Method and culture medium for generating embryo from pluripotent stem cells in vitro

Abstract

Disclosed herein are methods, compositions, and media for the in vitro generation of synthetic embryos from mammalian pluripotent stem cells, such as pluripotent embryonic stem cells. In some embodiments, the method may include co-culturing wild-type mammalian pluripotent stem cells and modified mammalian pluripotent stem cells under conditions of a culture medium that cause the pluripotent stem cells to self-organize into post-implantation embryo structures, the modified mammalian pluripotent stem cells comprise one or more genes encoding transcription factors that can drive the production of extraembryonic cells or extraembryonic sample cells, such as the GATA6 gene, the SOX17 gene, the GATA3 gene and / or the TFAP2C gene. In some embodiments, the pluripotent embryonic stem cells are human pluripotent embryonic stem cells, and the generated synthetic embryo is a human embryo.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 403,684, filed on September 2, 2022, and U.S. Provisional Patent Application No. 63 / 457,670, filed on April 6, 2023. The content of these related applications is hereby incorporated by reference in its entirety for all purposes.

[0003] Reference to Sequence Listing

[0004] This application is accompanied by a sequence listing in electronic format. The sequence listing is provided as a file named 30KJ-302455-WO-SeqList, created on August 24, 2023, and having a size of 33 kilobytes. The electronic format information of the sequence listing is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to the field of cell culture, and more particularly to the culture of embryonic and stem cells. Background Art

[0006] Mammalian embryos, especially human embryos, undergo morphogenetic transformations after implantation in the uterus. However, due to the inability to observe embryos in vivo, understanding of this critical stage is limited. Embryonic models derived from stem cells are important tools for exploring developmental events and tissue crosstalk during these stages. Summary of the Invention

[0007] Disclosed herein is an in vitro method for generating mammalian synthetic embryos. In some embodiments, the method includes co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene under culture medium conditions that cause the ESCs to self-organize into post-implantation embryonic structures. In some embodiments, the first modified mammalian ESCs comprise an inducible GATA6 gene, an inducible SOX17 gene, or both. In some embodiments, the first modified mammalian ESCs comprise an inducible GATA6 gene and an inducible SOX17 gene. In some embodiments, the second modified mammalian ESCs comprise an inducible GATA3 gene, an inducible TFAP2C gene, or both. In some embodiments, the second modified mammalian ESCs comprise an inducible GATA3 gene and an inducible TFAP2C gene.

[0008] In some embodiments, the method may further include contacting the first modified mammalian ESCs and / or the second modified mammalian ESCs with an inducer. The inducer may be, for example, doxycycline. In some embodiments, the inducer is supplied to the culture medium, optionally for about 1 to 7 days. In some embodiments, the method may further include adjusting the induction intensity, optionally by increasing or decreasing the concentration of the inducer, or increasing or decreasing the duration of the inducer in the culture medium. In some embodiments, the inducer is supplied to the culture medium throughout the co-culture process. In some embodiments, the wild-type mammalian ESCs and / or the modified mammalian ESCs are naive ESCs or primed ESCs. In some embodiments, the wild-type mammalian ESCs and / or the modified mammalian ESCs are pre-implantation naive hESCs, peri-implantation-like pluripotent naive hESCs, or post-implantation primed hESCs. In some embodiments, the pre-implantation naive hESCs are cultured in PXGL medium before co-culture, the peri-implantation-like pluripotent hESCs are cultured in RSeT medium before co-culture, and the post-implantation-like primed hESCs are cultured in mTeSR1 medium before co-culture. In some embodiments, the wild-type mammalian ESCs and the modified mammalian ESCs are peri-implantation-like pluripotent hESCs, optionally cultured in RSeT medium before co-culture. In some embodiments, the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 and / or SOX17 genes, and the second modified mammalian ESCs comprising the GATA3 and / or TFAP2C genes are provided in a ratio of about 1:1:1 to 1:1:5, optionally in a ratio of about 1:1:1 to 1:1:2. In some embodiments, the ESCs are cultured on a substrate, optionally wherein the substrate includes a culture dish, a U-shaped plate, a flask, or a microplate. In some embodiments, the ESCs are cultured in an inverted pyramid-shaped micropore. In some embodiments, one or more or each of the inverted pyramid-shaped micropores has a size of about 400 μm or about 800 μm, optionally a diameter of about 400 μm or about 800 μm. In some embodiments, the co-culture includes co-culturing the ESCs in a stem cell proliferation medium for about 5 days, optionally passaging the ESCs in the stem cell proliferation medium at least twice. In some embodiments, the stem cell proliferation medium is a serum-free medium. In some embodiments, the stem cell proliferation medium contains Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F12 (DMEM / F12), Neurobasal (a neurobasal medium), N2, B27, L-glutamine or analogs thereof, a reducing agent, an antibiotic, or a combination thereof. The reducing agent may be or may include β-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof.

[0009] In some embodiments, the stem cell proliferation medium is N2B27 medium. In some embodiments, the N2B27 medium comprises DMEM / F12, Neurobasal, B27, N2, GlutaMax, β-mercaptoethanol, penicillin / streptomycin, or a combination thereof. In some embodiments, the N2B27 medium comprises DMEM / F12 and Neurobasal A in a 1:1 ratio, 0.5×B27, 0.5×N2, 100 μM β-mercaptoethanol, 1×GlutaMAX, and 1× penicillin-streptomycin.

[0010] In some embodiments, the ESCs aggregate after co-culturing in the stem cell proliferation medium for up to 24 hours. In some embodiments, the aggregated ESCs exhibit a distinction between an inner cell mass and an outer cell mass. In some embodiments, the co-culture includes co-culturing the ESCs in the post-implantation medium for at least 2 days after co-culturing in the stem cell proliferation medium. In some embodiments, the ESCs are co-cultured in the post-implantation medium approximately 2 days after the ESCs aggregate. In some embodiments, the post-implantation medium comprises Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or a serum substitute thereof, an antibiotic, an antimicrobial agent, L-glutamine or an analogue thereof, insulin, an insulin analogue or an insulin receptor agonist, an estrogen analogue or an estrogen receptor agonist, progesterone, a progesterone analogue or a progesterone receptor agonist, or any combination thereof. In some embodiments, the non-human serum or serum substitute includes fetal bovine serum, bovine serum albumin, KnockOut TM Serum Substitute, or any combination thereof. In some embodiments, the antibiotic includes penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof. The estrogen receptor agonist can be or can include, for example, β-estradiol, estrone, estriol, and estetrol, or any analogue thereof.

[0011] In some embodiments, the insulin receptor agonist is selected from IGF-I, IGF-II, an analogue thereof, or any combination thereof. In some embodiments, the post-implantation medium comprises an antimicrobial agent, optionally the antimicrobial agent is sodium lactate. In some embodiments, the post-implantation medium comprises transferrin, sodium selenium, ethanolamine, or any analogue thereof. In some embodiments, the post-implantation medium comprises DMEM / F12, fetal bovine serum, GlutaMax, non-essential amino acids, essential amino acids, insulin-transferrin-selenium-ethanolamine (ITS-X), penicillin and / or streptomycin, glucose, sodium lactate, β-estradiol, progesterone, or any combination thereof.

[0012] In some embodiments, the post-implantation medium comprises DMEM / F12, about 20% fetal bovine serum, about 1× GlutaMax, about 1× non-essential amino acids, about 1× essential amino acids, about 1× ITS-X, about 25 U / mL penicillin and / or streptomycin, about 1.8 nM glucose, about 0.22% sodium lactate, about 8 nM β-estradiol, about 200 ng / ml progesterone, or any combination thereof. In some embodiments, co-culture comprises transferring the ESCs from one substrate to another substrate. In some embodiments, the post-implantation embryonic structure comprises an inner epiblast-like domain, an outer monolayer of trophoblast-like cells, and an intermediate hypoblast-like domain between the epiblast-like domain and the outer monolayer of trophoblast-like cells. In some embodiments, the inner epiblast-like domain is SOX2 positive and contains a central cavity, the outer monolayer of trophoblast-like cells is GATA3 positive, and the intermediate hypoblast-like domain is GATA6 positive. In some embodiments, the post-implantation embryonic structure expresses N-cadherin and SOX17 in the hypoblast-like domain, CDX2 in the trophoblast-like cells, and / or SOX2, NANOG, and E-cadherin in the epiblast-like domain. In some embodiments, the inner epiblast-like domain exhibits pluripotency and epithelial identity similar to that of a human embryo.

[0013] In some embodiments, the post-implantation embryonic structure comprises cell clusters similar to late embryonic epiblast, amnion, mesoderm, extraembryonic mesenchyme, and / or hypoblast / visceral endoderm. In some embodiments, the post-implantation embryonic structure expresses TDGF1, SOX2, NANOG, TFAP2A, ID1, ISL1, TFAP2C, VTCN1, GRHL1, MEIS1, TBXT, MESP1, MIXL1, CER1, SNAI1, EOMES, POSTN, COL6A3, IGF2, TBX20, BMP6, CDH2, HNF1B, FOXA2, or a combination thereof. In some embodiments, the post-implantation embryonic structure gives rise to amnion and primordial germ cells. In some embodiments, the efficiency of wild-type mammalian ESCs, first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene to form post-implantation embryos is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or higher.

[0014] In some embodiments, the method does not include any in vivo steps. In some embodiments, during co-culture, wild-type mammalian ESCs, first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene are not present in an in vivo environment, and optionally wherein the in vivo environment comprises a tissue, an organ, an organism, or a combination thereof. In some embodiments, the method does not include culturing trophoblast stem cells, hypoblast stem cells, or both alone or in combination with the ESCs. In some embodiments, the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene are human ESCs. In some embodiments, the post-implantation embryonic structure is a human embryonic structure. In some embodiments, the post-implantation embryonic structure is similar to a post-implantation human embryo at about 8-9 days after fertilization. In some embodiments, the method does not include using exogenous signaling pathway factors, and optionally the culture medium does not contain or provide exogenous signaling pathway factors. In some embodiments, the exogenous signaling factors include a WNT signaling pathway activator, a TGFβ superfamily member, or both.

[0015] Synthetic embryos obtained by any of the methods disclosed herein are disclosed. In some embodiments, the synthetic embryo is a human embryo, and optionally the synthetic embryo is similar to a post-implantation human embryo at about 8-9 days after fertilization.

[0016] Also disclosed herein is a method of studying mechanisms involved in embryogenesis, comprising any of the methods disclosed herein. Also disclosed herein is a method of identifying a compound useful for treating a disease, comprising contacting a synthetic embryo obtained by any of the methods disclosed herein with the compound.

[0017] Also disclosed herein is a method for diagnosing or treating a disease or disorder of a subject. In some embodiments, the method comprises generating a synthetic embryo by any of the methods disclosed herein; and transplanting the synthetic embryo into the subject. In some embodiments, the wild-type mammalian ESCs and the modified mammalian ESCs are obtained from the subject, or are derived from ESCs obtained from the subject.

[0018] Also disclosed herein is a method of elucidating the role of a candidate gene in embryonic development, comprising: obtaining wild-type mammalian ESCs, first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene, wherein the candidate gene has been modified or knocked out; and culturing the mammalian ESCs using any of the in vitro methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application document shall include at least one drawing in color. The Patent Office will provide a copy of the patent or patent application publication and the drawing upon the applicant's request and payment of the necessary fees.

[0020] Figure 1A-1F Depicts non-limiting exemplary embodiments and data related to the verification of extraembryonic-like induction. Figure 1A Depicts the generation and verification of inducible GATA6 (iG6) hESCs and / or inducible SOX17 (iS17) hESCs after adding doxycycline to basal N2B27 for 24 hours. iG6 (n = 551), iS17 (n = 550), and iG6-S17 (n = 707) cells are from 3 independent experiments. Figure 1B Depicts the generation and verification of inducible GATA3 (iG3) hESCs and / or inducible AP2Y (iAP2Y) hESCs after adding doxycycline to basal N2B27 for 24 hours. iG3 (n = 1456), iAP2 (n = 1456), and iG3-AP2Y (n = 782) cells are from 3 independent experiments. Figure 1C Depicts the uniform manifold approximation and projection (UMAP)-based dimensionality reduction of wild-type (RSeTWT), inducible GATA6-SOX17 (day 3 iG6-S17) RseT hESCs, and inducible GATA3-AP2Y (day 3 iG3-AP2Y) RseT hESCs after 3 days of doxycycline induction. Figure 1D Depicts the logistic regression analysis and comparison of the cells with the post-implantation human embryo population. The human embryo data from a previous report was used as training data, and the cell line data was used as test data. Figure 1E Depicts the differentially expressed genes selected from RNA sequencing (left) and the predicted differential motif accessibility by chromVAR scoring of ATAC sequencing (right) of wild-type, inducible GATA6-SOX17 RseT hESCs, and inducible GATA3-AP2Y RseT hESCs after 3 days of doxycycline induction. Figure 1F Depicts the verification of wild-type, inducible GATA6-SOX17 RseT hESCs, and inducible GATA3-AP2Y RseT hESC co-cultures under two dimensions (N = 3 independent experiments). In Figure 1A , Figure 1B and Figure 1F , the scale bar = 100 μm. For Figure 1A-1B , the mean ± SEM is plotted.

[0021] Figure 2A-2HDepicts non-limiting exemplary embodiments and data related to the generation of induced post-implantation human embryoids. Figure 2A Depicts an overview of the protocol for generating induced human embryoids by combining wild-type RseT hESCs with inducible GATA6-SOX17 (iG6-S17) cells and inducible GATA3-AP2 (iG3-AP2Y) cells. Extraembryonic-like cells aggregate on day 0 after 3 days of induction. Figure 2B Shows that 96 hours after aggregation, the structures exhibit distinct self-organization. Figure 2C Depicts the size of cell aggregates between day 1 and day 3 after aggregation. Structures on day 1 (n = 175), day 2 (n = 171), and day 3 (n = 91) are from 5 independent experiments. The lengths of all individual embryoids are plotted. Symbols (orange crosses, orange triangles, green triangles, blue circles, purple squares) each represent an independent experiment. Figure 2D Depicts the quantification of embryoid formation across starting pluripotent states. RSeT (n = 952), mTeSR (n = 30), PXGL (n = 207) structures are from 5 independent experiments. Statistical analysis was performed using one-way ANOVA and Holm-Sidak multiple comparison tests, showing P = 0.0347 for RSeT vs. mTeSR and P = 0.0283 for RSeT vs. PXGL. Unlabeled pairwise comparisons were not significant (n.s.; p > 0.05). Figure 2E Depicts the quantification of cell type proportions in properly organized embryoids. N = 16 embryoids, from 3 independent experiments. Figure 2F Depicts representative images of human embryos cultured in vitro for 9 days after fertilization, showing a clear cavitated SOX2 domain surrounded by a layer of GATA6-positive cells. A subset of GATA6-positive cells expresses the previous hypoblast marker CER1. These images represent 3 independent experiments. Figure 2G Depicts a hypoblast-like domain expressing N-cadherin, SOX17, and GATA4, and an epiblast-like domain maintaining the expression of pluripotency factors SOX2, OCT4, and NANOG. Cells derived from inducible GATA3-AP2Y expressing GFP show a clear external localization. The images represent 2 experiments respectively. Figure 2H Depicts induced human embryoids, showing clear apical-basal polarity, and quantification of induced human tissues. In Figure 2H , in the upper right figure, 1 is the cavity, 2 is the ECM, and 3 is SOX2 + cavity + ECM. For cavity and ECM efficiency, n = 506 structures are from 3 independent experiments. For the number of cavities, N = 27 embryoids are from 2 independent experiments. In Figure 2A , Figure 2B and Figure 2F-2HIn which, scale = 100 μm. * indicates P < 0.05. In Figure 2C the lengths of all individual embryoids were plotted. For Figure 2D-Figure 2E and Figure 2H , the mean ± SEM was plotted. The inner domain of the embryoid is surrounded by a dashed line.

[0022] Figure 3A-Figure 3I Non-limiting exemplary embodiments and data related to the differentiation of extraembryonic mesenchyme, amnion, and primordial germ cells are depicted. Figure 3A A schematic diagram of the expansion culture protocol of induced human embryoids and the sampling of combined single-cell RNA and single-cell ATAC sequencing using the 10x platform is depicted. Twelve embryoids each at 4 days, 6 days, and 8 days after aggregation were sequenced. Figure 3B Cell annotation based on transcriptional projections of multiple human and non-human primate embryo datasets using scmap combined with RNA velocity and chromatin velocity is depicted. Figure 3C Differentially expressed genes selected in the RNA sequencing data (top) and predicted differentially accessible motifs scored by chromVAR on the ATAC sequencing data (bottom) in cell clusters are depicted. Figure 3D Induced human embryoids with downregulated SOX2 and upregulated CDX2, ISL1 at day 6 and upregulated VTCN1 at day 8 are depicted, indicating robust amnion differentiation and maturation. In some rare cases, dorsal-ventral and / or anterior-posterior axis patterning can be observed. These images represent 3 experiments. Figure 3E Module scores of primordial germ cell marker genes are depicted. Figure 3F A Nebulosa plot visualizing the joint expression density of key primordial germ cell genes in induced human embryoids is depicted. Figure 3G A heatmap of the expression of selected primordial germ cell genes in cell clusters is depicted. Figure 3H Quantification of SOX17 / NANOG / AP2Y triple-positive (+) cells at day 4 (n = 10 embryoids) and day 6 (n = 10 embryoids) is depicted. N = 2 independent experiments were performed. Fig. 3I Immunofluorescence identification of SOX17 / NANOG / AP2 triple-positive primordial germ cell-like cells in induced human embryoids highlighted by arrows is depicted. The mean ± SEM is plotted. Scale = 100 μm. The inner domain of the embryoid is surrounded by a dashed line.

[0023] Figure 4A-4H Non-limiting exemplary embodiments and data related to BMP signal-driven amnion specification in induced human embryos are depicted. Figure 4A Expression of downstream targets ID1-4 of BMP signaling in embryoids is depicted. Figure 4BDepicts chromVAR-based motif accessibility scores for SMAD5 and SMAD2::SMAD3::SMAD4 (effectors of BMP and NODAL signaling, respectively). Figure 4C Depicts representative images and quantification of OCT4-positive and GATA6-positive cells from representative induced human embryoids on day 4 (n = 60 cells each; P < 0.0001) and day 6 (n = 40 cells each; P < 0.0001) from N = 3 independent experiments. Figure 4D Depicts representative images and quantification of SMAD2.3 in OCT4-positive and GATA6-positive cells from representative induced human embryoids on day 4 (n = 40 cells each; P = 0.0004) and day 6 (n = 40 cells each; P < 0.0001) from n = 2 independent experiments. Figure 4E Indicates that inhibition of BMP signaling prevents pluripotency exit and blocks upregulation of the amnion markers AP2Ya and CDX2. Figure 4F Depicts quantification of the percentage of the inner domain expressing SOX2 and CDX2 on day 4 (control: n = 147; LDN treatment: n = 126; BMP4 treatment: n = 57; Act-A treatment: n = 60 embryoids, from 5 independent experiments). For the SOX2+ / CDX2- domain, P = 0.0002 for control vs. LDN; P = 0.0433 for control vs. Act-A; P = 0.1753 for control vs. BMP4. Figure 4G Indicates that inhibition of BMP reduces the number of primordial germ cell-like cells in embryoids. Figure 4H Depicts quantification of the number of SOX17 / NANOG / AP2Y triple-positive primordial germ cell-like cells (PGCLCs) on day 4 (control: n = 45; LDN treatment: n = 30; BMP4 treatment: n = 48; Act-A treatment: n = 36 embryoids, from 6 independent experiments). P = 0.0011 for control vs. LDN. P > 0.99 for control vs. BMP4. P = 0.98 for control vs. Act-A. Unlabeled comparisons to control are n.s. Figure 4C and Figure 4D The statistical method used in Figure 4F is two-sided Mann-Whitney. In Figure 4H it is RM two-way ANOVA with Holm-Sidak multiple comparison test. Figure 4C 、 Figure 4D and Figure 4F the mean ± SEM is plotted. In Figure 4HIn it, the box includes the 25th to 75th percentile range, with the minimum and maximum values shown as whiskers. The center line represents the median, and the symbol + represents the mean. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The inner domain of the embryoid body is surrounded by a dashed line.

