI for 3 minutes at room temperature. The embryos were imaged after being pressed with a coverslip.

[0083] Karyotype analysis

[0084] XEN 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. XEN 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 XEN was dropped on a pre-cooled slide, dried and stained with Giemsa. Karyotype was analyzed under a 100X microscope.

[0085] Bulk RNA sequencing and analysis

[0086] A total of 1 μg RNA was used for library construction, sequencing and analysis. UltraTM RNA LibraryPrep Kit for Generate sequencing libraries. Library preparations were sequenced on the Illumina NovaSeq PE150 platform (Illumina, USA). RNA-seq reads were trimmed using Trim Galore and then mapped to the susScr11.1 reference genome using HISAT2, and expression was calculated using featureCount. HISAT2 was used to map to the susScr11.1 reference genome reference 13, and expression was calculated using featureCount reference 14.

[0087] High-throughput sequencing (ATAC-seq) and analysis of chromatin accessibility using transposases

[0088] The library was constructed using the Hyperactive ATAC-Seq Library Prep Kit for Illumina (VazymeBiotech Co. Ltd, TD711). The quality of the raw data was assessed using FastQC, the RNA-seq reads were corrected using Trim Galore, and then the data were mapped to the susScr11.1 reference genome using bwa, the normalized signals were generated using deeptools, the accessible chromatin regions were identified using MACS3, and the number of peaks was calculated using featureCount. Among them, the method for mapping the data to the susScr11.1 reference genome using bwa was referenced in 15, the method for generating normalized signals using deeptools was referenced in 16, the method for identifying accessible chromatin regions using MACS3 was referenced in 17, and the method for calculating the number of peaks using featureCount was referenced in 14.

[0089] Injection of porcine XEN into mouse embryos

[0090] 10-15 ZSGREEN-labeled pig XENs were injected into mouse 4-8 cell embryos or blastocysts, cultured in vitro until E4.5, and immunofluorescence staining was used to detect SOX17 expression. The embryos were transplanted into mice, and the chimerism was detected at E6.5 days of development. The method of embryo transplantation into mice is referenced in references 18 and 19.

[0091] Data analysis

[0092] 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.

[0093] Schematic diagram of the isolation of XEN from porcine blastocysts and the two-step conversion of porcine ESCs into XEN Figure 6 . Specific embodiments

[0095] Example 1 Transformation of porcine ESCs into XEN

[0096] In order to transform porcine ESCs into XEN, the applicant established a two-step method to achieve transformation. In the first stage, the cells were treated with 4FY medium for 3 days, and the cell morphology changed from dense clones ( Figure 1A ) gradually becomes flat ( Figure 1B In the second stage, the applicant selected growth factors human EGF and human FGF-basic, combined with small molecules Y-27632, SB431542 and CHIR99021 to form 2FYSC culture medium, digested the cells and replaced them with 2FYSC culture medium to form porcine XEN, which was alkaline phosphatase negative ( Figure 1C ). 3.3-6.6×10 per square centimeter. 3 porcine ESCs can be efficiently transformed into XEN ( Figure 1D ). In order to detect the expression of HYPO or XEN marker genes, the applicant conducted real-time quantitative PCR, and the results showed that the pluripotency genes POU5F1, SOX2, NANOG, and OTX2 in pig XEN showed a down-regulated expression trend, and the HYPO marker genes SOX17, GATA4, GATA6, etc. showed an up-regulated expression trend ( Figure 1E ). Immunofluorescence staining results showed that SOX17, GATA4, and GATA6 proteins were expressed in XEN, but the pluripotency marker SOX2 was not expressed ( Figure 1F ). XEN transformed from porcine ESCs have normal karyotype ( Figure 1G ). Under the conditions of embryoid body culture medium, embryoid bodies can be suspended and formed ( Figure 1H ), the visceral endoderm (VE) and parietal endoderm (ParE) markers APOE, CLDN6, AFP, and SPARC showed an up-regulated expression trend in the formed embryoid bodies ( Fig. 1I ). The above results show that the combined use of 4FY and 2FYSC culture medium can achieve the transformation of porcine ESCs into XEN.