[0024] Figure 5A-5H Depicts non-limiting exemplary embodiments and data related to the antagonistic effect of SOX17 induction on anterior hypoblast specification. Figure 5A Depicts the expression of CER1 and LEFTY1 in HYPO / VE in embryoid bodies. Figure 5B Depicts the analysis of GATA6 and SOX17 regulator activities by SCENIC and co-expression scores of SOX17 and CER1 in post-implantation human hypoblasts (9 - 11 days after fertilization). Data are from a previous report. Figure 5C Depicts a representative example of an embryoid body, showing CER1-positive cells generated from hypoblast-like cells induced with inducible GATA6 (iG6) instead of inducible GATA6-SOX17 (iG6-S17) dual induction. CER1 expression can also be observed if doxycycline is withdrawn on day 3 after aggregation. Figure 5D Depicts a representative image on day 4, indicating reduced pSMAD1.5 in the epiblast-like domain of the structure with a CER1-positive cell population. Figure 5E Depicts Figure 5C The proportion of embryoid bodies expressing CER1. iG6-S17: n = 78; iG6: n = 25; iS17: n = 26; iG6-S17-Dox. Day 1: n = 54; iG6-S17-Dox. Day 3: n = 65 embryoid bodies from 7 independent experiments. P<0.0001 for iG6-S17 compared to iG6. P<0.0001 for iG6-S17 compared to iG6-S17-Dox. Day 3. P = 0.87 for iG6-S17 compared to iS17. P = 0.87 for iG6-S17 compared to iG6-S17-Dox. Day 1. Fig. 5F Depicts the quantification of pSMAD1.5 levels in SOX2-positive cells in CER1-negative (CER1-) and CER1-positive (CER1+) iG6-S17 embryoid bodies on day 4. CER1-: n = 108 cells; CER1+: n = 123 cells, from 8 embryoid bodies, each from 2 independent experiments. In Fig. 5F it, P<0.0001. Figure 5G Depicts Figure 5D The quantification of Brachyury expression in Figure 5HDepicts representative images of BRY / TBXT expression in inducible human embryoids generated with iG6, iS17, or iG6-S17 cells (maintaining doxycycline, cells removed on day 1 or day 3 after aggregation). iG6-S17: n = 34; iG6: n = 15; iS17: n = 16; iG6-S17-Dox. Day 1: n = 16; iG6-S17-Dox. Day 3: n = 20 embryoids, from 6 independent experiments. iG6-S17 vs iG6, P = 0.0225. iG6-S17 vs iG6-S17-Dox. Day 3, P = 0.0002. iG6-S17 vs iS17, P = 0.69. iG6-S17 vs iG6-S17-Dox. Day 1, P = 0.81. Figure 5E and Figure 5G The statistical method used in Fig. 5F was RM two-way ANOVA with Holm-Sidak multiple comparison test. Figure 5E In Fig. 5F and Figure 5G , the two-way Mann-Whitney test was used. Scale bar = 100 μm. For

[0025] Figure 6A-6H Depicts non-limiting exemplary embodiments and data related to the selection of transgenes to drive extraembryonic-like cells. Fig. 6A Depicts Uniform Manifold Approximation and Projection (UMAP), showing a combined human pre-implantation to post-implantation dataset colored by the original publication. Figure 6B Depicts UMAP of a combined human dataset colored by embryonic stage (d.p.f. is an abbreviation for days post-fertilization). Figure 6C Depicts UMAP of a combined human dataset colored by cell type. Fig.6D Depicts major cell type gene expression in UMAP of a human dataset. Fig. 6E Depicts single-cell RNA sequencing plots of key marker gene expression in a human dataset divided by cell type (n = 10223 cells). Fig. 6F Depicts putative epiblast, hypoblast, and trophoblast gene regulatory networks generated by SCENIC during peri-implantation human embryonic development. Candidate factors are marked with boxes (TFAP2C, GATA3, GATA6, and SOX17). Figure 6G Depicts regulator activities scored by SCENIC for hypoblast markers GATA6, SOX17, and TrB markers GATA3 and TFAP2C (n = 10223 cells). Figure 6HDepicts the qRT-PCR analysis of a single inducible cell line. The doxycycline-inducible constructs were inserted into Shef6 hESCs using the piggybac transposase (inducible GATA6, SOX17, GATA6-SOX17, GATA3, AP2Y, and GATA3-AP2Y). After single-cell seeding and proliferation, colonies were manually isolated. After adding 1 μg / mL doxycycline for 72 hours under basal N2B27 conditions, appropriate transgene expression was verified by RT-qPCR. N = 3 technical replicates were included. Clones selected for further analysis were marked with boxes. For box plots, the box includes the 25th to 75th percentile interval, the minimum and maximum values are shown as whiskers, and the center line represents the median.

[0026] Figure 7A-7F Depicts non-limiting exemplary embodiments and data related to immunofluorescence analysis of the major marker genes of hypoblast and trophoblast between different pluripotent states after doxycycline induction. Fig. 7A Depicts the qRT-PCR analysis of induced GATA6 (iG6), induced SOX17 (iS17), or induced GATA6-SOX17 (iG6-S17) alone and together 3 days after doxycycline induction from three pluripotent states. Figure 7B Depicts the qRT-PCR analysis of induced GATA3 (iG3), induced AP2Y (iAY), or induced GATA3-AP2Y (iG3-AY) 3 days after doxycycline induction from multiple pluripotent starting states. For Figure 7A-7B , n = 3 technical replicates were from 3 independent experiments. Figure 7C Depicts the immunofluorescence analysis of iG6, iS17, or iG6-S17 cells 3 days after induction from multiple pluripotent states. Fig.7D Depicts Figure 7C Quantification of the immunofluorescence levels. Fig. 7E Depicts the immunofluorescence analysis of iG3, iAY, or iG3-AY 3 days after induction from multiple pluripotent states. Figure 7F Depicts Fig. 7E Quantification of the immunofluorescence levels. For Figure 7C-7F , n = 3 technical replicates were from 2 independent experiments. Cells were initially cultured in mTeSR, RSeT, or PXGL conditions and then cultured for 3 days under the same conditions or alternatively transferred to basal N2B27 medium or basal N2B27 medium supplemented with doxycycline. The induced transgenes were marked with boxes. Scale bar = 100 μm.

[0027] Figure 8A-8D Depicts non-limiting exemplary embodiments and data related to the comparison of transcription factor-mediated induction with published directed differentiation methods. Fig. 8A Depicts the comparison and quantification of GATA6, SOX17, and SOX2 after directed differentiation of yolk sac-like cells (Activin-A, CHIR99021, and LIF), doxycycline-mediated induction in inducible GATA6-SOX17 cells, or both. Cells were differentiated under RSeT conditions. Fig. 8A The legend of the graph in the bottom panel of Figure 8B is the same as the legend of the graph in the bottom panel of Figure 8B Depicts the comparison and quantification of EOMES, N-cadherin, and OTX2 after directed differentiation of yolk sac-like cells (Activin-A, CHIR99021, and LIF), doxycycline-mediated induction in inducible GATA6-SOX17 cells, or both. For Figure 8A-B , N2B27: n = 717; ACL: n = 1211; N2B27+Dox.: n = 522; ACL+Dox.: n = 544 cells from 3 fields of view obtained from 2 independent experiments. Figure 8C Depicts the comparison and quantification of GATA3, AP2Yα, and SOX2 after directed differentiation in PA (PD0325901 and A83-01) or PAL (PD0325901, A83-01, and LPA), doxycycline-mediated induction in inducible GATA3-AP2Y cells, or both. Figure 8C The legend of the graph in the bottom panel of Fig.8D is the same as the legend of the graph in the bottom panel of Fig.8D Depicts the comparison and quantification of GATA2, KRT7, and AP2Y after directed differentiation in PA (PD0325901 and A83-01) or PAL (PD0325901, A83-01, and LPA), doxycycline-mediated induction in inducible GATA3-AP2YRseT cells, or both. For Figure 8C-Figure 8D , N2B27: n = 443; PA: n = 487; PAL: n = 371; N2B27+Dox.: n = 357; PA+Dox.: n = 412; PAL+Dox.: n = 287 cells from 3 fields of view obtained from 2 independent experiments. Scale bar = 100 μm. For Figure 8A-8D , mean ± SEM is plotted. hESCs were differentiated under RSeT conditions.

[0028] Figure 9A-9G Depicts non-limiting exemplary embodiments and data related to the assessment of extraembryonic-like induction from RSeT cells. Fig.9A Depicts a quality control plot of 10x multiome sequencing data for a cell line (n = 5328 cells). Violin plots from minimum to maximum. Fig. 9BDepicts a logistic regression framework for assessing similarity between clusters, applied to cell line RNA sequencing data, using published in vitro blastoid and directed differentiation protocols as training data. Fig. 9C Depicts gene expression of selected genes at 3 days after doxycycline induction from sequencing of wild-type, inducible GATA6-SOX17 (iG6-S17), and inducible GATA3-AP2Y (iG3-AP2Y) RseT hESC populations, visualized by uniform manifold approximation and projection (UMAP). Visualization of sample distribution in UMAP is as Figure 1C shown. Fig.9D Depicts immunofluorescence images of human cell-mouse embryo chimeras at the late blastocyst stage, showing the transition of human cells labeled with human nuclear antigen (HuNAg) contributing to the SOX2-positive epiblast to SOX17-positive primitive endoderm upon iG6-S17 induction. Fig.9E Depicts quantification of the contribution of HuNAg-positive cells stained for SOX2 and SOX17. Control: n = 12 embryos, iG6-S17: n = 30 embryos, from 3 independent experiments. Fig.9F Depicts immunofluorescence images of human cell-mouse embryo chimeras at the late blastocyst stage, showing the transition of human cells from the SOX2-positive epiblast to the GATA3-positive trophectoderm upon iG3-AP2Y induction. Figure 9G Depicts quantification of the contribution of HuNAg-positive cells stained for SOX2 and GATA3. Control: n = 10 embryos, iG6-AP2Y: n = 27 embryos, from 3 independent experiments. hESCs were induced under RSeT conditions. For box plots, the box encompasses the 25th to 75th percentile interval, the minimum and maximum are shown as whiskers, and the center line represents the median. Scale bar = 100 μm.

[0029] Figures 10A-10F Depicts non-limiting exemplary embodiments and data related to the identification of clusters of post-implantation human embryo-like models. Fig. 10A Depicts embryoids generated from the second hESC cell line RUES2 on day 4. N = 371 structures from 2 independent experiments. Fig. 10B Depicts bright-field images of inducible human embryoids selected for sequencing on days 4, 6, and 8 (n = 12 for each stage). Note the presence of an inner domain surrounded by two concentric domains. Fig. 10C Depicts quality control plots of embryoid sequencing data on days 4, 6, and 8 after aggregation (n = 5217 cells). Violin plots from minimum to maximum. Fig. 10DDepicts the projection of induced human embryoid body cells onto datasets covering the peri-implantation to gastrulation stages of cynomolgus macaque (M. fasicularis) and human (Homo sapiens) using scmap. Fig. 10E Depicts the expression of major marker genes for epiblast, endoderm, mesoderm, trophectoderm, and amnion in a stem cell-derived model. Fig.10F Depicts a stream plot showing the contribution of embryoid bodies on day 4, day 6, or day 8 to the specified cell types. Scale bar = 100 μm. The inner domain of the embryoid body is surrounded by a dashed line.

[0030] Figures 11A-11D Depicts non-limiting exemplary embodiments and data related to the clustering comparison of embryoid bodies with human and cynomolgus macaque datasets. Fig.11A Depicts a logistic regression analysis comparing the annotated cluster groups (training data) of datasets from cynomolgus macaque (M. fasicularis) and human (H. sapiens) from the peri-implantation to gastrulation stages with the post-implantation human embryo-like model cluster groups (test data). Cynomolgus macaque data are from a previous report. Fig. 11B Depicts a logistic regression analysis comparing in vitro human embryo-like model and directed differentiation dataset (training data) with induced human embryoid bodies (test data). Fig. 11C Depicts the projection of an induced human embryoid body dataset onto an in vitro dataset using scmap. The in vitro dataset is from 3 previous reports. Fig.11D Depicts a violin plot of gene expression in GFP-negative and GFP-positive cells derived from induced GATA3-AP2Y (iG3-AP2Y) cells from an induced human embryoid body sequencing dataset (n = 5217 cells). The violin plot ranges from the minimum to the maximum.

[0031] Figure 12A-12J Depicts non-limiting exemplary embodiments and data related to the extraembryonic mesenchymal trajectory and wild-type cell differentiation ability. Fig. 12A Depicts the immunofluorescence of HAND1, showing its expression in GATA6-positive cells (presumptive extraembryonic mesenchyme) and upregulation in presumptive amnion (AP2Y-positive) from day 4 to day 6. These images represent 2 experiments. Fig. 12B Depicts the expression of HAND1 in an induced human embryoid body single-cell sequencing dataset. Fig. 12C Depicts the immunofluorescence of TBX20, demonstrating high expression in a subset of GATA6-positive cells (presumptive extraembryonic mesenchyme). These images represent 5 experiments. Fig.12D Depicts the expression of TBX20 in an induced human embryoid body single-cell sequencing dataset, demonstrating its enrichment in the extraembryonic mesenchymal cell cluster. Fig.12EDepicts the differentiation of ISL1-positive amnion and GATA6 / TBX20-positive extraembryonic mesenchyme in structures derived from the second cell background, RUES2. These images represent 2 experiments. Fig.12F Depicts the differentiation of primordial germ cell-like cells in embryoid bodies derived from the second cell background, RUES2. These images represent 2 experiments. Figure 12G Depicts examples and quantification of day 4 embryoid bodies. The embryoid bodies exhibit an outer layer of GFP-positive induced GATA3-AP2Y (iG3-AP2Y) cells, an inner domain composed of mKate2-positive wild-type hESCs, and an interstitial GATA6-positive population mainly composed of unlabeled induced GATA6-SOX17 (iG6-S17) cells. N = 9 embryoid bodies from 2 independent experiments. Fig.12H Depicts ISL1-positive amnion-like cells overlapping with mKate2-positive wild-type cells. These images represent 3 experiments. Fig.12I Depicts the expression of GATA6 and TBX20-positive extraembryonic mesenchyme-like cells overlapping with mKate2-positive wild-type cells. These images represent 3 experiments. Fig.12J Depicts the expression of AP2Y, SOX17, and NANOG triple-positive primordial germ cell-like cells overlapping with mKate2-positive wild-type cells. These images represent 3 experiments. Scale bar = 100 μm. For Figure 12G , mean ± SEM is plotted. The inner domain of the embryoid body is surrounded by a dashed line.

[0032] Figures 13A-13H Depicts non-limiting exemplary embodiments and data related to the role of BMP and induced GATA3-AP2Y cells in generating induced human embryoid bodies. Fig.13A Depicts the fitting of ID1-4 expression over time, colored by cell type assignment. Fig. 13B Depicts the motif accessibility scores of SMAD5 and SMAD2::SMAD3::SMAD4 fitted over time by ChromVar, colored by cell type assignment. Fig. 13C Depicts predicted ligand-receptor pairings in induced human embryoid bodies generated by CellPhoneDB. Fig.13D Depicts the predicted interactions of induced GATA6-SOX17 (G6-S17) and induced GATA3-AP2Y (G3-AP2Y) cells with wild-type RseT hESCs 3 days after induction, where wild-type RseT hESCs are the cell type aggregated to generate induced human embryoid bodies. Fig.13EDepicted is the failure of inducible human embryoid bodies to form if inducible GATA3-AP2Y cells are not included or if the BMP signaling antagonist LDN193189 (LDN) is added from day 0 to day 2. Fig.13F Depicted Fig.13E Quantification of embryoid body formation efficiency. N = 535 GloESC+iG6-S17 and 500 LDN-treated constructs from 4 independent experiments. Figure 13G Depicted is the quantification of embryoid body size after addition of LDN193189 from day 0 to day 2. n = 105 structures per condition per day from 3 independent experiments. The statistical test used was a two-sided Mann-Whitney between control and LDN at each time point (day 1, P < 0.0001, day 2, P = 0.0019, day 3, P < 0.0001). Fig.13H Depicted is an overview of a whole Aggrewell demonstrating the effects of BMP inhibition, BMP4 addition, and NODAL activation. These images are representative of 5 experiments. Note the significant increase in well-organized structures expressing SOX2 following BMP inhibition. Scale bar = 100 μm. **P<0.01. ****P<0.0001. For Fig.13F , mean ± SEM are plotted. Figure 13G , all individual data points are plotted.

[0033] Figure 14A-Figure 14C Depicted are non-limiting exemplary embodiments and data related to downregulation of CER1 expression in inducible human embryoid bodies following prolonged culture. Fig.14A Depicted are the efficiencies of embryoid body formation generated under different conditions. Note that the highest efficiencies were achieved using standard conditions described herein with consistent addition of doxycycline and the use of inducible GATA6-SOX17 cells (iG6-S17). iG6-S17: n=224; induced GATA6 (iG6): n=276; induced SOX17 (iS17): n=247; iG6-S17 with doxycycline removed at day 1: n=370; iG6-S17 with doxycycline removed at day 3: n=410 constructs from 2 independent experiments. Fig. 14B Immunofluorescence for CER1 and SOX2 at day 6 post-aggregation is depicted, showing that both SOX2 and CER1 are downregulated at this stage in constructs generated using inducible iG6 or iG6-S17 hypoblast-like cells (doxycycline was continuously added, or removed early at day 3 post-aggregation), as well as wild-type ESCs and inducible GATA3-AP2Y (iG3-AP2Y) cells. These images are representative of 3 experiments. Fig. 14CShown, embryoid bodies generated from Shef6-mKate2 ESCs demonstrated that when doxycycline was removed early on day 3 after aggregation, both the ISL1-positive and BRY-positive cell populations in structures generated from iG6 or iG6-S17 hypoblast-like cells differentiated from wild-type cells. In Fig. 14C In, the images show DAPI, mKate2 (WT ESC), ISL1, BRY, and merged views of mKate2, ISL1, and BRY from top to bottom. These images represent 3 experiments. Scale bar = 100 μm. The inner domain of the embryoid body is enclosed by a dashed line. Detailed Description

[0034] In the following detailed description, reference will be made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols generally represent like components unless the context otherwise indicates. The exemplary embodiments described in the detailed description, the drawings, and the claims are not intended to limit the present invention. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter described herein. It is readily understood that the various aspects of the disclosure, as generally described and illustrated in the drawings herein, can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are explicitly contemplated herein and form a part of the disclosure herein.

[0035] With respect to the related art, all patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety.

[0036] definition

[0037] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, N.Y., 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y., 1989). For the purposes of this disclosure, the following terms are defined as follows.

[0038] As used herein, the term "about," when referring to a measurable value (e.g., amount of a compound, dose, time, temperature, etc.), is intended to cover variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0039] As used herein, the term "stem cell" refers to a cell that can maintain a constant differentiation potential even after cell division. Examples of stem cells include: pluripotent embryonic stem cells derived from fertilized eggs or cloned embryos; epiblast stem cells; trophoblast stem cells; extraembryonic endoderm (XEN) stem cells; adult stem cells and multipotent stem cells present in the tissues of an organism (e.g., hepatic stem cells, dermal stem cells, and germline stem cells that underlie the corresponding tissues); multipotent stem cells derived from germline stem cells; pluripotent stem cells obtained by nuclear reprogramming of somatic cells; totipotent stem cells and non-totipotent stem cells, etc.

[0040] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the potential to differentiate into all cells except the placenta. Pluripotent stem cells have the potential to differentiate into any one of the following three germ layers: endoderm (forming structures such as the gastrointestinal and respiratory systems), mesoderm (forming structures such as the musculoskeletal system, vascular system, and urogenital system), or ectoderm (forming epidermal tissues and the nervous system).

[0041] As used herein, the term "embryonic stem cell" (ES cell or ESC) refers to a pluripotent stem cell derived from the inner cell mass of a blastocyst, which is an early pre-implantation embryo. It is expected that such cells can express genes involved in the naive pluripotency network (Oct4 / Nanog, Sox2, Klf4, etc.). Such cells can also have Oct4 proximal enhancer activity. They can contribute to all embryonic tissues in a chimera. ES cells can be derived from mammalian embryos, obtained from iPS cells, or obtained from a suitable cell line. Non-limiting examples of such stem cells include: embryonic stem cells of mammals, etc. produced by culturing early pre-implantation embryos; embryonic stem cells produced by culturing early embryos prepared by nuclear transfer of somatic cell nuclei; induced pluripotent stem cells (iPS cells) produced by transferring multiple different transcription factors into somatic cells; and pluripotent stem cells produced by genetically engineering the genes on the chromosomes of embryonic stem cells or iPS cells. More specifically, embryonic stem cells include: embryonic stem cells produced from the inner cell mass that constitutes an early embryo; embryonic stem cells produced from primordial germ cells; cells isolated from a cell population with the pluripotency of an early pre-implantation embryo (e.g., primitive ectoderm); and cells obtained by culturing these cells.

[0042] As used herein, the term "trophoblast stem cell" refers to stem cells derived from the trophoblast lineage of an embryo. Trophoblast stem cells are preferably extraembryonic cells derived from two cell types of the human placental precursor: cytotrophoblast and syncytiotrophoblast. These cells can be derived from the late pre-implantation stage or the early post-implantation stage, but the resulting cell line is equivalent to the stem cell compartment present in the extraembryonic ectoderm of the post-implantation mouse egg cylinder. Transcription factors such as Cdx2, Tead4, Gata3, Elf5, Eomes, and Tfap2C are characteristic of this lineage. TS cells can also be regarded as cells that are precursors of placental differentiated cells. In mice, TS cells can be derived from the polar trophoblast of the blastocyst or outgrowths of the extraembryonic ectoderm, where the extraembryonic ectoderm is derived from the polar trophoblast after implantation.

[0043] As used herein, the term "extraembryonic endoderm stem cell" (XEN stem cell) refers to stem cells derived from the extraembryonic endoderm of an embryo. The extraembryonic endoderm is generally a derivative of hypoblast cells that migrate into the blastocoel (starting on day 8 of human embryonic development) and line the blastocoel, forming the primary yolk sac and the definitive yolk sac. The extraembryonic endoderm fills the remaining part of the blastocoel.

[0044] As used herein, the term "differentiation" can refer to the process by which non-specialized ("non-committed") or less specialized cells acquire the characteristics of specialized cells (such as neuronal cells). Differentiated cells are cells that are in a more specialized ("committed") position in the cell lineage. The term "commitment" when used in the context of the differentiation process refers to the stage at which a cell has progressed in the differentiation pathway to the point where under normal circumstances it will continue to differentiate into a specific cell type or subpopulation of cell types and under normal circumstances cannot differentiate into other cell types or revert to a less differentiated cell type. As used herein, a cell lineage defines the heredity of a cell, i.e., from which cells that cell is derived and into which cells it can differentiate. A cell lineage places a cell within the genetic program of development and differentiation. As used herein, a "lineage-specific marker" can refer to a characteristic that is specifically associated with the cell phenotype of a lineage of interest and can be used to assess the differentiation of uncommitted cells into the lineage of interest.

[0045] As used herein, the terms "marker", "lineage marker", or "lineage-specific marker" can refer to nucleic acid or polypeptide molecules that are differentially expressed in cells of interest. Differential expression can refer to an increased level of a positive marker and a decreased level of a negative marker compared to undifferentiated cells. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or sufficiently lower in the cells of interest than in other cells such that the cells of interest can be identified and distinguished from other cells using any method known in the art. In some embodiments, the marker can be enriched. As used herein, the term "enriched" shall have its ordinary meaning and can also refer to a statistically significant increase in the level of a gene product (e.g., mRNA and / or protein) under one condition compared to another condition (e.g., in one cell layer compared to another cell layer).

[0046] As used herein, the term "concentration" shall have its ordinary meaning and can also refer to (a) mass concentration, molar concentration, volume concentration, mass fraction, molar fraction, or volume fraction, or (b) the ratio of the mass or volume of one component in a mixture or solution to the mass or volume of another component in the mixture or solution (e.g., ng / ml). In some embodiments, the concentration can refer to the fraction of active units per volume (e.g., U / ml).

[0047] As used herein, the term "analogue" refers to a compound that can be structurally related to a relevant molecule. The term "agonist" as used herein can refer to a compound that can be structurally unrelated to a relevant molecule. For example, an agonist can activate a relevant receptor by altering the conformation of the receptor. However, in both cases, the use of these two terms in this specification refers to a compound or molecule that can mimic, replicate, or otherwise generally substitute for the specific biological activity of a relevant molecule.

[0048] The phrase "culture medium" as used herein refers to a liquid substance used to support the growth and development of stem cells and embryos. According to some embodiments of the present invention, the culture medium used can be a water-based culture medium that contains a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, and / or proteins (e.g., cytokines, growth factors, and hormones) required for cell growth and embryo development.

[0049] Provided herein are methods, compositions, and culture media for generating post-implantation embryo models of mammals (e.g., humans) that include embryonic and extra-embryonic tissues. Two types of extra-embryonic-like cell types generated by overexpression of transcription factors are combined with wild-type embryonic stem cells and self-organize into structures that mimic various aspects of a post-implantation embryo (e.g., a post-implantation human embryo). The self-organized structure contains a pluripotent epiblast-like domain surrounded by hypoblast-like tissue and trophoblast-like tissue.

[0050] The present disclosure provides an in vitro method for generating synthetic embryos from embryonic stem cells. In some embodiments, the method comprises co-culturing wild-type mammalian embryonic stem cells (ESCs), a first modified mammalian ESC comprising the GATA6 gene and / or the SOX17 gene, and a second modified mammalian ESC comprising the GATA3 gene and / or the TFAP2C gene under culture conditions that cause the ESCs to self-organize into post-implantation embryonic structures. The present disclosure also provides synthetic embryo structures obtained by the methods disclosed herein.

[0051] The present disclosure also provides a method for studying mechanisms involved in embryogenesis according to the in vitro methods disclosed herein. The present disclosure also provides a method for identifying a compound useful for treating a disease, comprising contacting a synthetic embryo obtained by the in vitro methods disclosed herein with the compound. The present disclosure also provides a method for diagnosing or treating a disease or disorder in a subject. The method can comprise generating a synthetic embryo according to the methods disclosed herein and transplanting the synthetic embryo into the subject. The present disclosure also provides a method for elucidating the role of a candidate gene in embryonic development. The method can comprise obtaining wild-type mammalian ESCs, a first modified mammalian ESC comprising the GATA6 gene and / or the SOX17 gene, and a second modified mammalian ESC comprising the GATA3 gene and / or the TFAP2C gene, wherein the candidate gene has been modified or knocked out; and culturing the mammalian ESCs using the in vitro methods disclosed herein.

[0052] Generating synthetic embryonic structures from pluripotent stem cells

[0053] Human reproductive efficiency is extremely low, with an estimated 60% of pregnancies failing within the first two weeks after fertilization. Since the advent of in vitro fertilization technology, the first week of human embryonic development has been studied. However, the second week, including the implantation uterus and the preparation for gastrulation, remains a "black box". The human blastocyst at 5-6 days post-fertilization consists of the outermost trophectoderm (placental precursor) and the inner cell mass, which develops into the epiblast and the yolk sac precursor (hypoblast) of the embryo. At 7-8 days post-fertilization, the blastocyst implants into the endometrium, the epiblast polarizes and transitions from a naive pluripotent state to a primed state. The central amniotic cavity forms within the epiblast, separating the dorsal amniotic epithelium from the ventral epiblast, maintaining its pluripotency and ultimately forming the embryo. The trophectoderm develops into several trophoblast subtypes after implantation, while the hypoblast forms the primary yolk sac, which is subsequently replaced by the secondary yolk sac. A subset of cells in the hypoblast maintains the expression of NODAL, BMP, and WNT inhibitors, protecting the future anterior epiblast from posteriorizing signals during primitive streak formation, which is marked by upregulation of BRY / TBXT. Another extraembryonic tissue, the extraembryonic mesenchyme, lies between the inner cell mass-derived tissues and the trophoblast, yet the origin of these cells remains unclear.

[0054] Recent studies on mouse embryos have established conditions suitable for in vitro culture of human embryos until implantation, uncovering this developmental black box for the first time. This system has been used to characterize major developmental events, including the formation of the anterior hypoblast domain, the determination of trophoblast subtypes, and the transition of epiblast pluripotency states. However, mechanistic studies in human embryos remain challenging. Therefore, stem cell-based human embryo models will become important complementary tools for understanding this critical developmental period. Some research groups have reported the generation of blastocyst-like structures derived from human embryonic stem cells (hESCs). These blastocysts are similar to pre-implantation embryos but develop poorly towards the post-implantation stage. Other models, including gastruloids, two-dimensional micropatterns, and embryoid bodies, can mimic various aspects of post-implantation development. However, these models are entirely derived from hESCs, lack extra-embryonic tissues, and cannot reproduce embryonic morphology. A recent study combined epiblast-like spheres with hESCs treated with BMP4 and expressing multiple extra-embryonic markers, marking a step towards an integrated model of the post-implantation embryo. However, this model did not show self-organization of epiblast-like compartments in extra-embryonic tissues until after lumen formation, and the BMP4-treated hESCs were not associated with the target extra-embryonic lineages.

[0055] Several protocols have been developed to derive trophoblast and hypoblast cells from hESCs. Importantly, the pluripotency state affects the developmental trajectories of differentiated cells. The derivation of lineage-specific cell lines provides the possibility of mimicking these tissues in vitro. However, generating a modular integrated model system containing both embryonic and extra-embryonic tissues has proven challenging. This may be because the signaling pathway regulators required for hESC culture, hypoblast-like cell differentiation, and trophoblast-like cell differentiation are entirely different. In addition, while cross-tissue communication is an advantage of the integrated model system, generating embryoid bodies in media containing exogenous factors may impair tissue-driven self-organization.

[0056] To overcome these limitations, the present disclosure provides methods, compositions, and culture media for generating synthetic embryos (e.g., human embryos) from pluripotent stem cells (e.g., pluripotent embryonic stem cells (“ESCs”)), the methods being based on methods of expressing (e.g., overexpressing) transcription factors that can drive pluripotent embryonic stem cells to generate extraembryonic-like cells (including trophoblast-like cells and hypoblast-like cells). The in vitro embryonic models based on pluripotent stem cells described herein are generated using the embryonic and extraembryonic lineages of fully pluripotent embryonic stem cells (e.g., human ESCs or “hESCs”). Extraembryonic trophoblast-like cells and hypoblast-like cells can be generated by overexpressing transcription factors in wild-type embryonic stem cells. The present disclosure demonstrates that aggregates of induced extraembryonic-like lineages and wild-type ESCs (e.g., human ESCs) are capable of self-organizing into embryo-like structures that mimic several features of post-implantation development, including lumen formation, amnion formation, primordial germ cell formation, and anterior hypoblast specification. These induced embryoids are modular, independent of exogenous signaling factors, and susceptible to genetic perturbation. In some embodiments, the induced embryoids generated herein are human embryoids. Some of the methods, compositions, and culture media disclosed herein are also described in “Weatherbee, B.A.T. et al. Pluripotent stem cell-derived model of the post-implantation human embryo. Nature (2023), published online: June 27, 2023, doi.org / 10.1038 / s41586-023-06368-y”, the entire content of which is incorporated herein by reference.

[0057] The present disclosure provides an in vitro method for generating synthetic embryos from mammalian pluripotent stem cells (e.g., pluripotent embryonic stem cells or ESCs). The method can include co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene under culture conditions that cause the ESCs to self-organize into post-implantation embryo structures. In some embodiments, the pluripotent embryonic stem cells are human pluripotent embryonic stem cells (hESCs), and the generated synthetic embryos are human embryos. The pluripotent embryonic stem cells or ESCs can be ESCs across the pluripotency spectrum, including, for example, naive ESCs, formative ESCs, or primed ESCs. For example, the ESCs used herein can be pre-implantation naive ESCs, peri-implantation-like pluripotent ESCs, or post-implantation primed ESCs. In some embodiments, the ESCs used herein are peri-implantation-like pluripotent ESCs, such as peri-implantation-like pluripotent hESCs (e.g., RseT hESCs). In some embodiments, compared to embryonic stem cells in other pluripotent states (e.g., primed cells), the level of amnion-specific genes expressed by the ESCs used herein is lower during trophoblast-like cell induction. Embryonic stem cells in different pluripotent states can be pre-cultured using media / conditions known to those of skill in the art. For example, pre-implantation naive hESCs can be generated by culturing in PXGL medium prior to co-culture. Peri-implantation-like pluripotent hESCs can be generated by culturing in RSeT medium prior to co-culture. Post-implantation-like primed hESCs can be generated by culturing in mTeSR1 medium prior to co-culture.

[0058] Modified mammalian pluripotent stem cells, such as ESCs, can contain one or more genes encoding one or more transcription factors that can drive the generation of extraembryonic cells or extraembryonic-like cells. In some embodiments, the modified mammalian ESCs are inducible mammalian ESCs containing one or more inducible genes encoding one or more transcription factors described herein. The inducible ESCs can express one or more inducible genes upon induction. For example, the modified mammalian ESCs can contain only the inducible GATA6 gene. Alternatively or additionally, the modified mammalian ESCs can contain only the inducible SOX17 gene. In some embodiments, the modified mammalian ESCs contain the inducible GATA6 gene and the inducible SOX17 gene. The pluripotent stem cells containing the inducible GATA6 gene and / or the inducible SOX17 gene can be in any pluripotent state. For example, the modified mammalian ESCs containing only the inducible GATA6 gene can be in a pre-implantation naive state or an intermediate peri-implantation-like state. The modified mammalian ESCs containing only the inducible SOX17 gene can be in a pre-implantation naive state, an intermediate peri-implantation-like state, or a post-implantation primed state. The modified mammalian ESCs containing the inducible GATA6 gene and the inducible SOX17 gene can be in a pre-implantation naive state, an intermediate peri-implantation-like state, or a post-implantation primed state.

[0059] The modified mammalian ESCs can contain only the inducible GATA3 gene. Alternatively or additionally, the modified mammalian ESCs can contain only the inducible TFAP2C gene. In some embodiments, the modified mammalian ESCs contain the inducible GATA3 gene and the inducible TFAP2C gene. The pluripotent stem cells containing the inducible GATA3 gene and / or the inducible TFAP2C gene can be in any pluripotent state. For example, the modified mammalian ESCs containing only the inducible TFAP2C gene can be pre-implantation naive pluripotent stem cells (e.g., PXGL cells) or peri-implantation pluripotent stem cells (e.g., RSeT cells). The modified mammalian ESCs containing only the inducible GATA3 gene can be in a pre-implantation naive state, an intermediate peri-implantation-like state, or a post-implantation primed state. The modified mammalian ESCs containing the inducible GATA3 gene and the inducible TFAP2C gene can be in a pre-implantation naive state, an intermediate peri-implantation-like state, or a post-implantation primed state.

[0060] In some embodiments, GATA6 and / or SOX17 can drive the development of pluripotent stem cells (e.g., ESCs) into hypoblast-like cells. In some embodiments, GATA3 and / or TFAP2C can drive the development of pluripotent stem cells (e.g., ESCs) into trophoblast-like cells.

[0061] In some embodiments described herein, the modified mammalian ESCs are induced mammalian ESCs that express or overexpress one or more genes encoding one or more of the transcription factors described herein. In some embodiments, the modified ESCs (e.g., hESCs) can overexpress one or more of the transcription factors described herein. For example, modified ESCs containing the GATA6 gene and / or the SOX17 gene exhibit an increase in the expression of GATA6 and / or SOX17 mRNA to at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 140-fold, 160-fold, 180-fold, 200-fold, 220-fold, 240-fold, 260-fold, 270-fold, 280-fold, 300-fold or more compared to wild-type ESCs. In some embodiments, modified ESCs containing the GATA3 gene and / or the TFAP2C gene exhibit an increase in the expression of GATA3 and / or TFAP2C mRNA to at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 140-fold, 160-fold, 180-fold, 200-fold, 220-fold, 240-fold, 260-fold, 270-fold, 280-fold, 300-fold or more compared to wild-type ESCs.

[0062] The method can further include contacting an inducible mammalian ESC with an inducer (e.g., doxycycline) to generate an induced mammalian ESC. Induction can be performed during or prior to co-culturing the modified ESC with the wild-type ESC. The contacting can be performed for any duration to increase the mRNA expression of transcription factors (e.g., GATA6, SOX17, GATA3, and / or TFAP2C) to a desired level. In some embodiments, the inducer can be provided to the culture medium during the co-culture process. For example, the inducer can be supplied or applied to the culture medium containing wild-type ESC and modified inducible ESC for about, at least, at least about, at most, or at most about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer. In some embodiments, the inducer is supplied throughout the co-culture process. Alternatively or additionally, the modified mammalian ESC can be induced prior to co-culture. For example, the inducible ESC can be induced (e.g., in the presence of an inducer) prior to co-culturing with the wild-type ESC to generate induced ESC that express or overexpress one or more transcription factors (e.g., GATA6, SOX17, GATA3, and TFAP2C). The induced ESC is then co-cultured with the wild-type ESC under conditions that allow the ESC to self-organize into an aggregated structure. The duration of induction and / or the concentration of the inducer can be adjusted, for example, by increasing or decreasing the concentration or amount of the inducer in the culture medium, or by increasing or decreasing the duration of the inducer in the culture medium. In some embodiments, induction can be terminated by removing the inducer from the culture medium, e.g., by replacing or supplementing the culture medium with fresh medium without the inducer.

[0063] The number or amount of wild-type ESCs and modified ESCs can have any suitable ratio, which can vary in different embodiments. In some embodiments, the provided ESCs containing the GATA6 gene and / or the SOX17 gene and / or the ESCs containing the GATA3 gene and / or the TFAP2C gene exceed the wild-type ESCs. In some embodiments, the provided ESCs containing the GATA3 gene and / or the TFAP2C gene exceed the ESCs containing the GATA6 gene and / or the SOX17 gene. The provided amount or cell count of the ESCs containing the GATA3 gene and / or the TFAP2C gene can be at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold that of the wild-type ESCs and / or the ESCs containing the GATA6 gene and / or the SOX17 gene. In some embodiments, the provided amounts of the ESCs containing the GATA6 gene and / or the SOX17 gene and the wild-type ESCs are approximately the same (e.g., a ratio of 1:1). In some embodiments, the ratio of wild-type ESCs, ESCs containing the GATA6 gene and / or the SOX17 gene, and ESCs containing the GATA3 gene and / or the TFAP2C gene is from about 1:1:2 to about 1:1:10, such as 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, or higher. In some embodiments, the ratio of wild-type ESCs, ESCs containing the GATA6 gene and / or the SOX17 gene, and ESCs containing the GATA3 gene and / or the TFAP2C gene is about 1:1:2. In some embodiments, the ratio between wild-type ESCs, ESCs containing the GATA6 gene and / or the SOX17 gene, and ESCs containing the GATA3 gene and / or the TFAP2C gene is about 1:1:1.

[0064] In some embodiments, the duration of co - culturing wild - type ESCs, ESCs comprising the GATA6 gene and / or the SOX17 gene, and ESCs comprising the GATA3 gene and / or the TFAP2C gene is about, at least, at least about, at most, or at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, the co - culturing comprises co - culturing wild - type ESCs, ESCs comprising the GATA6 gene and / or the SOX17 gene, and ESCs comprising the GATA3 gene and / or the TFAP2C gene in a medium suitable for pluripotent stem cell proliferation (e.g., N2B27 medium). In some embodiments, the culturing in the pluripotent stem cell proliferation medium lasts about 5 days, and optionally the ESCs are passaged at least twice in the stem cell proliferation medium. During culturing in the stem cell proliferation medium, the ESCs can aggregate and form an aggregated structure that exhibits a distinction between an inner cell domain and an outer cell domain, e.g., after 3 days of culturing in the stem cell proliferation medium. After aggregation, the ESCs can be further cultured in the pluripotent stem cell proliferation medium for about 2 days and then transferred to a post - implantation medium. The method further comprises co - culturing the ESCs in a post - implantation medium (e.g., post - implantation human embryo medium, such as hIVC1) after co - culturing in the pluripotent stem cell proliferation medium. The culturing in the post - implantation medium can last about, at least, at least about 1 day, 2 days, 3 days, 4 days, 5 days, or longer. Co - culturing the ESCs in the post - implantation medium can start about 2 days after ESC aggregation. In some embodiments, co - culturing the ESCs comprises transferring the ESCs from one substrate to another substrate.

[0065] Thus, in some embodiments, the method comprises: co - culturing wild - type mammalian embryonic stem cells (ESCs), a first modified mammalian ESC comprising the GATA6 gene and / or the SOX17 gene, and a second modified mammalian ESC comprising the GATA3 gene and / or the TFAP2C gene in a medium suitable for pluripotent stem cell proliferation (e.g., N2B27 medium) under conditions that cause the ESCs to form an aggregated structure. The method can further comprise: culturing the aggregated structure in a post - implantation medium under conditions that cause the aggregated structure to develop into a synthetic embryo mimicking a post - implantation embryo structure. The cell aggregates formed from wild - type ESCs, ESCs comprising the GATA6 gene and / or the SOX17 gene, and ESCs comprising the GATA3 gene and / or the TFAP2C gene can self - organize into a synthetic embryo structure mimicking the embryo structure at the post - implantation stage. The post - implantation embryo structure can be a human embryo structure. In some embodiments, the post - implantation embryo structure is similar to a post - implantation human embryo about 8 - 9 days after fertilization.

[0066] Synthetic embryo structures generated using the methods and culture media described herein comprise embryonic and extra-embryonic tissues and recapitulate embryonic morphology. Post-implantation embryo structures (e.g., human post-implantation embryo structures) described herein can comprise a pluripotent epiblast-like domain surrounded by hypoblast-like and trophoblast-like tissues. In some embodiments, the post-implantation embryo structure comprises a SOX2-positive epiblast-like domain containing a central cavity; an outer monolayer of GATA3-positive putative trophoblast-like cells; and an intermediate putative hypoblast-like domain of GATA6-positive cells between the inner cavitating domain and the outer layer.

[0067] The efficiency of co-culturing wild-type ESCs and modified ESCs to form post-implantation embryo structures can vary in different embodiments depending on various factors such as the pluripotency state of the ESCs, the expression levels of one or more of the transcription factors described herein, the cell culture time, the induction time and induction intensity, and / or other factors that can be determined by those skilled in the art upon reading this disclosure. For example, in some embodiments, using ESCs in an intermediate pluripotency state such as peri-implantation pluripotent ESCs (e.g., RseT hESCs) can generate post-implantation embryos with higher efficiency compared to using ESCs in other pluripotency states (e.g., pre-implantation naive ESCs or post-implantation primed ESCs). In some embodiments, the efficiency of co-culturing wild-type ESCs, ESCs comprising the GATA6 gene and / or the SOX17 gene, and ESCs comprising the GATA3 gene and / or the TFAP2C gene to form post-implantation embryos is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35% or higher. In some embodiments, the efficiency of forming post-implantation embryos from the ESCs described herein can be greater than 20% (e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or higher).

[0068] The synthetic embryo structures described herein are ESC-derived embryo models that are capable of forming a pluripotent epiblast-like domain surrounded by hypoblast-like and trophoblast-like tissues. Specifically, trophoblast and hypoblast cells are derived or programmed from ESCs (e.g., hESCs). Thus, the method does not include culturing trophoblast cells, hypoblast cells, or both. Specifically, the method does not include co-culturing trophoblast cells and / or hypoblast cells with pluripotent embryonic stem cells.

[0069] In some embodiments, the methods disclosed herein do not include any in vivo steps. In some embodiments, in any of the culture steps disclosed herein, wild-type ESCs, ESCs comprising the GATA6 gene and / or the SOX17 gene, ESCs comprising the GATA3 gene and / or the TFAP2C gene, and synthetic embryos are not present in an in vivo environment. An in vivo environment can include tissues, organs, organisms, or combinations thereof.

[0070] The methods, compositions, and culture media described herein also eliminate the need for exogenous signaling pathways that can compromise tissue-driven self-organization. In some embodiments, the methods, compositions, and culture media for generating synthetic embryos (e.g., human synthetic embryos) described herein do not include the use of exogenous signaling pathway factors (e.g., WNT signaling pathway activators and / or TGFβ superfamily members). For example, any culture media used herein do not contain or are supplemented with exogenous signaling pathway factors.