[0097] Example 2 Isolation and establishment of porcine XEN from porcine blastocysts using 2FYSC medium

[0098] In order to isolate and obtain pig XEN from blastocysts, the applicant used 2FYSC medium to inoculate the 6th day blastocysts onto feeder cells, gradually forming cell derivatives ( Figure 2A ), with the passage, the formation of pig XEN ( Figure 2A ). Alkaline phosphatase staining of porcine XEN was negative ( Figure 2B). In order to detect the expression of HYPO or XEN marker genes, the applicant performed real-time quantitative PCR, and the results showed that compared with ESCs, the pluripotency genes POU5F1, SOX2, and NANOG in blastocyst-derived pig XEN showed a down-regulated expression trend, while the HYPO or XEN marker genes SOX17, GATA4, GATA6, etc. showed an up-regulated expression trend ( Figure 2C ). Immunofluorescence staining results showed that the blastocyst-derived pig XEN expressed SOX17, GATA4, and GATA6 proteins, but did not express the pluripotency marker SOX2, nor did it express the trophectoderm markers GATA3 and CDX2 ( Figure 2D ). XEN isolated from blastocysts have normal karyotype ( Figure 2E ). Under the conditions of embryoid body culture medium, pig XEN can be suspended to form embryoid bodies ( Figure 2F ), the VE and ParE markers APOE, CLDN6, AFP, etc. in the formed embryoid bodies showed an up-regulated expression trend ( Figure 2G ). The above results show that pig XEN lines can be isolated and established from pig blastocysts using 2FYSC medium.

[0099] Example 3 Analysis of XEN transcriptional properties obtained from porcine ESCs and blastocysts

[0100] In order to clarify the transcriptional characteristics of XEN obtained from pig ESCs and blastocysts, the applicant conducted batch RNA sequencing and analysis. The results of principal component analysis and correlation analysis showed that there were significant differences between pig ESCs and XEN, and that XEN derived from pig blastocysts and ESCs had similarities ( Figure 3A -B). Comparison and analysis with in vivo early embryo data (see reference 20 for details) showed that XEN from pig blastocysts and ESCs was similar to sph HYPO in vivo ( Figure 3C ). Pluripotency genes such as SOX2, ZIC5, and OTX2 showed a downward expression trend as ESCs transformed into XEN ( Figure 3D ); During early pig embryonic development, these pluripotency genes are highly expressed in EPI compared to HYPO ( Figure 3D HYPO-related genes such as SOX17, GATA4, and GATA6 showed an up-regulated expression trend as ESCs transformed into XEN cells ( Figure 3E ); During early pig embryonic development, these HYPO-related genes were highly expressed in HYPO compared with EPI ( Figure 3E These results indicate that XEN obtained from porcine blastocysts and ESCs have similar transcriptional properties, similar to sph HYPO in vivo.

[0101] Example 4 Analysis of XEN Chromatin Accessibility from Porcine ESCs and Blastocysts

[0102] In order to clarify the dynamic changes of chromatin in XEN obtained from pig blastocysts and ESCs, the applicant conducted ATAC-seq analysis. The results of principal component analysis and correlation analysis showed that there were differences between pig ESCs and XEN, and that XEN derived from pig blastocysts and ESCs had similarities ( Figure 4A -D). We further compared the peaks of each site in the cells and divided them into three groups, namely, closed in ESCs but open in XEN (close to open, CO), open in ESCs but closed in XEN (open to close, OC), and permanently open in both ESCs and XEN (permanently open, PO) ( Figure 4C ). Approximately 51,717 OC peaks and 40,833 CO peaks are distributed on chromosomes ( Figure 4E ). Among these peaks, genes unique to the OC and CO groups were further identified, and the results showed that some common pluripotency genes POU5F1 and NANOG and HYPO-related genes GATA4, GATA6, PDGFRA and other sites were always in an open state ( Figure 4F ); genes highly expressed in early embryonic EPI (for details, see reference 20) SOX2, DNMT3B, PHC1 and other loci are gradually closed as ESCs transform into XEN ( Figure 4F ), and its expression showed a down-regulation trend ( Figure 4G ); genes highly expressed in early embryos (see reference 20) such as HNF4A, APOC3, and LMNA are gradually opened as ESCs transform into XEN ( Figure 4F ), and its expression showed an up-regulated trend ( Figure 4G ). These results indicate that during the conversion of porcine ESCs to XEN, HYPO or XEN-related signals are activated in the cells, ESCs or pluripotency maintenance-related signals are inhibited, and the cells undergo different transcriptional programs.

[0103] Example 5 XEN obtained from pig ESCs and blastocysts has chimerism to mouse extraembryonic tissue characteristics

[0104] XEN has the property of chimerism in extraembryonic tissues. In order to evaluate whether pig XEN can chimerism in mouse extraembryonic tissues, the applicant injected ZSGREEN-labeled pig XEN into mouse 4-8 cell embryos or blastocysts, and cultured the chimeric embryos in vitro until E4.5. Immunofluorescence staining of E4.5 chimeric embryos showed that ZSGREEN-labeled pig XEN cells existed in the chimeric embryos and expressed the HYPO marker SOX17 ( Figure 5A-B), indicating that porcine XEN is involved in the HYPO development of E4.5 chimeric embryos. The chimeric embryos were transplanted into recipient mice. In E6.5 embryos, porcine XEN can be chimeric to both VE and ParE lineages ( Figure 5C ). These results indicate that porcine XEN has the ability to integrate into mouse extraembryonic tissues.

[0105] The literature information involved in the background technology and embodiments of the present invention is as follows:

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[0126] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells, comprising 4FY culture medium and 2FYSC culture medium, characterized in that: The 4FY medium comprises a basal medium and an additional component added to the basal medium, wherein the basal medium is a Neurobasal medium and a DMEM / F12 medium mixed in a ratio of 1:1 (v / v); the additional components include: N2 supplement, B27 supplement, NEAA, GlutaMAX, penicillin / streptomycin, fetal bovine serum, serum replacement, 2-mercaptoethanol, 2-phospho-L-ascorbate, IL-6, sIL-6 receptor α, activin A, IGF1 and Y-27632; The 2FYSC culture medium comprises a basal culture medium and supplementary components added to the basal culture medium, wherein the basal culture medium is a mixture of Neurobasal culture medium and DMEM / F12 culture medium in a 1:1 (v / v) ratio; the supplementary components include: N2 supplement, B27 supplement, NEAA, GlutaMAX, penicillin / streptomycin, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, human EGF, human FGF-basic, Y-27632, SB431542 and CHIR99021.

2. A culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells according to claim 1, characterized in that: In the 4FY medium, the concentration of activin A is 15-30 ng / mL; The concentration of IGF1 is 10-100 ng / mL; The concentration of IL-6 is 10-30 ng / mL; The concentration of the sIL-6 receptor α is 10-30 ng / mL; The concentration of Y-27632 is 1-10 μM; The content of KOSR is 1-10%; The concentration of the 2-mercaptoethanol is 50-200 μM; The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL.

3. A culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells according to claim 2, characterized in that: In the 4FY medium, the concentration of activin A is 17-23 ng / mL; The concentration of IGF1 is 40-60 ng / mL; The concentration of IL-6 is 16-24 ng / mL; The concentration of the sIL-6 receptor α is 16-24 ng / mL; The concentration of Y-27632 is 3-7 μM; The content of KOSR is 3-7%; The concentration of the 2-mercaptoethanol is 75-150 μM; The concentration of the 2-phospho-L-ascorbate is 30-70 μg / mL.