[0071] Pluripotent embryonic stem cells and embryonic development

[0072] Methods, compositions, and culture media for culturing synthetic embryos in vitro from mammalian pluripotent stem cells (e.g., ESCs) are disclosed herein. In some embodiments, the mammalian pluripotent ESCs are hESCs. In some embodiments, the method comprises co-culturing wild-type mammalian embryonic stem cells (ESCs), a first modified mammalian ESC comprising the GATA6 gene and / or the SOX17 gene, and a second modified mammalian ESC comprising the GATA3 gene and / or the TFAP2C gene, under culture media conditions that cause the ESCs to self-organize into synthetic embryos that mimic post-implantation embryo structures. In some embodiments, the generated post-implantation embryo structures represent a multi-lineage stem cell-derived model of a human post-implantation embryo that undergoes lumenogenesis in its epiblast-like domain and differentiation events that reflect the interaction between extraembryonic-like tissues and embryo-like tissues.

[0073] While mammalian embryogenesis has some common features in all species, it is understood that the developmental patterns and rates vary among different mammalian species. Generally, a fertilized egg undergoes multiple cleavage steps (going through the two-cell stage, four-cell stage, and eight-cell stage), then compresses to form a solid ball of cells called a morula, and the cells continue to divide within the morula. Eventually, the inner cells of the morula form the inner cell mass, and the outer cells form the trophectoderm. The morula then develops into a blastocyst, which is surrounded by the trophectoderm and contains a fluid-filled vesicle, with the inner cell mass located at one end of the blastocyst.

[0074] As used herein, the term "embryo" refers to a mammalian organism at the single-cell stage. The embryos described herein are produced by culturing embryonic stem cells in vitro under appropriate conditions and are similar or mimic natural embryos generated in vivo at the corresponding stage, e.g., having similar morphology, length, weight, cell type composition, and expression of developmental marker genes.

[0075] The developmental stages of an embryo can be defined by the development of specific structures and can be used to define corresponding stages in the development of other species. In some embodiments, the developmental stages of an embryo can be defined according to the "Carnegie stages", which is a standardized system for providing a unified developmental chronology of vertebrate embryos. The earliest Carnegie stages are shown in Table 1 below.

[0076] Table 1: Carnegie stages of development

[0077]

[0078] In some embodiments, the methods, compositions, and culture media described herein can enable culturing that continues through post-implantation stages corresponding to Carnegie stage (a), stage 5(b), stage 5(c), stage 6, stage 7, stage 8, stage 9 and above, and corresponding stages in other species.

[0079] The methods, compositions, and culture media described herein can be applied to embryos of any suitable mammalian species, including human and non-human, such as: primates, including humans, great apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, New World monkeys; rodents (e.g., mice, rats, guinea pigs, pigs, hamsters); cats; dogs; lagomorphs (including rabbits); cattle; sheep; goats; horses; pigs; and any other livestock, agricultural, laboratory, or domesticated mammals. The methods, compositions, and culture media described herein can be applied to human embryos. Thus, any culture medium embodiment defined herein can support in vitro development of human embryos from pre-implantation to post-implantation developmental stages on a substrate.

[0080] As used herein, the term "pre-implantation stage" can be used to refer to developmental stages earlier than the stage corresponding to Carnegie stage 5(a) and corresponding stages in other species. As used herein, the term "post-implantation stage" can refer to developmental stages later than the stage corresponding to Carnegie stage 5(a) and corresponding stages in other species. The "post-implantation stage" can be determined by detecting upregulation of one or more genes in the embryo. For example, this stage can be determined by detecting one or more of the following changes: upregulation of Fgf5 in the epiblast; differentiation of the primitive endoderm into visceral endoderm that upregulates Cer1 in a subset of cells (the anterior visceral endoderm); upregulation of Eomes in the visceral endoderm; upregulation of Hand1 in the trophectoderm.

[0081] Stem cells (e.g., mammalian pluripotent stem cells such as embryonic stem cells) can be cultured using the culture media, kits, and methods described herein. In the embodiments described herein, stem cells include pluripotent stem cells (PSCs). PSCs can be obtained from fertilized eggs, cloned embryos, germline stem cells, or tissue stem cells. Also included are cells having differentiation pluripotency similar to embryonic stem cells, which are artificially obtained by transferring several different genes into somatic cells (also known as induced pluripotent stem cells or iPS cells). Induced pluripotent stem cells can be derived from any suitable source (e.g., hair follicles, skin cells, fibroblasts, etc.). Pluripotent stem cells can be prepared by methods known in the art. Any stem cells defined herein can be derived from diseased or non-diseased tissue. Stem cells can be from any suitable mammalian species, such as: primates, including humans, anthropoid apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, New World monkeys; rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; Lagomorpha (including rabbits); cattle; sheep; goats; horses; pigs; and any other livestock, agricultural, laboratory, or domesticated mammals. The methods disclosed in the present invention can be applied to stem cells of any non-human mammal (including but not limited to the mammals described above). In some embodiments, the non-human mammal is a rodent. In some embodiments, the PSC cells disclosed herein are mammalian embryonic stem cells (ESCs).

[0082] The pluripotent stem cells (e.g., ESCs) used in this article can be in different pluripotent states. Pluripotent embryonic stem cells can be naive ESCs, primed ESCs, or have an intermediate pluripotent state (e.g., formative ESCs) between naive and primed pluripotent stem cells. Pluripotent stem cells can be pre-implantation naive stem cells, peri-implantation pluripotent stem cells, or post-implantation primed stem cells. Naive ESCs contain cells that exhibit naive characteristics, such as overall low DNA methylation, expression of naive pluripotency markers (e.g., Klf4, Tfcp2l1, Esrrb, Klf2, Tbx3, Prdm14, and Dppa3), active mitochondria, reduced glucose dependence, and the ability to proliferate by single-cell enzymatic digestion, as understood by those skilled in the art. Markers highly expressed in naive ESCs can be silenced in formative ESCs and primed ESCs. Formative ESCs can have upregulated Otx2, Dnmt3b, Fgf5, Zic2 / 5, Etv1 / 4, Oct6, and Grhl2, which are reported to play important roles in the transition from the naive state to the primed state. For example, when cells exit naive pluripotency, increased Otx2 binds to many enhancer regions of formative genes (which can or cannot be pre-bound by Oct4) and activates related genes. Thus, the Oct4–Otx2 regulatory axis actively establishes a new regulatory chromatin landscape to exit naive pluripotency and transition to the formative state. Primed pluripotent stem cells have upregulated Sox11, Zic23, Dusp6, and Cd24 and downregulated or silenced naive markers (e.g., silenced Klf4). For example, more information related to the pluripotent states of mammalian cells (e.g., mouse and human cells) can be found in Genes (Basel). 2022 Aug; 13(8):1459, doi:10.3390 / genes13081459, the content of which is incorporated herein by reference in its entirety.

[0083] In some embodiments, the pluripotent stem cells used herein to generate synthetic embryos are naive cells (e.g., naive ESCs or naive hESCs). In some embodiments, the pluripotent stem cells used herein are intermediate stem cells with formative pluripotency at the pre-implantation or early post-implantation stage. In some embodiments, compared to naive cells (e.g., PXGL cells), the pluripotent stem cells used herein can be easily differentiated into peri-implantation and post-implantation yolk sac-like endoderm cells. In some embodiments, the ESCs used herein can be pre-implantation naive hESCs, peri-implantation-like pluripotent hESCs, or post-implantation primed hESCs. In some embodiments, the ESCs are peri-implantation pluripotent hESCs. Peri-implantation pluripotent hESCs can exhibit a naive-like state, such as tightly packed, dome-shaped colonies with refractive edges, and show increased expression of gene markers such as Klf2, Klf4, and Tfcp2L1. Compared to other ESCs in a pluripotent state (e.g., primed cells), the ESCs used herein can express low levels of amnion-specific genes during the trophoblast-like cell induction process.

[0084] Pluripotent stem cells (e.g., ESCs) in different pluripotent states can be pre-cultured using suitable media / conditions that can be determined by those skilled in the art. For example, pre-implantation naive hESCs can be generated by culturing in PXGL medium before co-culture. Peri-implantation-like pluripotent hESCs can be generated by culturing in RSeT medium before co-culture. Post-implantation-like primed hESCs can be generated by culturing in mTeSR1 medium before co-culture.

[0085] The conversion between different pluripotent states can be achieved in vitro. In some embodiments, those skilled in the art should understand that under suitable culture conditions, primed pluripotent stem cells (e.g., hESC) can be converted into formative cells or naive cells. For example, to convert primed pluripotent stem cells (e.g., primed hESC) into peri-implantation pluripotent stem cells (e.g., RSeT cells), the primed hESC can be passaged in a medium to mitomycin C-inactivated CF-1 MEF, and the medium contains DMEM / F12 containing KnockOut Serum Replacement, a reducing agent, non-essential amino acids, antibiotics, L-glutamine or its analogs, a member of the fibroblast growth factor family (FGF), and a ROCK inhibitor. To culture peri-implantation pluripotent stem cells (e.g., RSeT cells), the medium can be switched to a medium suitable for culturing naive-like pluripotent stem cells, such as RSeT medium. RSeT medium is a chemically defined cell culture medium used to reverse primed human pluripotent stem cells (hPSC) to a naive-like state and maintain naive-like hPSC under feeder-dependent and hypoxic conditions. In some embodiments, RSeT medium does not contain bFGF or TGFβ. RSeT medium is applicable to human embryonic stem cells and human induced pluripotent stem cells. More information about the culture conditions of naive cells (e.g., PXGL cells) can be found in the Examples section and "Bredenkamp, N. et al., Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency. Stem cell reports 13, 1083-1098 (2019). / / doi.org:10.1016 / j.stemcr.2019.10.009", the content of which is incorporated herein by reference in its entirety.

[0086] Those skilled in the art should understand that pluripotent stem cells (e.g., ESC) can be obtained from a stem cell bank (e.g., the UK Stem Cell Bank) where human stem cell lines are available for research, or from a fertility center. Preferably, embryonic stem cells are or can be obtained by methods that do not involve the destruction of human or non-human animal embryos.

[0087] Synthetic embryonic structure

[0088] The present disclosure provides methods, compositions, and culture media for mimicking mammalian embryonic development by culturing pluripotent stem cells (e.g., embryonic stem cells). The methods, compositions, and culture media disclosed herein can generate synthetic embryonic structures by co-culturing wild-type embryonic stem cells and extraembryonic-like cells, which are generated by overexpressing transcription factors in wild-type embryonic stem cells. Accordingly, the present disclosure also provides mammalian embryonic structures comprising embryonic and extraembryonic tissues. In some embodiments, the mammalian embryonic structures generated using the methods, compositions, and culture media described herein are post-implantation embryonic model structures, such as human embryoids. In some embodiments, the synthetic embryonic structures represent self-organizing aggregates that comprise a pluripotent epiblast-like domain surrounded by extraembryonic-like tissue, wherein the epiblast-like domain can differentiate into amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP signaling.

[0089] In some embodiments, the wild-type pluripotent stem cells and modified pluripotent stem cells used herein can form cell aggregates within 24 hours of culturing in a culture medium (e.g., stem cell proliferation medium). Approximately four days after aggregation, the cell aggregates can self-organize into structures that comprise a SOX2-positive epiblast-like domain containing a central cavity, an outer monolayer of GATA3-positive putative trophoblast-like cells, and an intermediate putative hypoblast-like domain of GATA6-positive cells between the inner cavity domain and the outer layer. The synthetic embryonic structures can comprise aggregates that comprise an organized SOX2-positive domain surrounded by concentric layers of GATA6-positive and GATA3-positive cells. In some embodiments, the aggregates do not undergo a blastocyst-like morphological transition prior to forming post-implantation-like structures.

[0090] In some embodiments, the synthetic embryonic structures generated using the methods and compositions described herein can reach the post-implantation stage (e.g., the post-implantation gastrulation stage). In some embodiments, the synthetic embryonic structures generated herein can reach the early gastrulation stage. In some embodiments, the synthetic embryos generated herein can reach the late gastrulation stage. As used herein, in the context of an embryo, the term "gastrulation" refers to an embryo after the expanded blastocyst stage and before the somitogenesis stage, which is characterized by the formation of the primitive streak and epithelial-mesenchymal transition to form three germ layers. The gastrulation process is generally considered to be the process by which a bilaminar embryonic disc transforms into a trilaminar embryonic disc as intraembryonic mesoderm appears between the ectoderm and endoderm. The gastrulation stage can be an early gastrulation stage, a mid-gastrulation stage, or a terminal or late gastrulation stage.

[0091] In some embodiments, the embryonic structures generated using the methods and culture media described herein include post-implantation embryos, e.g., post-implantation pre-gastrulation embryonic structures. In the context of mammalian embryos (e.g., human embryos), the term "post-implantation pre-gastrulation" as used herein refers to an embryo after the blastocyst implantation stage and before the early gastrulation stage, which is characterized by an ovoid cylindrical shape prior to symmetry breaking.

[0092] The embryonic stage of synthetic embryonic structures generated using the methods and culture media disclosed herein can be evaluated by comparing them in a variety of ways to their in vivo natural embryonic counterparts at the same developmental stage, including but not limited to morphology, length, weight, cell type composition, chromatin accessibility patterns, measuring the expression of developmental marker genes using specific antibodies or primers (e.g., Oct4, Nanog, Sox2, Klf4, Cdx2, Gata4, Gata6, Brachyury, Otx2, Fgf5, and other genes described in the Examples and known in the art), or transcriptomic analysis, single-cell RNA sequencing, and other methods further described in the Examples section.

[0093] In some embodiments, the post-implantation embryonic structures generated herein contain an internal epiblast-like domain, an outer single layer of trophoblast-like cells, and an intermediate hypoblast-like domain between the epiblast-like domain and the outer single layer of trophoblast-like cells. The internal epiblast-like domain can be SOX2 positive and contain a central cavity, the outer single layer of trophoblast-like cells can be GATA3 positive, and the intermediate hypoblast-like domain can be GATA6 positive. In some embodiments, the post-implantation embryonic structure expresses N-cadherin and SOX17 in the hypoblast-like domain, CDX2 in the trophoblast-like cells, and / or SOX2, NANOG, and E-cadherin in the epiblast-like domain. The internal epiblast-like domain can exhibit pluripotency and epithelial identity similar to that of a human embryo.

[0094] In some embodiments, the post-implantation embryonic structures generated herein contain cell populations similar to late embryonic epiblast, amnion, mesoderm, extraembryonic mesenchyme, and / or hypoblast / visceral endoderm. In some embodiments, the post-implantation embryonic structure expresses TDGF1, SOX2, NANOG, TFAP2A, ID1, ISL1, TFAP2C, VTCN1, GRHL1, MEIS1, TBXT, MESP1, MIXL1, CER1, SNAI1, EOMES, POSTN, COL6A3, IGF2, TBX20, BMP6, CDH2, HNF1B, FOXA2, VTCN1, HAND1, TBX20, CDX2, PRDM1, OCT4, or a combination thereof.

[0095] In some embodiments, post-implantation embryonic structures can give rise to amnion and primordial germ cells. Post-implantation embryonic structures can generate primordial germ cell-like cells that express the pluripotency marker NANOG as well as the primordial germ cell markers PRDM1 (BLMP1) and NANOS3.

[0096] BMP signaling can play a role in the differentiation of the epiblast-like domain. In some embodiments, the human embryonic structures generated herein can express one or more of the downstream BMP-responsive genes ID1, ID2, ID3, or ID4 (see, e.g., Example 4). In some embodiments, phosphorylation of (p)SMAD1.5 in the OCT4-positive epiblast-like domain at day 4 and day 6 after aggregation indicates active BMP signaling.

[0097] By introducing one or more inducible genes into pluripotent stem cells, one or more of the gene markers described herein can be upregulated or downregulated in the generated synthetic embryonic structures. For example, compared to overexpressing GATA6 alone, induction of SOX17 alone or in combination with GATA6 can result in a decreased ability to upregulate CER1 (see, e.g., Example 5). Alternatively or additionally, compared to structures with continuous induction of GATA6-SOX17 or SOX17, synthetic embryonic structures generated by induction of GATA6 alone or with reduced induction intensity and / or induction duration (e.g., by withdrawing doxycycline after a certain period, such as after day 3) show increased expression of the primitive streak marker BRY / TBXT at day 6 after aggregation. Thus, in some embodiments, the in vitro synthetic embryonic structures generated herein can be used as modular embryoid models to study gene marker regulation and the interaction between embryonic and extra-embryonic tissues, to explore the role of specific tissues and tissue-specific gene requirements, and to study the mechanisms involved in embryogenesis.

[0098] In some embodiments, the synthetic embryonic structures generated using the methods and culture conditions described herein are mammalian embryonic structures. In some embodiments, the mammalian embryonic structures are human embryonic structures, such as human embryoids. The human embryo-like structures generated herein can exhibit an organizational structure similar to that of a human embryo at approximately 8-9 days post-fertilization.

[0099] In vitro culturing of embryonic cells from stem cells (e.g., pluripotent ESCs) can be carried out until the post-implantation stage or any developmental stage in between. In some embodiments, the synthetic embryos generated using the methods and media described herein cannot mimic the stages after primitive streak formation. In some embodiments, the synthetic embryos generated using the methods and media described herein may not contain all cell types of a gastrulating embryo. In some embodiments, the synthetic embryos generated using the methods and media described herein may not be able to further develop into viable human embryos.

[0100] The culture conditions for generating synthetic embryos, including substrates, culture media, etc., are described in the following sections and in the specific embodiments of human embryos in the Examples section.

[0101] The embryonic stage of the synthetic embryos described herein can be evaluated by comparing them in a variety of ways to in vivo or natural embryo counterparts at the same developmental stage, including but not limited to morphology, length, weight, using specific antibodies or primers to assay the expression of developmental marker genes, transcriptional analysis, etc., as further described below and in the Examples section.

[0102] Morphological assessment of embryonic development can be carried out based on previously determined morphological characteristics or according to the number of embryonic days, such as the morphological characteristics described in the Carnegie stages of development (see, for example, Table 1; Stages of Human Embryonic Development. R. O'Rahilly and F. Müller (eds.), Carnegie Institution of Washington, Washington, D.C., 1987).

[0103] In some embodiments, one or more of the developmental markers described herein can be used to evaluate the developmental stage of synthetic embryo structures. There are a variety of methods in the art for detecting the presence or amount of marker gene products (e.g., mRNA and / or protein), as well as their localization or subcellular localization (e.g., nucleus and / or cytoplasm) in embryo structures. Marker expression can be evaluated by any of a variety of well-known methods for detecting transcriptional molecules or protein expression. Non-limiting examples of such methods include immunological methods, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification and sequencing methods for detecting secreted proteins, cell surface proteins, cytoplasmic proteins, or nuclear proteins.

[0104] In some embodiments, the activity of a specific gene is characterized by measuring the gene transcript (e.g., mRNA), measuring the amount of translated protein, or measuring the activity of the gene product. Marker expression can be monitored in a variety of ways, including detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity); alternatively, it can be a qualitative assessment of the level of gene expression, particularly in comparison to a control level. The type of level detected will be clear from the context.

[0105] In another embodiment, detecting or determining the expression level of a marker and its functionally similar homologs (including fragments or genetic alterations thereof, e.g., in its regulatory or promoter regions) includes detecting or determining the RNA level of the marker of interest. In some embodiments, one or more cells can be obtained from a synthetic embryo structure and RNA can be isolated from these cells. In some embodiments, RNA is obtained from a single cell. For example, cells can be isolated from a tissue sample by laser capture microdissection (LCM). Using this technique, cells can be isolated from tissue sections (including stained tissue sections), thus ensuring the isolation of the desired cells. Cells can also be obtained from, for example, synthetic embryo cells and cultured in vitro, e.g., to obtain a larger population of cells from which RNA can be extracted. Methods for establishing non-transformed cell cultures (i.e., primary cell cultures) are known in the art. In some embodiments, the cells can be dissociated (e.g., by enzymatic or mechanical means) and separated by methods known in the art (e.g., fluorescence-activated cell sorting, microfluidics, etc.).

[0106] When isolating RNA from, for example, synthetic embryo structures at different developmental stages and / or cells containing the synthetic embryo structures, it may be crucial to prevent any further changes in gene expression after removing tissue or cells from the subject. It is known that expression level changes can rapidly alter after perturbation (e.g., heat shock or activation with lipopolysaccharide (LPS) or other reagents). In addition, RNA in tissues and cells may rapidly degrade. Thus, in preferred embodiments, the tissue or cells obtained from the subject should be snap-frozen as soon as possible.

[0107] RNA can be extracted from cells by a variety of methods, e.g., guanidinium thiocyanate lysis followed by CsCl centrifugation. Methods for obtaining RNA from single cells are also known in the art. Subsequently, the RNA sample can be subjected to enrichment for specific substances. In some embodiments, poly(A)+RNA is isolated from the RNA sample. Typically, this purification takes advantage of the poly-A tail on mRNA. Specifically, as described above, oligo(dT) nucleotides can be immobilized on a solid support as an affinity ligand for mRNA. Kits for this purpose are commercially available, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY). In some embodiments, the RNA population is subjected to enrichment for marker sequences. Enrichment can be performed, for example, by primer-specific cDNA synthesis or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription.

[0108] RNA populations, whether or not enriched for a particular substance or sequence, can be further amplified. The "amplification process" as defined herein increases the copy number of polynucleotides (e.g., RNA). For example, when the RNA is mRNA, amplification methods such as RT-PCR can be used to amplify the mRNA so that the signal can be detected or the detection is enhanced. Such amplification methods are particularly beneficial when the size or volume of a biological, tissue or tumor sample is small.

[0109] A variety of amplification and detection methods can be used. For example, within the scope of the disclosed methods, mRNA can be reverse transcribed into cDNA and then polymerase chain reaction (RT-PCR) can be performed; alternatively, as described in U.S. Patent No. 5,322,770, a single enzyme can be used for the above two steps; or, mRNA can be reverse transcribed into cDNA and subsequently symmetric gap ligase chain reaction (RT-AGLCR) can be performed, as described by R.L. Marshall et al. in PCR Methods and Applications 4:80-84 (1994). Real-time PCR can also be used. Other known amplification methods that can be used herein include, but are not limited to: the so-called "NASBA" or "3SR" techniques, which are described in PNAS USA 87:1874-1878 (1990) and also in Nature 350 (No. 6313):91-92 (1991); Q-beta amplification, as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (such as described by G.T. Walker et al., Clin. Chem. 42:9-13 (1996) and European Patent Application No. 684315); target-mediated amplification, as described in PCT Publication WO9322461; PCR; ligase chain reaction (LCR) (e.g., see Wu and Wallace, Genomics 4,560 (1989), Landegren et al., Science 241,1077 (1988)); self-sustained sequence replication (SSR) (e.g., see Guatelli et al., Proc. Nat. Acad. Sci. USA, 87,1874 (1990)); and transcription amplification (e.g., see Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989)). At the current state of the art, there are many techniques for determining the absolute and relative levels of gene expression, and common techniques applicable to the methods disclosed herein include Northern blot analysis, RNase protection assay (RPA), microarray, and PCR-based techniques, such as quantitative PCR and differential display PCR. For example, Northern blotting involves electrophoresing an RNA preparation on a denaturing agarose gel and transferring it to a suitable support (e.g., activated cellulose membrane, nitrocellulose membrane, glass membrane, or nylon membrane). Subsequently, radiolabeled cDNA or RNA is hybridized to the preparation, washed, and analyzed by autoradiography.

[0110] In situ hybridization visualization techniques can also be employed, in which radiolabeled antisense RNA probes are hybridized to thin sample sections, washed, lysed with ribonuclease, and exposed to a photographic emulsion for autoradiography. The sample can be stained with hematoxylin to show the histological composition of the sample, and then dark field imaging can be performed with a suitable filter to show the developed emulsion. Non-radiolabeled markers, such as digoxigenin, can also be used. In some embodiments, the probe is labeled with a fluorescent moiety.

[0111] Alternatively, mRNA expression can be detected on a DNA array, chip, or microarray. Labeled nucleic acids from a test sample obtained from a subject can be hybridized to a solid surface containing marker DNA. A positive hybridization signal is obtained with a sample containing the marker transcript. Methods for preparing DNA arrays and their uses are well known in the art (see, for example, U.S. Patent Nos. 66,186,796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485). Serial analysis of gene expression (SAGE) can also be performed (see, for example, U.S. Patent Application No. 20030215858). In some embodiments, next-generation sequencing (e.g., RNA-seq) can be used to analyze total mRNA expression from one or more cells (e.g., single-cell RNA-seq). Nucleic acid target molecules labeled with barcodes (e.g., source-specific barcodes) can be sequenced with the barcode, thereby generating single reads and / or contigs containing the sequence or portions thereof of the target molecule and the barcode. Exemplary next-generation sequencing techniques include, for example, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing, among others. Methods for constructing sequencing libraries are known in the art.

[0112] In some aspects of the present disclosure, single-cell sequencing is high-throughput single-cell RNA sequencing. In certain embodiments, single-cell sequencing is low-cost high-throughput single-cell RNA sequencing. Without being bound by any particular theory, single-cell RNA sequencing can efficiently and cost-effectively sequence thousands to tens of thousands of single cells. In certain embodiments, single-cell RNA sequencing includes: pairing single cells in droplets with oligonucleotides for reverse transcription, wherein the oligonucleotides are configured to provide cell-source specific barcodes that uniquely identify transcripts from each cell and unique molecular identifiers (UMIs) that uniquely identify each transcript. In certain embodiments, single-cell RNA sequencing includes pairing single cells in droplets with single microparticle beads coated with oligonucleotides for reverse transcription, wherein the oligonucleotides contain bead-specific barcodes that uniquely identify each bead and unique molecular identifiers (UMIs) that uniquely identify each primer. In some aspects of the present disclosure, unbiased classification of cells in a biological sample includes sequencing the transcriptomes of thousands of cells, preferably tens of thousands of cells (e.g., greater than 1000 cells, or greater than 10,000 cells).

[0113] The activity or level of a lineage marker protein can be detected and / or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a variety of methods well known to those skilled in the art. Any method known in the art for detecting polypeptides can be used. These methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination reactions, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, and the like.

[0114] Non-limiting examples of techniques are described below that can be used to detect a marker protein based on the present disclosure according to the preference of the practitioner. One such technique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)), in which a suitably processed sample is run on an SDS-PAGE gel and subsequently transferred to a solid support (e.g., a nitrocellulose filter). Then, an anti-marker protein antibody (unlabeled) is contacted with the support and assayed using a secondary immunological reagent (e.g., labeled protein A or anti-immunoglobulin, suitable labels include 125 I, horseradish peroxidase, alkaline phosphatase, fluorophore). Chromatographic detection can also be used.

[0115] Immunohistochemistry can be used to detect the expression of a marker protein. A suitable antibody is contacted with, for example, a thin layer of cells, washed, and then contacted with a labeled secondary antibody. The labeling can be carried out by a fluorescent marker, an enzyme (such as peroxidase), avidin, or a radioactive label. The assay is visually scored using a microscope.

[0116] Antibodies against the marker protein (such as intracellular antibodies) can also be used for imaging purposes, such as detecting the presence of the marker protein in cells or embryos. Suitable labels include radioisotopes, iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 mTc), fluorescent labels (such as fluorescein and rhodamine), and biotin.

[0117] Antibodies that can be used to detect the marker protein include any antibody, whether natural or synthetic, full-length antibody or its fragment, monoclonal antibody or polyclonal antibody, as long as it can bind the marker protein to be detected sufficiently strongly and specifically. The Kd of the antibody can be at most about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M. The phrase "specifically binds" means that, for example, an antibody binds to an epitope, antigen, or antigenic determinant in such a way that the binding can be displaced or competed for by a second preparation having the same or a similar epitope, antigen, or antigenic determinant. The antibody can preferentially bind the marker protein relative to other proteins (such as related proteins).

[0118] Antibodies are commercially available or can be prepared according to methods known in the art. Antibodies and their derivatives that can be used include polyclonal or monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, primatized antibodies (CDR-grafted antibodies), veneered antibodies or single-chain antibodies, as well as functional fragments of antibodies, i.e., marker protein-binding fragments. For example, antibody fragments that can bind to a marker protein or a portion thereof can be used, including but not limited to Fv, Fab, Fab′, and F(ab′)2 fragments. Such fragments can be produced by enzymatic cleavage or recombinant techniques. For example, papain or pepsin cleavage can produce Fab or F(ab′)2 fragments, respectively. Other proteases with the desired substrate specificity can also be used to produce Fab or F(ab′)2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons are introduced upstream of the natural termination site. For example, a chimeric gene encoding a portion of the F(ab′)2 heavy chain can be designed to contain a DNA sequence encoding the CH domain and hinge region of the heavy chain. In some embodiments, drugs other than antibodies that specifically bind to the marker protein are used, such as peptides. Peptides that specifically bind to the marker protein can be identified by any method known in the art. For example, a phage display peptide library can be used to screen for specific peptide binders to the marker protein.

[0119] Substrate for growing synthetic embryos

[0120] In some embodiments, the wild-type and modified pluripotent stem cells (e.g., ESCs), cell aggregates, post-implantation embryos, and / or synthetic embryos described herein are cultured in a substrate. In some embodiments, the method includes transferring ESCs and / or embryos from one substrate to another substrate. The substrates used in the methods disclosed herein can be the same or different. For example, mammalian ESCs (e.g., wild-type ESCs and modified ESCs) can be cultured in a first substrate to form an aggregated structure. The aggregated cells can be transferred to a second substrate to develop into a post-implantation embryo. The first substrate and the second substrate can be of the same type or different types. In some embodiments, the first substrate and the second substrate are of different types. In some embodiments, the first substrate and the second substrate can be a microwell plate including inverted pyramid-shaped microwells, such as AggreWell TM Microwell plate.

[0121] The substrates used herein may include Petri dishes, U-shaped plates, flasks, or microplates. The microplates may include inverted pyramid-shaped microwells. The size (e.g., depth and / or diameter) of each inverted pyramid-shaped microwell may vary. The size of each inverted pyramid-shaped microwell may be about 400 μm or about 800 μm. The diameter of each inverted pyramid-shaped microwell may be about 400 μm or about 800 μm. In some embodiments, the size and / or diameter of each inverted pyramid-shaped microwell may be about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mM, or a value or range between any two of these values. The depth of each microwell (e.g., container) may be about 250 μm to about 400 μm, such as about 300 μm to about 350 μm. Additionally or alternatively, the average depth of the plurality of containers may be about 250 μm to about 400 μm, such as about 300 μm to about 350 μm. Especially when the containers are wells, they may be arranged in an array on the substrate, i.e., in a grid pattern with regular spacing in substantially orthogonal directions. Regardless of the topography of the substrate, the substrate can carry one or more embryos. When the substrate includes one or more containers, each such container can independently accommodate one or more embryos, such as 2, 3, 4, 5, 6, 7, or 8 embryos or more. In some embodiments, each embryo structure is located in a different respective well. In alternative embodiments, each container contains a plurality of embryos, such as 2, 3, 4, 5, 6, 7, or 8 embryos or more.

[0122] The methods disclosed herein can be applied to any suitable culture volume. For example, the culture volume for each embryo can be about 50 μl to about 10 ml, optionally about 100 μl to about 5 ml, optionally about 250 μl to about 5 ml, optionally about 1 ml to about 5 ml. The culture volume for each embryo can be about 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl, 700 μl, 750 μl, 800 μl, 850 μl, 900 μl, 950 μl, 1000 μl, 1500 μl, 2000 μl, or more.

[0123] Culture medium

[0124] In some embodiments, the method includes co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene in a medium suitable for pluripotent stem cell proliferation (e.g., N2B27 medium) under conditions that cause the ESCs to form an aggregated structure. The method may further include culturing the aggregated structure in a post-implantation medium under conditions that cause the aggregated structure to self-organize into a synthetic embryonic structure that mimics a post-implantation embryo structure.

[0125] In some embodiments, the method includes co-culturing mammalian pluripotent stem cells (e.g., ESCs) in a stem cell proliferation medium and optionally passaging the ESCs in the stem cell proliferation medium at least twice (e.g., 2, 3, 4 times or more). The mammalian pluripotent stem cells may be cultured in the stem cell proliferation medium for 1 day, 2 days, 3 days, 4 days, or 5 days. In some embodiments, the pluripotent stem cells aggregate after co-culturing in the stem cell proliferation medium for about 3 days.

[0126] The method may further include co-culturing the pluripotent stem cells in a post-implantation medium for at least 2 days (e.g., 2 days, 3 days, 4 days, 5 days, 6 days or more) after co-culturing in the stem cell proliferation medium. In some embodiments, after culturing in the stem cell proliferation medium for about 5 days, the pluripotent stem cells are cultured in the post-implantation medium for at least 2 days.

[0127] In some embodiments, the method includes partially replacing a quantity of the stem cell proliferation medium (e.g., at least half of the medium) with fresh stem cell proliferation medium or post-implantation medium. The replacement may be performed every 20 to 28 hours (e.g., every 24 hours). In some embodiments, the method includes partially replacing a quantity of the post-implantation medium (e.g., at least half of the medium) with fresh post-implantation medium.

[0128] The culture media disclosed in this article, including a stem cell proliferation medium and a post-implantation medium, may contain a basal medium. The basal medium may contain water, salts, amino acids, carbon sources, vitamins, lipids, and buffers. Those skilled in the art can obtain suitable carbon sources from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. The basal medium is commercially available. For example, it can be purchased under the trade names Advanced DMEM / F12 (Gibco, 12634-010) and CMRL-1066 (Invitrogen or Sigma). The basal medium may include Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F-12 (DMEM / F12), Roswell Park Memorial Institute (RPMI) Medium 1640, A, Connaught Medical Research Laboratories 1066 (CMRL-1066), or any combination thereof.

[0129] The basal medium may contain Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F-12 (DMEM / F12), non-human serum or its serum substitute, antibiotics, L-glutamine or its analogs (e.g., GlutaMAX TM ), or any combination thereof.

[0130] The non-human serum or serum substitute may include fetal bovine serum, bovine serum albumin, rat serum, KnockOut TM serum substitute, or any combination thereof. The antibiotics may include penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof.

[0131] The concentration or content of one or more components in the solution or culture medium may vary. For example, non-human serum or its serum substitute, antibiotics, reducing agents, and / or L-glutamine (e.g., GlutaMax TM) content can vary, and in some embodiments, those skilled in the art can adjust it as needed. In some embodiments, the amount of non-human serum or its serum substitute can be about 0.01% to about 40% of the culture medium by volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) (e.g., about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or a value or range between any two of these values). In some embodiments, the amount of antibiotic can be about 0.01% to about 10% of the culture medium by volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) (e.g., about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a value or range between any two of these values). For example, the amount of reducing agent can vary. For example, in some embodiments, the concentration of the reducing agent in the composition can be about 0.1 μM to about 1 mM (e.g., about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 10 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, or a value or range between any two of these values). The amount of L-glutamine (e.g., GlutaMAXTM) can vary. For example, in some embodiments, the concentration of L-glutamine in the culture medium can be about 0.1 mM to about 40 mM, about 0.2 mM to about 20 mM, about 0.5 mM to about 10 mM, about 1 mM to about 5 mM, or about 1.5 mM to about 2.5 mM, e.g., about 2 mM. Unless otherwise specified, when percentages of agents, components, and compounds are provided, they can be % w / w, % w / v, or % v / v relative to the entire formulation.

[0132] Each component in the culture media described herein can be present in an amount such that the culture medium is suitable for supporting the self-organization of stem cells (e.g., ESCs) into post-implantation embryonic structures and / or the further development of such post-implantation embryonic structures.

[0133] In the embodiments described herein, the culture media and compositions used herein do not contain or are not supplemented with exogenous signal transduction pathway factors. In some embodiments, the culture media and compositions used herein do not contain a WNT signal transduction pathway activator (e.g., a WNT agonist or a WNT signal agonist). Exemplary WNT signal transduction pathway agonists include, but are not limited to, CHIR99021, derivatives of CHIR99021 (e.g., salts of CHIR99021, such as trihydrochloride, hydrochloride of CHIR99021), recombinant Wnt3a protein, glycogen synthase kinase 3 (GSK3) inhibitors (e.g., 3F8, A1070722, AR-A014418, BIO, BIO-acetoxime, FRATide, 10Z-Hymenialdisine, indirubin-3'-oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC693868, SB216763, SB415286, TC-G24, TCS2002, TCS21311, TWS119), and analogs or derivatives of any of the foregoing.

[0134] In some embodiments, the culture media or compositions used herein do not contain TGFβ superfamily members. The "TGFβ superfamily" refers to proteins having the structural and functional characteristics of known members of the TGFβ family. The TGFβ protein family has been well characterized both structurally and functionally. It includes TGFβ family of proteins, inhibins (including inhibin A and inhibin B), activins (including activin A, activin B, and activin AB), Müllerian inhibiting substance (MIS), bone morphogenetic proteins (BMPs), dpp (decapententaplegic), Vg-1, MNSF (monoclonal non-specific suppressor factor), etc. The activity of this protein family is based on specific binding to certain receptors on various cell types. The family members share regions of sequence identity related to their function, especially at the C-terminus. The TGFβ family includes more than a hundred different proteins, all of which share at least one region of amino acid sequence identity. TGF-β superfamily members (e.g., BMP4) can be natural or recombinant. Exemplary TGFβ superfamily members include, but are not limited to, growth differentiation factor 8 (GDF8) (GenBank accession number EAX10880), growth differentiation factor 11 (GDF11) (GenBank accession number AAF21630), activin A, Nodal, activin A, activin B, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4), and any functional fragments thereof.

[0135] The mammalian pluripotent stem cells described herein (e.g., wild-type ESCs and modified ESCs overexpressing transcription factors) can be cultured alone in a suitable medium suitable for the proliferation of stem cells and pluripotent stem cells prior to the co-culture described herein, as will be understood by those skilled in the art. For example, ESCs can be cultured in a serum-free, substantially serum-free or essentially serum-free medium. The medium can comprise a serum replacement medium. Such serum replacement media are commercially available under the trade names KSR (KnockOut TM Serum Replacement, Invitrogen, 10828-010) and N2B27 (e.g., Invitrogen, ME100137L1). The serum replacement medium can be included in the medium at about 5% to about 60%, about 10% to about 50%, about 15% to about 45% or about 20% to about 40%. Exemplary media include, but are not limited to, RSeT medium, PXGL medium, cRM-1 medium, mTeSR1 medium, and other media recognizable to those skilled in the art.

[0136] In some embodiments, the culture medium can be supplemented with an inhibitor of Rho-associated protein kinase (ROCK) (also referred to herein as a ROCK inhibitor). Exemplary ROCK inhibitors include, but are not limited to, N-[(1S)-2-hydroxy-1-phenylethyl]-N′-[4-(4-pyridyl)phenyl]-urea (AS1892802), fasudil hydrochloride (also known as HA1077), -[3-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide (GSK269962), 4-[4-(trifluoromethyl)phenyl]-N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide (GSK429286), (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H 1152 dihydrochloride), (S)-(+)-4-glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (glycyl-H 1152 dihydrochloride), N-[(3-hydroxyphenyl)methyl]-N′-[4-(4-pyridyl)-2-thiazolyl]urea dihydrochloride (RKI1447 dihydrochloride), (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxine-2-carboxamide dihydrochloride (SR3677 dihydrochloride), and trans-4-[(R)-1-aminoethyl]-N-4-pyridylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (Thiazovivin), Rock inhibitor (an isoquinoline sulfonamide compound, Rho kinase inhibitor), N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea (Rho kinase inhibitor II), 3-(4-pyridyl)-1H-indole (Rho kinase inhibitor III, Rockout), and 4-pyrazoleboronic acid pinacol ester;Rock antibodies commercially available from Santa Cruz Biotechnology, selected from Rock-1 (B1), Rock-1 (C-19), Rock-1 (H-11), Rock-1 (G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2 (D-11), Rock-2 (N-19), Rock-2 (H-85), Rock-2 (30-J); ROCK CRISPR / Cas9 knockout plasmids, selected from Rock-1 CRISPR / Cas9 KO plasmid (h), Rock-2 CRISPR / Cas9 KO plasmid (h), Rock-1 CRISPR / Cas9 KO plasmid (m), Rock-2 CRISPR / Cas9 KO plasmid (m); ROCK siRNA, shRNA plasmids and / or shRNA lentiviral particle gene silencers, selected from Rock-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA (r): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433, Rock-2 siRNA (r): sc-108088. In some embodiments, the ROCK inhibitor includes Y-27632. The ROCK inhibitor can be provided in an effective amount at a concentration of about 0.1 μM to about 100 μM. In some embodiments, the culture medium contains a ROCK inhibitor at a concentration of about 10 μM. In some embodiments, the culture medium does not contain a ROCK inhibitor.;

[0137] The culture media described herein may contain other components, or analogs thereof. The term "analog" as used herein may refer to a bioactive analog of any component in the culture medium. Such analogs may be natural or synthetic.

[0138] The specific bioactive ligands and compounds used in the culture media defined herein, such as insulin, progesterone, etc., are for illustrative purposes only. However, those skilled in the art will readily recognize that analogs of such ligands and compounds can equally be used as substitutes as long as the relevant bioactivity is retained. Those skilled in the art can identify other bioactive compounds suitable for use as substitutes in a conventional manner. For example, these can be naturally occurring compounds or compounds prepared by synthetic or semi-synthetic methods.

[0139] Stem Cell Proliferation Medium

[0140] In some embodiments, the pluripotent stem cell proliferation medium used herein is serum-free or substantially serum-free. Alternatively, the stem cell proliferation medium can be supplemented with KSR, optionally about 5%-15% KSR. In some embodiments, the stem cell proliferation medium is a chemically defined in vitro medium that is serum-free or substantially serum-free and comprises a basal medium containing water, salts, amino acids, carbon sources, vitamins, lipids, and buffers. In some embodiments, the stem cell proliferation medium can further comprise sodium pyruvate. Sodium pyruvate can be included in the medium at a concentration of about 0.05 mM to about 10 mM, about 0.1 mM to about 2 mM, or about 0.2 mM to about 1 mM. In some embodiments, the stem cell proliferation medium comprises Neurobasal medium (e.g., Neurobasal or Neurobasal A from ThermoFisher Scientific). The stem cell proliferation medium can also comprise or be supplemented with B-27 supplement and N-2 supplement. Those skilled in the art will understand that the B-27 supplement is a chemically defined mixture of antioxidant enzymes, proteins, vitamins, and fatty acids combined in an optimized ratio to support the survival of neurons in culture. The N2 supplement is a chemically defined serum-free supplement that can be used for the growth and expression of post-mitotic neurons in neuroblastoma as well as in primary cultures from the peripheral and central nervous systems.

[0141] The stem cell proliferation medium can comprise an effective amount of L-glutamine or an analogue thereof. The concentration of L-glutamine in the medium can be about 0.1 mM to about 40 mM, about 0.2 mM to about 20 mM, about 0.5 mM to about 10 mM, about 1 mM to about 5 mM, or about 1.5 mM to about 2.5 mM, such as about 2 mM. In some embodiments, the concentration of L-glutamine included in the stem cell proliferation medium is about 2 mM.

[0142] The stem cell proliferation medium may further comprise or be supplemented with an effective amount of a reducing agent. The reducing agent may include β-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof. In some embodiments, the concentration of the reducing agent in the stem cell proliferation medium may be from about 0.1 μM to about 1 mM (e.g., about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 10 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, or a value or range between any two of these values. In some embodiments, the concentration of the reducing agent in the stem cell proliferation medium is about 0.1 mM. In some embodiments, the stem cell proliferation medium comprises β-mercaptoethanol (BME) at a concentration of about 0.1 mM.