4. A culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells according to claim 1, characterized in that: In the 2FYSC medium: The concentration of human EGF is 1-100 ng / mL; The concentration of human FGF-basic is 1-100 ng / mL; The concentration of Y-27632 is 1-20 μM; The concentration of SB431542 is 0.1-2 μM; The concentration of CHIR99021 is 0.1-5 μM; The content of KOSR is 1-10%; The concentration of 2-mercaptoethanol is 50-200 μM; and / or, The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL.

5. A culture medium for obtaining extraembryonic endoderm stem cells from embryonic stem cells according to claim 4, characterized in that: In the 2FYSC medium: The concentration of human EGF is 25-75 ng / mL; The concentration of human FGF-basic is 10-50 ng / mL; The concentration of Y-27632 is 5-15 μM; The concentration of SB431542 is 0.5-1.5 μM; The concentration of CHIR99021 is 1.0-4.0 μM; The content of KOSR is 3-7%; The concentration of the 2-mercaptoethanol is 75-150 μM; and / or, The concentration of the 2-phospho-L-ascorbate is 30-70 μg / mL.

6. A culture medium for obtaining extraembryonic endoderm stem cells from porcine blastocysts, characterized in that: The 2FYSC medium comprises a basal medium and an additional component added to the basal medium, wherein the basal medium is a Neurobasal medium and a DMEM / F12 medium mixed in a ratio of 1:1 (v / v); the additional components include: N2 supplement, B27 supplement, NEAA, GlutaMAX, penicillin / streptomycin, KOSR, 2-mercaptoethanol, 2-phospho-L-ascorbate, human EGF, human FGF-basic, Y-27632, SB431542 and CHIR99021; In the 2FYSC medium: The concentration of human EGF is 1-100 ng / mL; The concentration of human FGF-basic is 1-100 ng / mL; The concentration of Y-27632 is 1-20 μM; The concentration of SB431542 is 0.1-2 μM; The concentration of CHIR99021 is 0.1-5 μM; The content of KOSR is 1-10%; The concentration of 2-mercaptoethanol is 50-200 μM; and / or, The concentration of the 2-phospho-L-ascorbate is 10-100 μg / mL.

7. A method for obtaining extraembryonic endoderm stem cells from porcine embryonic stem cells, characterized in that: The porcine embryonic stem cells are first cultured in the 4FY culture medium described in claim 1; the cells are then digested and transferred into the 2FYSC culture medium described in claim 1 to obtain porcine extraembryonic endoderm stem cells.

8. The method for obtaining extraembryonic endoderm stem cells from porcine embryonic stem cells according to claim 7, characterized in that: The porcine embryonic stem cells are first cultured in 4FY medium for at least 1, 2, 3, 4 or 5 days; When porcine embryonic stem cells were first cultured in 4FY medium, the cell density of porcine ESCs was 1-20×10 per square centimeter. 3 When porcine embryonic stem cells were first cultured in 4FY medium, the cells were cultured at 38.5°C, 5% O2 and 5% CO2.

9. A method for obtaining extraembryonic endoderm stem cells from porcine blastocysts, characterized in that: The porcine blastocysts were cultured in 2FYSC medium to obtain porcine extraembryonic endoderm stem cells; 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; then, the oocytes were exposed to 60V pulses for 30μs in activation medium and subsequently incubated in PZM-3 medium; whole parthenogenetic blastocysts were digested with 0.5% pronase to remove the zona pellucida, and then transferred onto mouse embryonic fibroblast feeder cells treated with mitomycin C and cultured in 2FYSC medium until the blastocysts attached; TrypLE TM Derivatives observed within 7-10 days were express dissociated and passaged onto mitomycin C-treated mouse embryonic fibroblast feeder cells in 2FYSC-containing medium.

10. The method according to claim 7 or 9, characterized in that: After obtaining porcine extraembryonic endoderm stem cells, porcine XEN were maintained on mitomycin C-treated mouse embryonic fibroblast feeder cells and passaged every 3-4 days by enzymatic digestion; TrypLE TM Express cells were dissociated into single cells, centrifuged, resuspended, and inoculated into 2FYSC medium at a ratio of 1:

3. The cells were cultured at 38.5°C, 5% O2, and 5% CO2.

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