[0143] In some embodiments, the stem cell proliferation medium comprises Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F-12 (DMEM / F12), A, N2, B27, L-glutamine or analogs thereof, a reducing agent, an antibiotic or a combination thereof, wherein the amounts of the respective components are such that the medium is capable of supporting the proliferation of pluripotent stem cells on a substrate. In some embodiments, the stem cell proliferation medium comprises DMEM / F12, A, B-27, N-2, GlutaMax TM , β-mercaptoethanol, penicillin / streptomycin or a combination thereof. In some embodiments, the stem cell proliferation medium is N2B27 medium. The N2B27 medium may comprise a 1:1 mixture of DMEM / F12 and Neurobasal A, 0.5× B-27, 0.5× N-2, 100 μM β-mercaptoethanol, 1× GlutaMAX, and 1× penicillin-streptomycin.

[0144] Postimplantation medium

[0145] The methods described herein further comprise: after co-culturing in the stem cell proliferation medium, co-culturing pluripotent stem cells (e.g., cell aggregates formed from ESCs) in a post-implantation medium. In some embodiments, the post-implantation medium is a post-implantation human embryo medium (e.g., hIVC1).

[0146] The post-implantation culture medium may contain non-human serum. The non-human serum in the post-implantation culture medium may vary. In some embodiments, the post-implantation culture medium may contain from about 5% to about 40% (e.g., 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or a value or range between any two of these values) of non-human serum (e.g., fetal bovine serum) by volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w). In some embodiments, the post-implantation culture medium contains from about 15% to about 30% of non-human serum (e.g., fetal bovine serum). In some embodiments, the post-implantation culture medium contains about 20% fetal bovine serum. In some embodiments, the fetal bovine serum is inactivated.

[0147] The post-implantation culture medium may further contain (a) insulin, an insulin analog, or an insulin receptor agonist; (b) estrogen, an estrogen analog, or an estrogen receptor agonist; and (c) progesterone, a progesterone analog, or a progesterone receptor agonist.

[0148] The amount of insulin, estrogen, progesterone, or an analog or receptor agonist thereof present in the post-implantation culture medium can vary. For example, in some embodiments, the post-implantation culture medium can contain from about 1 ng / ml to about 100 mg / ml (such as about 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml, 34 ng / ml, 35 ng / ml, 36 ng / ml, 37 ng / ml, 38 ng / ml, 39 ng / ml, 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml, 50 ng / ml, 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, 59 ng / ml, 60 ng / ml, 61 ng / ml, 62 ng / ml, 63 ng / ml, 64 ng / ml, 65 ng / ml, 66 ng / ml, 67 ng / ml, 68 ng / ml, 69 ng / ml, 70 ng / ml, 71 ng / ml, 72 ng / ml, 73 ng / ml, 74 ng / ml, 75 ng / ml, 76 ng / ml, 77 ng / ml, 78 ng / ml, 79 ng / ml, 80 ng / ml, 81 ng / ml, 82 ng / ml, 83 ng / ml, 84 ng / ml, 85 ng / ml, 86 ng / ml, 87 ng / ml, 88 ng / ml, 89 ng / ml, 90 ng / ml, 91 ng / ml, 92 ng / ml, 93 ng / ml, 94 ng / ml, 95 ng / ml, 96 ng / ml, 97 ng / ml, 98 ng / ml, 99 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml,One or more hormones (such as progesterone) and / or one or more growth factors (such as insulin or insulin-like growth factor) at 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 1 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml or a numerical value or range between any two of these values). In some embodiments, the post-implantation medium can contain from about 0.5 nM to about 1 mM (e.g., about 0.5 nM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 86 nM, 87 nM, 88 nM, 89 nM, 90 nM, 91 nM, 92 nM, 93 nM, 94 nM, 95 nM, 96 nM, 97 nM, 98 nM, 99 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 0.5 mM, 1 mM or a numerical value or range between any two of these values of hormones (such as estrogen) and / or insulin or insulin-like growth factor.

[0149] In some embodiments, the insulin receptor agonist is selected from IGF-I, IGF-II, analogs thereof, or any combination thereof. The estrogen receptor agonist can be selected from β-estradiol, estrone, estriol, and estetrol, or any analogs thereof. The post-implantation medium can contain transferrin, sodium selenite, ethanolamine, or any analogs thereof. The post-implantation medium can contain insulin-transferrin-selenium-ethanolamine (ITS-X). In some embodiments, the post-implantation medium further contains an agonist of the activin type 1 or type 2 receptor. In some embodiments, the post-implantation medium does not contain a reducing agent.

[0150] In some embodiments, the post-implantation medium can contain a basal medium as defined above (e.g., Advanced DMEM / F12), which is supplemented with an insulin receptor agonist such as insulin (e.g., from about 2 mg / ml to about 25 mg / ml), transferrin (e.g., from about 1 mg / ml to about 10 mg / ml), selenium such as sodium selenite (e.g., from about 0.001 mg / ml to about 0.01 mg / ml), ethanolamine (e.g., from about 0.5 mg / ml to about 10 mg / ml), an estrogen receptor agonist such as estradiol (e.g., from about 5 nM to about 10 nM), and a progesterone receptor agonist such as progesterone (e.g., from about 50 ng / ml to about 500 ng / ml).

[0151] The post-implantation medium can further contain an effective amount of non-essential amino acids selected from L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The post-implantation medium can further contain an effective amount of essential amino acids selected from L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine. The non-essential amino acids and / or essential amino acids can have an effective amount, e.g., by volume / volume (%v / v), weight / volume (%w / v), or weight / weight (%w / w), accounting for about 0.1% to about 2% of the medium (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a value or range between any two of these values. In some embodiments, the post-implantation medium contains about 1% of non-essential amino acids and / or essential amino acids.

[0152] The post-implantation medium can contain L-glutamine. The concentration of L-glutamine in the medium can be from about 0.1 mM to about 40 mM, from about 0.2 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 1 mM to about 5 mM, or from about 1.5 mM to about 2.5 mM, such as about 2 mM. In some embodiments, the concentration of L-glutamine in the medium is about 2 mM.

[0153] Penicillin can be included in the post-implantation medium at a concentration of from about 1 unit / ml to about 500 units / ml, from about 2 units / ml to about 250 units / ml, from about 5 units / ml to about 100 units / ml, from about 10 units / ml to about 50 units / ml, or from about 20 units / ml to about 30 units / ml (such as about 25 units / ml). Streptomycin can be included in the medium at a concentration of from about 1 μg / ml to about 500 μg / ml, from about 2 μg / ml to about 250 μg / ml, from about 5 μg / ml to about 100 μg / ml, from about 10 μg / ml to about 50 μg / ml, 25 μg / ml or from about 20 μg / ml to about 30 μg / ml, such as about 25 μg / ml. The medium can contain penicillin at a concentration of about 25 units / ml and / or streptomycin at a concentration of about 25 μg / ml. In some embodiments, the post-implantation medium can further contain an antimicrobial agent, such as sodium lactate.

[0154] The post-implantation medium can further contain an effective amount of glucose. The concentration of glucose contained in the medium can be from about 0.5 mM to about 5 mM, from about 1 mM to about 4 mM, from about 1.5 mM to about 3 mM, such as about 2 mM. In some embodiments, the concentration of glucose contained in the medium is about 1.8 mM.

[0155] In some embodiments, the post-implantation medium contains DMEM / F12, fetal bovine serum, GlutaMax, essential and non-essential amino acids, ITS-X, β-estradiol, progesterone, glucose, sodium lactate, penicillin and / or streptomycin, or any combination thereof. In some embodiments, the post-implantation medium contains DMEM / F12, about 20% fetal bovine serum, about 1×GlutaMax, about 1× non-essential amino acids, about 1× essential amino acids, about 1×ITS-X, about 25 U / mL penicillin and / or streptomycin, about 1.8 nM glucose, about 0.22% sodium lactate, about 8 nM β-estradiol, about 200 ng / ml progesterone, or any combination thereof.

[0156] application

[0157] The present disclosure also provides synthetic embryo models obtainable by the in vitro methods described herein for use in methods of diagnosing, preventing, or treating diseases in patients in need thereof. For example, the embryonic cells obtainable by the present invention can be used in stem cell therapies, such as treating cancer, tissue replacement, reconstructive surgery, tissue repair, wound healing, bone marrow transplantation, stroke, baldness, blindness, deafness, diabetes, heart disease, bowel disease, arthritis, bone injury, tooth replacement, neuronal diseases, and any other condition where replacement cell or tissue therapy may be beneficial. Those skilled in the art will appreciate that these cells can also be used to screen therapeutic compounds for efficacy and safety.

[0158] In some embodiments, the synthetic embryo structures for use in methods of diagnosing, preventing, or treating diseases in patients in need thereof, as described herein, can be used for transplantation into a patient. It is contemplated that in certain embodiments, the pluripotent stem cells used to obtain the embryo can initially be obtained from the patient, thereby reducing the likelihood of rejection by the patient's immune system. Thus, the pluripotent stem cells (e.g., embryonic stem cells) obtained from the patient can be cultured using the methods described herein to provide material for transplantation back into the patient to prevent or treat a condition. For example, the embryo can be used to grow replacement organs or tissues for the patient to restore the function of such organs or tissues after loss of function due to degeneration, aging, and / or disease.

[0159] The present disclosure also provides a method of providing a transgenic non-human animal, comprising gestating an embryo derived from cells cultured using the in vitro methods described herein. Such transgenic non-human animals can be used for drug screening or disease research. For example, model animals can be produced to study a particular disease. It is contemplated that the methods provided herein can be used to more efficiently develop transgenic embryos and chimeric embryos (which currently rely on labor-intensive processes such as harvesting blastocysts and manually replacing the inner cell mass).

[0160] The present disclosure provides methods for studying the effect of a test agent on embryonic development. In some embodiments, the method comprises: a) generating a synthetic embryo model using the methods described herein; b) contacting the synthetic embryo model with the test agent; and c) determining the effect of the test agent on the synthetic embryo model. In some embodiments, determining comprises comparing the phenotype or genotype of the synthetic embryo in the presence of the test agent with the phenotype or genotype of the synthetic embryo in the absence of the test agent. The method can comprise contacting mammalian pluripotent stem cells (e.g., wild-type ESCs and modified ESCs) with the test agent during or after step (a), and before or during or after step (b), and before or during or after step (c).

[0161] The method can include determining the effect on the formation of synthetic embryos at different developmental stages. The determination can be made using any method known in the art. For example, the method can include recording one or more images of the embryo structure.

[0162] Methods for studying mechanisms involved in embryogenesis are disclosed herein. In some embodiments, the method includes any in vitro method described herein for generating synthetic embryo structures at various developmental stages. Studying mechanisms involved in embryogenesis can include any method known in the art. For example, the study can include studying the effect of a test agent as described above on embryo development. In some embodiments, studying mechanisms involved in embryogenesis can include determining the effect of genetic perturbations in the embryo structure.

[0163] The method can include recording a plurality of images of the synthetic embryo structure. These plurality of images can be recorded over a pre-determined period of time to show the development of the embryo structure over time. The imaging device can include a microscope device, a suitable recording device, and optionally an image processing device.

[0164] Generally, fluorescent markers (such as fluorescent dyes or fluorescent marker proteins) are used for imaging embryo development. Such markers can be added to the culture system. For example, a fluorescent dye can be added to visualize specific molecules or cellular structures. For example, DAPI can be used to stain DNA, or MitoTracker (Invitrogen) can be used to stain mitochondria. Additionally or alternatively, the embryo structure can endogenously produce such fluorescent markers. For example, it can contain one or more cells that express a fluorescent marker protein. Such cells can have been genetically engineered to confer their ability to express such marker proteins. Thus, a fluorescence imaging device can be particularly suitable for the method. Thus, the imaging device can include a fluorescence microscope, such as a confocal microscope, which can include but is not limited to wide-field microscopes, scanning confocal microscopes, spinning disk confocal microscopes, and light sheet microscopes.

[0165] A confocal microscope can image a single point of a specimen at any given time, but can generate two-dimensional or three-dimensional images by scanning different points on the specimen in a conventional raster, thereby providing image data that can be combined into two-dimensional or three-dimensional images. For example, scanning the specimen in a single plane can generate a two-dimensional image of a slice of the entire specimen. Multiple or "stacked" such two-dimensional images can be combined into a three-dimensional image. Spinning disk confocal microscopes have several advantages over laser scanning confocal microscopes. Additionally, light sheet microscopes can also provide good imaging of embryo development.

[0166] The present disclosure also discloses a method for elucidating the role of genes in embryonic development, the method comprising: obtaining pluripotent stem cells in which the genes have been modified or knocked out, and culturing the pluripotent stem cells (e.g., ESCs) using the in vitro methods described herein and extraembryonic-like cells generated by overexpression of ESC transcription factors. Thus, these methods can contribute to the development of therapeutic methods for embryonic development-related diseases, such as fertility treatments.

[0167] The present disclosure also discloses a method for imaging an embryo during development, comprising: culturing mammalian pluripotent stem cells (e.g., ESCs) and mammalian extraembryonic-like cells or mammalian synthetic embryo structures generated by overexpression of ESC transcription factors using the methods described herein; and recording an image of the embryo using an imaging device. The image can be a two-dimensional image or a three-dimensional image. Multiple images of the same embryo can be recorded. The imaging device can include a microscope device and a suitable recording device. The imaging device can also include an image processing device. Additionally, the imaging device can include a fluorescence microscope. Additionally or alternatively, the imaging device can include a confocal microscope.

[0168] Example

[0169] Some aspects of the above-described embodiments are further disclosed in detail in the following examples, but these examples do not limit the scope of the present disclosure in any way.

[0170] Materials and methods

[0171] For Examples 1-5 below, the following experimental materials and methods were used.

[0172] Ethical Statement

[0173] The research work on human embryonic stem cells (Shef6) was carried out with the approval of the Steering Committee of the UK Stem Cell Bank (approval number SCSC21-38) and complied with the regulations of the UK Stem Cell Line Usage Operating Procedures. Human embryo research is regulated by the UK Human Fertilisation and Embryology Authority (HFEA) and carried out under licence R0193. Ethical approval was obtained from the Cambridge University Research Ethics Committee for Human Biology. Patients undergoing in vitro fertilisation at CARE Fertility, Bourn Hall Fertility Clinic, Herts&Essex Fertility Clinic and King's Fertility were given the option to continue to store, dispose of or donate their embryos for research (including specific project information), or to receive training at the end of their treatment. Patients received counselling, did not receive any financial benefit and could withdraw at any time before their embryos were used for research. Informed research consent for the donation of embryos was obtained from both gamete providers. Only blastocysts showing appropriate morphology (i.e., an expanded blastocoel and a healthy inner cell mass) were used in subsequent experiments. Embryos were not cultured beyond 14 days post-fertilisation or the first appearance of the primitive streak. Mice were housed in an animal room with a 12-hour:12-hour light-dark cycle and had free access to food and water. Mouse experiments were regulated by the Animal (Scientific Procedures) Act 1986 Amendment Regulations 2012 and carried out following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). The experiments were approved by the Home Office. This study used 6- to 45-week-old CD1 and F1 wild-type male mice and 6- to 18-week-old CD1 and F1 wild-type female mice. Animals were checked daily. Animals showing health problems were culled by cervical dislocation. All work with embryos and embryoids was carried out in the UK and complied with the 2021 ISSCR guidelines.

[0174] hESC culture

[0175] Cultivate Shef6 human embryonic stem cells (from the UK Stem Cell Bank) or RUES2 human embryonic stem cells (kindly provided by Ali Brivanlou of Rockefeller University) at 37 °C, 20% O2 and 5% CO2 in mTESR medium (05825, STEMCELL Technologies) on Matrigel-coated culture plates. Coat the culture plates with 1.6% growth factor-reduced Matrigel (356230, BD Biosciences) dissolved in DMEM / F12 (21331-020, Life Technologies) at 37 °C for 1 hour. Passage hESCs with TrypLE (12604013, Thermo Fisher Scientific). Add 10 μM ROCK inhibitor Y-27632 (72304, STEMCELL Technologies) within the first 24 hours after passage. Replace the medium every 24 hours. Regularly detect mycoplasma contamination of the cells by PCR (6601, Takara Bio) and verify by short tandem repeat analysis. To convert naive hESCs to RSeT or PXGL culture conditions, passage the cells in the medium onto mitomycin C-inactivated CF-1 MEF (3×10 3 cells / cm 2; in GSC-6101G, Amsbio), the medium consists of DMEM / F12 containing 20% Knockout Serum Replacement (10828010, ThermoFisher Scientific), 100 μM β-mercaptoethanol (31350-010, ThermoFisher Scientific), 1× GlutaMAX (35050061, ThermoFisher Scientific), 1× non-essential amino acids, 1× penicillin-streptomycin, and 10 ng / ml FGF2 (Department of Biochemistry, University of Cambridge) and 10 μM ROCK inhibitor Y-27632 (72304, STEMCELL Technologies). For RSeT cells, the medium was replaced with RSeT medium (05978, STEMCELL Technologies) after 24 hours. Cells were cultured in RSeT and passaged every 4 to 5 days as described above. For PXGL cells, transformation was performed as described above. Briefly, cells were cultured under conditions of 5% O2 and 7% CO2. The medium was replaced with Chemical Reset Medium 1 (cRM-1), which consists of N2B27 medium supplemented with 1 μM PD0325901 (Institute of Stem Cell, University of Cambridge), 10 ng / mL human recombinant LIF (300-05, Pepro Tech), and 1 mM valproic acid. N2B27 contains a 1:1 mixture of DMEM / F12 and Neurobasal A (10888-0222, ThermoFisher Scientific) supplemented with 0.5× B27 (10889-038, ThermoFisher Scientific), 0.5× N2 (self-made), 100 μM β-mercaptoethanol, 1× GlutaMAX, and 1× penicillin-streptomycin. The cRM-1 medium was changed every 48 hours for 4 days, after which the medium was changed to PXGL. PXGL medium consists of N2B27 supplemented with 1 μM PD0325901, 10 ng / mL human recombinant LIF, 2 μM (2285, Tocris) and 2 μM XAV939 (X3004, Merck). PXGL cells were passaged with TrypLE (12604013, ThermoFisher Scientific) for 3 minutes every 4 to 6 days. When passaging, 10 μM ROCK inhibitor Y-27632 and 1 μL / cm 2Geltrex (A1413201, ThermoFisher Scientific) was cultured for 24 hours. To differentiate into yolk sac-like cells or trophoblast cells, RSeT cells were passaged onto Matrigel-coated IBIDI chamber slides. After 24 hours, the medium was changed to 'ACL' (100 ng / ml Activin-A (Qk001, QKINE), 3 μM CHIR99021 (72052, STEMCELL Technologies), and 10 ng / ml human LIF) for hypoblast induction, or the medium was changed to 'PA' (1 μM PD0325901 and 1 μM A83-01 (72022, STEMCELL Technologies)) with or without 500 nM lysophosphatidic acid - LPA (3854, Tocris).

[0176] Generation of inducible hESC lines

[0177] To generate the Piggybac plasmid, the full-length coding sequence was amplified from human cell line cDNA with AttB overhangs using Phusion High-Fidelity DNA Polymerase (M0530S, New England BioLabs) according to the manufacturer's instructions. The amplicon was introduced into pDONR221 entry plasmid using BP Clonase (11789100, ThermoFisher Scientific), and subsequently introduced into the destination plasmid using LR Clonase (11791020, ThermoFisher Scientific) according to the manufacturer's instructions. Using the Neon transfection system, in addition to PB-CAG-rTTA3-Bsd or PB-CAG-rTTA3-Zeo and the pBase plasmid expressing PiggyBac transposase, hESCs were electroporated with GATA6-3XFLAG-TetOn-Zeo (entry plasmid 72922, Addgene) and / or SOX17-TetOn-Hygro or GATA3-EGFP-TetO-Hygro and / or TFAP2C-TetOn-G418, and the electroporation settings were as follows: 1200V, 20ms, and 2 pulses. Two days after transfection, antibiotics were used at 1 / 4 dose and gradually increased to the final concentration of 100 μg / mL Zeocin (bleomycin) (ant-zn-1, Invitrogen), 20 μg / mL blasticidin (A113903, ThermoFisher Scientific), 50 μg / mL G418 (10131035, ThermoFisher Scientific), or 50 μg / mL hygromycin B (10687010, ThermoFisher Scientific). Shef6-mKate2 hESCs were obtained as a gift. Single colonies were manually picked under a dissecting microscope to generate clones. The transgene was activated by adding 1 μg / mL doxycycline hydrochloride (D9891, Sigma). To select clones for downstream experiments, the isolated colonies that survived after manual picking were induced for 72 hours, and cell pellets were collected for qPCR or staining for immunofluorescence analysis. Immunofluorescence analysis was performed in naive hESCs. The expression changes of the transgene and another key lineage marker were evaluated compared to uninduced controls. Clones with robust transgene upregulation and downstream non-induced lineage marker upregulation were selected for subsequent experiments (e.g., 1 to 2 clones per transgene line). Note that AP2Y-inducible cells failed to reset under PXGL naive conditions.

[0178] qRT-PCR analysis

[0179] Collect cell pellets and extract RNA using the Qiagen RNeasy kit according to the manufacturer's instructions. Reverse transcription reaction was performed using 1 μg of RNA with random primers (C1181, Promega), dNTP (N0447S, New England BioLabs), RNAse inhibitor (M0314L, New England Biolabs), and M-MuLV reverse transcriptase (M0253L, New England Biolabs). RT-qPCR was performed using Power SYBR Green PCR Master Mix (4368708, ThermoFisher Scientific) on a Step One Plus real-time PCR machine (Applied Biosystems). The following program was used: preheat at 95 °C for 10 minutes; followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. A single melting curve was observed for all primers used in this study. The oligonucleotides used in this study are shown in Table 2 below.

[0180] Table 2: Oligonucleotides used in this study

[0181]

[0182]

[0183] Generation of hPSC-mouse embryo chimeras

[0184] For human cell-mouse embryo chimeras, at embryonic day 2.5 (E2.5), the oviducts and uterine horns were removed and flushed with M2 medium (self-made) supplemented with 4 mg / mL BSA (A9418, Sigma). The zona pellucida of the retrieved eight-cell stage embryos before compaction was then removed with acidic Tyrode's Solution. Human cells (wild-type, GATA6-SOX17 cells induced for 3 days, and GATA3-AP2YRseT cells induced for 3 days) were prepared by dissociating cells with TrypLE and washing as described above. The cells were resuspended in RSeT medium or N2B27 medium supplemented with 5% KSR and 1 μg / mL doxycycline. The resulting small cell aggregates were aggregated with eight-cell stage mouse embryos in the depressions of these media for 24 hours and then transferred to KSOM ± 1 μg / mL doxycycline medium for continued culture for 24 hours until embryonic day 4.5 (E4.5). As a negative control, embryos were cultured under the same conditions without the addition of human cells. The chimeric blastocysts were then fixed for immunofluorescence analysis. In embryos that successfully developed to the late blastocyst stage, the contribution of human nuclear antigen-positive cells to the SOX2, SOX17, and / or GATA3 populations was quantitatively analyzed.

[0185] Generation of induced human embryos

[0186] To generate a three-dimensional stem cell-derived model of the post-implantation embryo, RSeT cells at passages 2 to 6 after transfer to RSeT medium were passaged normally. The medium for extraembryonic-like cells (inducible GATA6, inducible GATA6-SOX17, or inducible GATA3-AP2Y) was changed to N2B27 containing 5% Knockout Serum Replacement and 1 μg / mL DOX the next day (day -3). This medium was changed every 24 hours for 3 days. On day 0 (the day of aggregation), Aggrewell plates (34415, STEMCELL Technologies) were pre-coated with an anti-adhesion solution (07010, STEMCELL Technologies) and centrifuged at 2000 g for 5 minutes. The wells were washed twice with PBS and then the experimental medium was added. This medium consisted of N2B27 containing 5% Knockout Serum Replacement, 1 μg / mL doxycycline, and 10 μM Y-27632. One hour after adding 10 μM Y-27632 to the wells containing cells for generating inducible human embryoids, the induced cells and wild-type ESCs were separated by enzymatic digestion. The separated cells were pelleted and the cell pellet was resuspended in the experimental medium and then placed in gelatin-coated wells for MEF depletion. After 15 to 30 minutes, the cells were counted, mixed, and plated into Aggrewell plates such that finally each microwell in the Aggrewell plate was seeded with 8 wild-type ESCs, 8 hypoblast-like cells, and 16 trophoblast-like cells. At 8 days post-fertilization, the ratio of epiblast cells:hypoblast cells:trophoblast cells in in vitro cultured human embryos was 32:24:228. However, importantly, many trophoblast cells did not contact the inner cell mass-derived tissue or were in a terminally differentiated state. In addition, after adding doxycycline, the proliferation rate of inducible GATA6-SOX17 cells in the culture was slower than that of the other two cell populations. Therefore, the initial seeding density should meet the following: (1) the total cell number should be similar to the cell number used for successful cell sorting in the mouse model; (2) the cell ratio should reflect the situation of the peri-implantation embryo; (3) reduce the number of inducible GATA3-AP2Y cells and increase the number of inducible GATA6-SOX17 cells.

[0187] On the first day, the culture medium was changed twice, and two-thirds of the culture medium was replaced each time with N2B27 medium supplemented with 5% Knockout Serum Replacement and 1 μg / mL doxycycline. On the second day, half of the culture medium in the Aggrewell was replaced with hIVC1 medium containing 25 ng / mL hIGF1 (78022.1, STEMCELL Technologies) and 1 μg / mL doxycycline. The hIVC1 medium consists of Advanced DMEM / F12 (12634-010, ThermoFisher Scientific), supplemented with 20% heat-inactivated FBS (10270106, ThermoFisher Scientific), 1× GlutaMax, 1× non-essential amino acids, 1× essential amino acids, 1× ITS-X, 25 U / mL penicillin / streptomycin, 1.8 mM glucose (G8644, Sigma-Aldrich), 0.22% sodium lactate (L7900, Sigma-Aldrich), 8 nM β-estradiol (50-28-2, Tocris), and 200 ng / mL progesterone (P0130, Sigma-Aldrich). Starting from day 3, half of the culture medium was replaced daily with this medium. On day 4, under a dissecting microscope, the aggregates were manually picked using a mouth pipette and transferred to individual wells of an ultra-low attachment 96-well plate (CLS7007, Corning) containing hIVC1 medium supplemented with IGF1 and doxycycline as described above for further culture.

[0188] Immunostaining and Image Analysis

[0189] Samples were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA; 1710, Electron Microscopy Sciences) for 20 minutes at room temperature. Samples were washed three times with PBS containing 0.1% (vol / vol) Tween-20 (PBST), and then incubated with a PBS solution containing 0.3% (vol / vol) Triton X-100 (T8787, Sigma Aldrich) and 0.1 mM glycine (BP381-1, ThermoFisher Scientific) for 30 minutes at room temperature. Samples were blocked with blocking buffer (PBST containing 5% (w / vol) BSA, A9418, Sigma), and then incubated with the primary antibody diluted with blocking buffer overnight at 4°C. A list of the primary antibodies is shown in Table 3 below. Samples were washed three times with PBST and incubated with a fluorescently conjugated Alexa Fluor secondary antibody (ThermoFisher Scientific, 1:500) and DAPI (D3571, ThermoFisher Scientific, 1 μg / mL) diluted with blocking buffer for 2 hours at room temperature. For the quantification of pSMAD1.5, OCT4-positive or GATA6-positive nuclei were isolated, and the fluorescence intensity of pSMAD1.5 was measured. For the quantification of SMAD2.3, the fluorescence intensity of OCT4-positive or GATA6-positive nuclei (excluding the outermost GFP+ cell layer) and the fluorescence intensity of the cytoplasm were measured. The data were expressed as the ratio of nuclear fluorescence intensity to cytoplasmic fluorescence intensity. Immunofluorescence images were analyzed using FIJI. The total cell number and the generated spot renderings in the embryoid bodies on day 4 were quantified using the spots tool with manual management in Imaris software (version 9.1.2, Oxford Instruments).

[0190] Table 3: Antibodies used in this study

[0191]

[0192]

[0193] Thawing and culture of human embryos

[0194] The method for thawing and culturing human embryos was as previously described. Briefly, according to the manufacturer's instructions, a Kitazato thawing kit (VT8202-2, Hunter Scientific) was used to thaw cryopreserved human blastocysts (day 5 or day 6 after fertilization). One day before thawing, the thawing solution (TS) was placed at 37 °C overnight. The next day, the in vitro fertilization (IVF) straw was immersed in 1 mL of pre-warmed TS for 1 minute. Subsequently, the embryos were transferred to the dilution solution (DS) for 3 minutes, in the wash solution 1 (WS1) for 5 minutes, and in the wash solution 2 (WS2) for 1 minute. These steps were performed using a STRIPPER micropipette (Origio) in a reproplate (REPROPLATE, Hunter Scientific). The embryos were incubated in pre-equilibrated human IVC1 medium supplemented with 50 ng / mL insulin-like growth factor-1 (IGF1) (78078, STEMCELL Technologies) covered with mineral oil at 37 °C, 5% CO2, and normoxic conditions for 1-4 hours to allow the embryos to recover. After thawing, the blastocysts were briefly treated with acidic Tyrode's solution (T1788, Sigma) to remove the zona pellucida, and then placed in pre-equilibrated post-implantation human embryo medium (hIVC1) in an 8-well μ-Slide tissue culture plate (80826, Ibidi) at a volume of approximately 400 μL per well per embryo. Half of the medium was changed every 24 hours.

[0195] Statistical analysis

[0196] Statistical analysis was performed using Graphpad Prism v9.4. The sample size was not pre-determined, and the researchers were also informed of the experimental conditions. All experiments were performed independently at least twice. The Shapiro-Wilk test was used to test the normality of the data. Normally distributed data were analyzed using parametric tests (unpaired t-test or ANOVA), and non-normally distributed data were analyzed using non-parametric tests (Mann-Whitney U test or Kruskal Wallis test), as shown in the figure legends. The sample numbers have been noted in the figure legends. All statistical tests were two-tailed tests. Each test used individual samples. Unless otherwise stated, repeated experiments were biological replicates. In the figures, all data are presented as mean ± SEM. For box plots, the box represents the interquartile range from the 25th percentile to the 75th percentile, the whiskers represent the minimum and maximum values, the center line represents the median, and the + symbol represents the mean. For multiple comparison tests, only comparisons with the control conditions were made. Unlabeled pairwise comparisons were not significant (p > 0.05).

[0197] Collection, generation, and sequencing of single-nucleus ATAC / RNA 10x libraries

[0198] To collect post-implantation embryo-like models for single-cell sequencing, embryoids with correct organization on days 4, 6, and 8 were visually selected and washed twice with PBS in a 4-well culture dish, then transferred to TrypLE. The samples were agitated by pipetting every 5 minutes for 10 - 20 minutes until dissociation. Enzyme activity was inactivated by adding a 2-fold volume of PBS solution with 20% fetal bovine serum (FBS). The cells were collected in a Falcon tube, pelleted, and the pellet was resuspended in cryopreservation buffer, which consisted of 50 mM Tris (pH 8.0) (15 - 567 - 027, Fisher Scientific), 25% glycerol (G5516, Sigma-Aldrich), 5 mM Mg(OAc)2 (63052, Sigma-Aldrich), 0.1 mM EDTA (15575020, ThermoFisher Scientific), 5 mM DTT (R0861, ThermoFisher Scientific), 1× protease inhibitor mixture (P8340, Sigma-Aldrich), and a 1:2500 dilution of Superasin (an RNase inhibitor, AM2694, Invitrogen). For cell lines, 10,000 cells were counted, pelleted, and the pellet was resuspended in the above cryopreservation medium, then slowly frozen at -80°C.

[0199] For nuclear isolation and library construction, a low-input nuclear isolation protocol from 10x Genomics was used. Briefly, the frozen cell pellet was thawed in a 37°C water bath for 30 seconds, then centrifuged (500 g for 5 minutes at 4°C) to pellet the cells. Subsequently, the supernatant was aspirated. The cell pellet was washed twice with 200 μL of 1× PBS containing 0.04% BSA, then centrifuged. The supernatant was aspirated between the two washes. Subsequently, cold lysis buffer (45 μL per sample) was added to the washed cell pellet. The cell pellet containing the lysis buffer was placed on ice for 3 minutes. Subsequently, wash buffer (50 μL per sample) was added. The washed isolated nuclei were resuspended in diluted nuclear buffer. The isolated nuclei were resuspended in 5 μL of diluted nuclear buffer and directly added to the transposition reaction. All subsequent steps followed the 10x Genomics single-cell multiome ATAC and gene expression protocol and its manufacturer's instructions and guidelines. The final library was loaded onto the NextSeq 2000 at a loading concentration of 650 pM using the P2 100-cycle kit, and paired-end sequencing was performed according to the sequencing reads recommended by 10x Genomics (28 / 10 / 10 / 90 cycles for the gene expression library and 50 / 8 / 24 / 49 cycles for the ATAC library).

[0200] Single-cell sequencing analysis

[0201] Processing and quality control

[0202] Analyze the raw reads using the CellRanger ARC pipeline to generate ATAC and RNA fastq files for each sample, and then align the genomic reads and transcriptomic reads. Subsequently, use the Read10X_h5 command to read the matrices into Seurat48 and Signac49. For ATAC-seq data, use the peaks from the standard chromosomes and additionally use macs2 to call peaks to add additional Signac detections. Cells with RNA UMI counts >500, mitochondrial reads <20%, ATAC reads >500, TSS enrichment >1, and identified as singlets using scDblFinder50 are retained for downstream analysis. For UMAP projection, use SCTransform for RNA counts and regress on the mitochondrial count percentage and cell cycle score. Use PCA and LSI plots to generate a weighted nearest neighbor (wnn) embedding model that incorporates both modalities. Run the chromVAR51 software to calculate motif accessibility scores in peak detection. Data visualization is performed using the DimPlot, FeaturePlot, VlnPlot, TSSPlot, and FragmentHist functions of Seurat and the 52do_Alluvialplot and do_Nebulosaplot functions of SCpubr.

[0203] Comparison with published datasets

[0204] The analysis tool scmap was used to project cell labels from other single-cell datasets onto the transcriptional data of post-implantation embryo-like models. All reference data used were publicly available and accompanied by published cell type annotations. The cynomolgus monkey gene names were converted to hgnc gene symbols using biomaRt. For data generated using smart-seq2 or other non-UMI-based single-cell sequencing methods, the scmapCluster method was used with a similarity threshold of 0.5. For UMI-based methods, scmapCell was first used, followed by the scmapCell2Cluster method with w = 2. Multiple datasets were used to draw conclusions using scmap. Transcriptionally similar clusters (e.g., trophoblasts or amnion) may be misprojected if they do not exist due to limited cell assignments in some datasets. After cell type assignment and processing of the sequenced cell lines, a previously reported and validated logistic regression framework was applied to project the cell line data onto the published single-cell data and project the published cluster annotations (e.g., training data) onto the post-implantation embryo-like model clusters (e.g., test data), resulting in a quantitative measure of predicted similarity. In this study, only differentially expressed genes were used (generated using the FindAllMarkers function of Seurat based on the coarse cell assignment results, where the amnion cluster and the mesoderm cluster were aggregated).

[0205] Multivelo RNA / Chromatin Speed

[0206] The recently published velocity calculation method Multivelo was adopted, which incorporates single-cell ATAC and RNA data simultaneously. Multivelo was run on all cells that passed the above quality control and processing. The analysis was based on available vignettes containing 1,000 highly variable genes and the "grid" method. Gene expression and chromvar were plotted against latent time using the switchde software package.

[0207] CellPhoneDB Analysis

[0208] CellPhoneDB 2.0 with default settings was used to evaluate potential tissue signal interaction communication. For simplicity, the coarse cell assignment method was used, where the separate amnion clusters (AM-1, AM-2, AM-3) and mesoderm clusters (MESO-1, MESO-2) were aggregated. Selected significant interactions were plotted as dot plots.

[0209] Reanalysis of the human in vitro cultured embryo dataset

[0210] The previously published data was realigned to the hg38 human genome using kallisto or kb-bustools. Datasets not sequenced using UMI-based technology were normalized to quasiumis using quminorm. Datasets were integrated using SCTransform-based integration to generate a single-cell RNA sequencing dataset of human embryos from fertilized eggs to 14 days post-fertilization. Cells were clustered and assigned identities based on previous annotations and canonical marker expression. This dataset showed good overlap with datasets having cell type separation and some temporal resolution. SCENIC with default settings in R was used, and new assays were generated in Seurat objects using the AUC-regulon table. Using this assay, the epiblast, hypoblast, and trophoblast lineages were subsequently compared using Seurat's FindMarkers function to perform Wilcoxon rank sum tests with Bonferroni correction to identify differentially active regulators predicted in pairs. Regulators enriched in both of two related comparisons (e.g., hypoblast vs. epiblast; hypoblast vs. trophoblast) were used as enriched active transcription factors (e.g., in the hypoblast) for subsequent analysis. The interaction map of these factors was subsequently drawn in Cytoscape.

[0211] Availability of data and code

[0212] For aligning sequencing data, GRCh38 (www.ncbi.nlm.nih.gov / assembly / GCF_000001405.26 / ) and GRCm38 (www.ncbi.nlm.nih.gov / assembly / GCF_000001635.20 / ) were used. The code used to analyze the data described in this article is available at / / github.com / bweatherbee / human_model.

[0213] Example 1

[0214] Induction of extraembryonic lineages

[0215] Factors capable of similarly upregulating the extraembryonic gene program in human ESCs were identified for the first time. Single-cell RNA sequencing data of human embryos cultured to gastrulation published previously ( Figure 6A-6E ) was integrated. The predicted activities of transcription factors enriched in the epiblast, trophoblast, or hypoblast were scored using the computational tool SCENIC ( Fig. 6F)。As expected, SOX2, NANOG, and POU5F1 (OCT4) showed high predicted activity in the epiblast. Transcription factors including GATA4, GATA6, SOX17, and FOXA2 were particularly active in the hypoblast, while GATA3, NR2F2, GATA2, and TFAP2C (AP2Y) showed enriched activity in the trophoblast ( Figure 6F-6G )。Studies have shown that overexpression of GATA6 or SOX17 can drive the endodermal gene program in primed hESCs. Therefore, GATA6 or SOX17 was selected as a candidate gene for programming hESCs into hypoblast-like cells. Similarly, it has been reported that GATA3 and TFAP2C have a high chromatin co-occupancy rate during the differentiation of hESCs into trophoblast stem cells. GATA3 and TFAP2C also showed high predicted activity in the trophoblast. Therefore, GATA3 and TFAP2C were selected as candidate genes for driving the differentiation of hESCs into trophoblast-like cells. For the transcription factors of interest, hESCs were generated and validated using doxycycline-inducible single or combined transgenes ( Figure 1A and Figure 1B , Figure 6H )。

[0216] Notably, the pluripotent state to some extent determines the potential for differentiation from ESCs. Therefore, to evaluate the ability of the selected candidate transcription factors to drive the development of hESCs towards an extraembryonic-like expression profile, the candidate transcription factors were overexpressed individually and in combination in cells with a naive to primed pluripotency spectrum. Three defined starting conditions were used for cell culture: PXGL that supports pre-implantation-like cells; RSeT that generates intermediate peri-implantation-like cells; and the conventional mTeSR1 condition for maintaining post-implantation-like cells. Using single or combined transgenes and starting from different pluripotent states, significant differences in extraembryonic gene induction were observed at both the protein and mRNA levels ( Figures 7A-7F )。In hypoblast-like induction, overexpression of GATA6 did not drive SOX17 expression under RSeT or PXGL conditions, but overexpression of SOX17 led to a significant upregulation of GATA6 under all starting pluripotent state conditions ( Fig. 7A 、 Figure 7C and 7D )。After combined induction of GATA6 and SOX17, FOXA2 expression was continuously upregulated under primed and RSeT conditions, but not under PXGL conditions ( Figure 7C and 7D )。These data indicate that although GATA6 and SOX17 can indeed drive the endodermal gene program, the regulation of specific downstream targets varies depending on the starting pluripotent state.

[0217] When driving the trophoblast-like gene program, AP2Y seems to be particularly effective in upregulating the expression of GATA2 and CK7. However, inducing AP2Y alone leads to cell death and loss of transgene expression in naive cells, but not in RSeT cells or PXGL cells ( Fig. 7E and Figure 7F ). Co-induction of GATA6 and SOX17 or GATA3 and AP2Y results in the continuous downregulation of pluripotency markers, including NANOG, SOX2, and OCT4 ( Figure 7A-7F ).

[0218] RseT hESCs may be the best starting cell type for generating the post-implantation embryo models disclosed herein because they: (1) represent the peri-implantation developmental stage; (2) have lower expression levels of amnion-specific genes after induction of GATA3 and AP2Y compared to naive cells ( Figure 7B , Fig. 7E and Figure 7F ); and (3) are known to be more easily differentiated into peri-implantation and post-implantation yolk sac-like endodermal cells compared to PXGL cells. For these reasons, and the synergistic effect of dual induction of candidate transcription factors, in subsequent experiments, RseT hESCs with inducible GATA6-SOX17 and inducible GATA3-AP2Y were used to induce hypoblast-like cells and trophoblast-like cells, respectively. In the basal medium, dual induction of GATA6 and SOX17 in RSeT cells induced the expression of endoderm genes comparable to the directed differentiation protocol under yolk sac-like cell differentiation conditions ( Fig. 8A and 8B ). In the basal medium, dual induction of GATA3 and AP2Y in RSeT cells induced the expression of trophoblast genes, although the expression levels were different compared to the directed trophoblast differentiation protocol ( Figure 8C and 8D ).

[0219] To further characterize RseT hESCs with inducible expression of GATA6-SOX17 or GATA3-AP2Y, single-cell 10x multiome sequencing was performed. The transcriptome and chromatin accessibility were evaluated simultaneously. Cells were clustered according to the sample source ( Figure 1C and Fig.9A ). Applying a logistic regression framework showed that wild-type hESCs, inducible GATA6-SOX17 RseT hESCs, and inducible GATA3-AP2Y RseT hESCs had the highest similarity to the epiblast, hypoblast, and cytotrophoblast of the post-implantation embryo, respectively ( Figure 1D)。In addition, compared with in vitro blastoid and directed differentiation models, RseT hESCs are similar to pluripotent populations; induced GATA6-SOX17 cells are similar to blastoid-derived hypoblasts; induced GATA3-AP2Y cells are similar to post-implantation-like trophoblast stem cells, but different from blastoid-derived trophectoderm-like cells ( Fig. 9B )。Analysis of differentially expressed genes and differentially accessible motifs revealed similar embryonic and extraembryonic dynamics ( Figure 1E and Fig. 9C )。Specifically, enriched expression and motif accessibility scores of pluripotency and epiblast markers were detected in RseT hESCs, enriched expression and motif accessibility scores of hypoblast markers in GATA6-SOX17-induced cells, and enriched expression and motif accessibility scores of trophoblast markers in GATA3-AP2Y-induced cells ( Figure 1E )。Collectively, these data indicate that transcription factor-mediated induction of extraembryonic cell fates in RseT hESCs can drive hypoblast-like or trophoblast-like gene programs without exogenous factors, although this induction is heterogeneous and there are some marker gene expression defects ( Fig. 9C )。The ability of induced cells to proliferate as stable cell lines in culture has not been tested. When aggregated with 8-cell stage mouse embryos, relative to wild-type controls, for induced GATA6-SOX17 and GATA3-AP2Y cells, the human cells tended to differentiate into SOX17-positive primitive endoderm and GATA3-positive trophectoderm, respectively ( Figure 9D-9G )。If this relative shift towards an extraembryonic identity is sufficient to allow self-organization, then the challenges associated with conflicting medium requirements when successfully co-culturing embryonic and extraembryonic-like cells will be overcome. Indeed, co-culture of wild-type RseT hESCs with induced GATA6-SOX17 RseT hESCs and induced GATA3-AP2Y RseT hESCs in a 1:1:1 ratio showed good viability and mixing properties ( Figure 1F )。

[0220] Example 2

[0221] Assembly of the 3D post-implantation model

[0222] Since all three RseT hESC-derived cell types (i.e., wild-type, GATA6-SOX17-induced, and GATA3-AP2Y-induced) can be co-cultured in N2B27 medium, doxycycline was used to induce the expression of the selected transcription factors for 3 days. Subsequently, the cell mixture was aggregated in Aggrewell plates ( Figure 2A)。Cell aggregation occurred within 24 hours. At 48 hours after aggregation, a clear distinction between the inner cell domain and the outer cell domain was observed using bright-field imaging( Figure 2A )。At 48 hours after aggregation, the culture medium was changed to post-implantation human embryo medium (hIVC1). Throughout the culture period, incubation with doxycycline was continued, and proliferation remained consistent across experiments( Figure 2A and Figure 2C )。Four days after aggregation, the cell aggregates self-organized into a structure with the following components: a SOX2-positive epiblast-like structure containing a central cavity; an outer single layer of GATA3-positive putative trophoblast-like cells; and an intermediate putative hypoblast-like domain of GATA6-positive cells between the inner cavitated domain and the outer layer( Figure 2B and Fig. 10A )。

[0223] Similar to the post-implantation mouse embryo model, the aggregates did not undergo a blastocyst-like morphological transition before forming the post-implantation-like structure. The efficiency of induced human embryoid body formation (defined as aggregates containing an organized SOX2-positive domain surrounded by concentric layers of GATA6-positive and GATA3-positive cells) was approximately 23%( Figure 2D )。In contrast, when using primed mTeSR1 or naive PXGL hESC as the starting pluripotent state for constitutive wild-type cells, GATA6-SOX17-induced cells, and GATA3-AP2Y-induced cells, the efficiency of organized multi-lineage structure formation was less than 5%( Figure 2D-Figure 2E )。The organized embryoid-like structures exhibited an organizational structure similar to that of human embryos at 8 - 9 days after fertilization( Figure 2F )。

[0224] The currently disclosed induced human embryoid bodies expressed several other lineage markers in an organized manner, including N-cadherin, SOX17, and GATA4 in the putative hypoblast-like compartment( Figure 2G )。Structures expressing SOX17 and / or GATA6 were also observed in the outer layer of GATA3-AP2Y-induced cells (labeled with eGFP), which may reflect the reported trend of peripheral cells in embryoid bodies to assume an endodermal identity. The epiblast-like inner compartment expressed SOX2, NANOG, and E-cadherin and maintained a pluripotent and epithelial identity similar to that of human embryos( Figure 2G )。In addition, this inner region exhibited apical-basal polarity, with laminin deposited on the substrate and PODLX, PARD6, and ZO-1 expressed apically( Figure 2H )。These data indicate that RseT hESC-derived embryoid-like structures can self-organize under minimal culture conditions.

[0225] Example 3

[0226] Differentiation in embryoid bodies

[0227] To gain insights into whether the currently disclosed human embryo-like models have formed gene expression and chromatin accessibility patterns that reflect those of natural human embryos, single-cell multi-omic RNA and assay for transposase-accessible chromatin sequencing (ATAC-seq) were performed at days 4, 6, and 8 post-fertilization ( Figure 3A ). Based on the development of the following three tissues, individual structures were selected for sequencing: (1) the internal epithelial domain; (2) the intermediate domain surrounding the central epithelial cells; and (3) the outer GFP-positive cell layer ( Fig. 10B and 10C ). To assign clusters without bias, the dataset obtained in this study was projected onto human and cynomolgus monkey datasets using scmap, covering the peri-implantation to gastrula stages ( Figure 3B and Fig. 10D ). This analysis allowed the projection of gene expression signatures of previously annotated cynomolgus monkey cell type clusters onto the currently disclosed human embryo models. Using multi-omic based velocity inference (multivelo), a good correlation was found between the inferred differentiation time and the structural transitions from day 4 to day 8 post-aggregation. These data, combined with the expression of canonical markers, enabled the annotation of cell types in the human embryo-like structures disclosed herein ( Figure 3B , Fig. 10E and 10F ). Cell clusters similar to late epiblast (L-EPI), amnion (AM-1, AM-2, and AM-3), mesoderm (MESO-1, MESO-2), extraembryonic mesenchyme (EXMC), and hypoblast / visceral endoderm (HYPO / VE) were identified ( Figure 3C , Fig. 10E and 10F ). The composition of these assigned cell clusters was shown to vary according to the date of sample collection, where the L-EPI cell cluster included only structures from days 4 and 6, and over time, gradually changed from AM-1 to AM-2 and AM-3, and similarly, from mesoderm to EXMC ( Fig.10F ).

[0228] Finally, the cell clusters described herein were directly compared with previously annotated datasets of cynomolgus monkey and human embryos at the peri-implantation and perigastrulation stages. This analysis demonstrated the similarity between the induced human embryoid clusters and primate embryos ( Fig.11A and Fig. 11B ). Similarly, the induced human embryoid clusters were consistent with in vitro hESC-derived models, including post-implantation amniotic blastoids (PASE), blastocysts, and recently identified extraembryonic mesenchyme-like cells generated during TSC-like directed differentiation ( Fig. 11B and Fig. 11C ). Compared with embryonic-like structures, there are significant similarities among the in vitro amnion, hypoblast, and extraembryonic mesenchymal populations. However, distinct trophoblast-like cell clusters derived from GFP-positive inducible GATA3-AP2Y cells were not identified, although they were present as an outer layer in inducible human embryoids ( Figure 3A and Figure 11D ). Given the abnormal upregulation of endodermal markers after aggregation, inducible GATA3-AP2Y-derived cells are unlikely to represent true trophoblasts. Nevertheless, when human embryos are cultured in vitro to the post-implantation stage, inducible human embryoids give rise to several cell types, including amnion and extraembryonic mesenchyme, but these cell types cannot differentiate robustly. In fact, immunofluorescence analysis showed that at day 6 after aggregation, the internal SOX2-positive domain upregulated amnion markers, including CDX2 and ISL1. At day 8 after aggregation, the inner layer domain expressed the mature amnion markers VTCN1 and HAND1 ( Figure 3D , Figure 11A and Figure 11B ), associated with the transition between AM-1, AM-2, and AM-3. The GATA6-positive domain also expressed HAND1, which supports the presence of extraembryonic mesenchyme ( Figure 12A and Figure 12B ). A subset of GATA6-positive cells showed high co-expression of TBX20, further emphasizing the presence of extraembryonic mesenchyme in this intermediate region ( Figure 12C and Figure 12D ). In most of the currently disclosed human embryoids, the entire epiblast-like domain differentiates towards the amnion fate. However, between day 6 and day 8 after aggregation, rare cases of embryonic-like structures showed broken dorsal-ventral and / or anterior-posterior symmetry, accompanied by regionalized expression of ISL1, SOX2, and BRACHYURY ( Figure 3D ).

[0229] Recent reports have speculated that amniotic cells and primordial germ cells (gamete precursors) are produced at least in part by bipotent progenitors. Therefore, it was evaluated whether such progenitors or their descendants are directed to differentiate in the currently disclosed human embryo-like models. First, the primordial germ cell module was scored based on the gene expression identified in in vitro differentiated human primordial germ cell-like cells. Cells with a transcriptome similar to that of primordial germ cell-like cells were identified ( Figures 3E - 3F) Cells with a primordial germ cell-like cell gene expression module score higher than 98% were labeled as putative primordial germ cell-like cells ("PGCs"). Primordial germ cell-like cells express TFAP2A (AP2a), which is a key marker for bipotent amniotic and primordial germ cell-like cell progenitors. Different from other cells in the AM-1 and AM-2 clusters, primordial germ cell-like cells express the pluripotency marker NANOG as well as the primordial germ cell markers PRDM1 (also known as BLIMP1) and NANOS3( Figure 3G ) Immunofluorescence analysis of a panel of typical human primordial germ cell markers41 confirmed that triple-positive primordial germ cell-like cells of AP2Y / SOX17 / NANOG could be observed 4 days after aggregation and increased in number on day 6( Figures 3H - 3I and Figure 12F ). These data indicate that robust specification of primordial germ cell-like cells occurs concomitantly with the formation of amniotic-like cells and within the inner epiblast-like compartment, supporting the existence of bipotent progenitors for these two lineages.

[0230] Next, Shef6-mKate wild-type ESCs were used to verify the differentiation trajectories within embryoid bodies. At day 4 after aggregation, most wild-type cells contributed to the inner SOX2-positive ectoderm-like domain and partially to the GATA6-positive cell population( Figure 12G ), which is consistent with the results of a small amount of early extraembryonic mesenchymal differentiation in the sequencing analysis( Figure 10F ). At day 6 after aggregation, wild-type cells contributed to the ISL1-positive amniotic-like domain and GATA6- and TBX20-positive extraembryonic mesenchymal-like cells( Figure 12H and Figure 12I ). Finally, triple-positive primordial germ cell-like cells of AP2Y / SOX17 / NANOG were confirmed to be derived from the mKate2-labeled wild-type cell population( Figure 12J ). These data confirm the differentiation of the epiblast-like domain into several post-implantation lineages.

[0231] Example 4

[0232] BMP - mediated epiblast differentiation

[0233] In primate embryos, the amnion, primordial germ cells, and extraembryonic mesenchyme are thought to differentiate in response to BMP signaling. To understand whether this is consistent with the human embryo-like model disclosed herein, the expression of the downstream BMP-responsive genes ID1-4 was examined. ID1 and ID4 were upregulated during amnion formation, while ID2 and ID3 were enriched in the amnion and extraembryonic mesenchymal trajectories, indicating that BMP signaling may be active( Figure 4A and Figure 13A)。In addition, the accessibility of the SMAD5 motif is high in both trajectories, while the accessibility score of the SMAD2::SMAD3::SMAD4 motif (a downstream target of Activin-NODAL signaling) is not high ( Figure 4B and Figure 13B ). Consistent with this observation, high BMP and low NODAL signaling environments have recently been implicated in amniotic differentiation of marmoset ESCs and hESCs during extraembryonic mesenchymal differentiation, suggesting that similar kinetics may drive the differentiation of these cell populations in induced human embryo-like structures.

[0234] To further understand the potential intertissue communication in the human embryo-like model disclosed herein, the computational tool CellPhoneDB was used to predict ligand-receptor pairs across clusters in single-cell sequencing data ( Figures 13C to 13D ). This analysis uses the expression of a curated set of receptor-ligand pairs across clusters to score potential intertissue communication. CellPhoneDB predicted that BMP2 / 6 derived from the hypoblast cluster and BMP4 secreted by extraembryonic mesenchyme may be mediators of intertissue communication. In contrast, predicted intertissue NODAL signaling was low, further supporting the presence of a high BMP, low NODAL signaling environment in the human embryo-like model. When CellPhoneDB was applied to the single-cell sequencing data of the three cell lines aggregated to generate the embryo-like model, induced GATA3-AP2Y cells were predicted to be the initial source of BMP ( Figure 13D ). Aggregation of only induced GATA6-SOX17 and wild-type RseT hESCs (i.e., without induced GATA3-AP2Y cells) or addition of the ALK1 / 2 / 3 / 6 (type I BMP receptor) inhibitor LDN193189 between day 0 and day 2 prevented the formation of organized structures ( Figures 13E - 13G ), demonstrating the requirement for BMP secreted by induced GATA3-AP2Y cells during embryoid body formation.

[0235] To verify the role of BMP signaling during the differentiation of the ectoderm-like domain, the expression of phosphorylated (p)SMAD1.5 was examined. Enrichment of pSMAD1.5 in the OCT4-positive epiblast-like domain on days 4 and 6 after aggregation indicated active BMP signaling ( Figure 4C ). In contrast, the nuclear-cytoplasmic ratio of total SMAD2.3 in these cells was low, reflecting low NODAL signaling within the epiblast-like domain ( Figure 4D), which is consistent with the prediction of CellPhoneDB. To functionally validate the role of BMP signaling in the differentiation of the inner layer domain, human embryonic-like models were treated with LDN193189 within 48 to 96 hours after aggregation. Compared with untreated controls or BMP4-treated structures, at day 4 and day 6 after aggregation, the treated structures showed increased maintenance of SOX2 expression in the inner layer domain and reduced upregulation of CDX2 and AP2a. Supplementation with activin-A, an agonist of SMAD2 / 3 signaling, produced a similar phenotype but to a lesser extent ( Figure 4E and 4F , Figure 13H ). In addition, LDN193189 treatment reduced the number of primordial germ cell-like cells, while BMP4 or activin-A treatment had minimal effect on the emergence of this cell population ( Figures 4G - 4H ). These data suggest that endogenous BMP and NODAL are key drivers for the differentiation of the epiblast-like domain into amnion and primordial germ cell-like cells in induced embryoids.

[0236] Example 5

[0237] SOX17 inhibits anterior hypoblast

[0238] Through antagonism by the anterior hypoblast, BMP signaling is localized to the posterior of the embryo, and this anterior hypoblast secretes inhibitors of BMP, WNT, and NODAL, including CER1 and LEFTY1 ( Figure 2F ). Recent studies have shown that these markers of the anterior hypoblast are expressed in both peri-implantation and post-implantation human embryos cultured in vitro. Neither CER1 nor LEFTY1 was significantly expressed in the HYPO / VE single-cell sequencing cluster ( Figure 5A ). Reanalysis of previously published 10x single-cell RNA sequencing data of post-implantation human embryos cultured in vitro showed that SOX17 regulatory activity was significantly enriched in the CER1-negative hypoblast subcluster ( Figure 5B ). These sequencing data were also published in Supplementary Data Table 8 of Molè et al. ("A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre," Nature Communications 2021, 12(1), 3679), the entire content of which is incorporated herein by reference. In fact, compared with overexpression of GATA6 alone, induction by SOX17 alone or in combination with GATA6 both led to a decreased ability to upregulate CER1 ( Figure 6H)。To test whether induction by GATA6 alone altered the properties of the hypoblast-like cell subpopulation in the human embryo-like model disclosed herein, hypoblast-like cells with inducible expression of GATA6 or SOX17 alone or in combination were generated. Compared with embryoids generated from inducible GATA6-SOX17 or SOX17 cells, an increased proportion of CER1-positive cells was observed in embryoids derived solely from inducible GATA6 cells ( Figure 5C and Figure 5E , Figure 14A ). To verify that SOX17 induction inhibits CER1 expression, doxycycline was withdrawn on day 1 or day 3 after aggregation. Withdrawal of doxycycline on day 3 (but not on day 1) promoted CER1 expression in embryoids ( Figure 5C and Figure 14A ). Co-staining of pSMAD1.5 and CER1 showed a significant decrease in pSMAD1.5 expression in the inner domain cells of structures with a CER1-positive cell population ( Figure 5D and Figure 5F ). On day 6 after aggregation, CER1 expression decreased in all embryoids regardless of the initial hypoblast induction protocol ( Figure 14B ). However, transient expression of CER1 in the anterior hypoblast-like cells affected the epiblast-like domain of the embryoids. Compared with structures with continuous GATA6-SOX17 or SOX17 induction, embryoids generated by GATA6 single induction or withdrawal of doxycycline on day 3 showed increased expression of the primitive streak marker BRY / TBXT on day 6 after aggregation ( Figures 5G - 5H and Figure 14C ).

[0239] Collectively, these data indicate that functional differences in the gene regulatory network during the differentiation of the hypoblast cell subpopulation were observed in embryoids. It also indicates that prolonged overexpression of SOX17 has an inhibitory effect on the identity of the CER1-positive anterior hypoblast. These experiments highlight the value of the modular embryoid model in studying the interactions between embryonic and extra-embryonic tissues.

[0240] Other considerations

[0241] In the present disclosure, a multi-lineage stem cell-derived human post-implantation embryo model was generated. The model underwent luminal genesis and differentiation of the epiblast-like domain and reflected the development-related interactions between extraembryonic-like tissues and embryonic-like tissues. The stem cell-derived human embryo induction model generated amnion-like cells in response to BMP signaling, and these amnion-like cells gradually matured. Similarly, primordial germ cell-like cells were prone to differentiation in the human embryonic stem cell model. Evidence suggests that these cells differentiated along the amnion differentiation trajectory and may be derived from a common AP2a-positive progenitor, as reported in other in vitro systems. Extraembryonic mesenchyme-like cells very similar to primate embryonic cells were also observed. Analyses demonstrated a developmental trajectory from a late epiblast-like population to mesoderm intermediates, which was consistent with recently reported in vitro differentiation protocols for extraembryonic mesenchyme, data from cynomolgus monkeys, and historical observations of rhesus monkey and human embryos.

[0242] Unexpectedly, transgenic regulation used to drive hypoblast-like identity altered the contribution balance of the hypoblast transitioning from CER1-negative hypoblast-like cells to CER1-positive hypoblast-like cells. This observation indicates that SOX17 overexpression blocked the formation of the CER1-positive anterior hypoblast. NODAL signaling is required for the formation of the CER1-positive mouse anterior visceral endoderm, and SOX17 may inhibit overly high NODAL activity and antagonize its targets. The low NODAL activity observed in inducible human embryoids, combined with SOX17 overexpression, may partially explain the lower level of anterior hypoblast formation in embryoids. In addition, the low NODAL environment, combined with the lack of anterior hypoblast, may prompt the differentiation of the epiblast-like cell population over time.

[0243] In the currently disclosed human embryo model with a CER1-positive hypoblast, although the CER1-positive cells were lost by day 6, a significant increase in the expression of the primitive streak-like BRACHYUYRY / TBXT was still observed. The transient presence of the anterior hypoblast-like cell population may protect the pluripotency of the epiblast-like domain for a longer time, thus allowing cells to exit pluripotency at the gastrulation-competent cell stage in the late development. These results contrast with embryoids lacking an anterior hypoblast-like cell population, which mainly generate amnion. These data suggest the possible existence of a unique intermediate pluripotent state that is capable of generating amnion and extraembryonic mesenchyme but not germ layer derivatives.

[0244] Modular generation of integrated embryoids from its components will help to explore the roles of specific tissues and tissue-specific gene requirements. However, generating extraembryonic tissues using overexpression of transcription factors may also lead to differentiation defects. For example, although GATA3-AP2Y induction drives a trophoblast-like gene program in two dimensions, after aggregation in a human embryo-like model, this cell population abnormally upregulates endodermal markers (including SOX17 and GATA6). Nevertheless, GATA3-AP2Y-induced cells are necessary for the successful organization of embryo-like structures and may serve as a key source of BMP. The initial pluripotent state is a key factor in inducing downstream gene regulatory networks. There is evidence that using peri-implantation hESCs rather than more naive hESCs can effectively form embryo models. However, given that the induction of trophoblast gene networks seems to be more robust in naive hESCs, using inconsistent pluripotent embryo models can better recapitulate embryonic development. Similarly, different combinations of transcription factors may be required for lineage-directed differentiation from different starting states. Therefore, further exploration of the epigenetic maps and binding sites of these factors may help to improve strategies for generating truly extraembryonic cells.

[0245] In summary, a modular model of post-implantation human development including embryo-like cells and extraembryo-like cells is proposed. This post-implantation embryo model can self-organize and shows axis formation in rare cases. Similar to other human integrated embryo-like models (such as blastoid), it needs to be further optimized to maximize the retention of all major lineages of the post-implantation embryo and their more complete developmental potential and embryo-like morphology. Since this model cannot implant, it cannot develop into the fetal stage and cannot simulate the developmental stages after primitive streak formation. It also does not contain all cell types of the gastrulating embryo. However, constructing these integrated models of post-implantation human embryos is an important step towards studying the post-implantation developmental mechanisms that cannot be carried out in in vivo human embryos.

[0246] Terms

[0247] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art should understand that various other omissions, additions, and modifications can be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0248] Regarding the use of almost all plural and / or singular terms herein, those skilled in the art can appropriately convert the plural to the singular and / or the singular to the plural according to the context and / or application. For clarity, various singular / plural permutations may be set forth explicitly herein. As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" also include plural referents. Any reference to "or" herein is intended to cover "and / or" unless otherwise indicated.

[0249] Those skilled in the art should understand that, generally speaking, the terms used herein, especially in the appended claims (e.g., the main body of the appended claims), are usually intended to be "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "containing" should be interpreted as "including but not limited to", etc.). Those skilled in the art should also understand that if an intention is to specifically recite a quantity in the introduced claims, such intention will be clearly stated in the claims, and if there is no such recitation, there is no such intention. For example, for the sake of understanding, the following appended claims may contain the use of introductory phrases "at least one" and "one or more" to introduce the claims. However, the use of such phrases should not be construed as implying that by introducing a claim recitation with the indefinite article "a" or "an", any particular claim containing such introduced claim recitation is limited to an embodiment containing only one such recitation, even if the same claim contains an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as "at least one" or "one or more"); the same is true for the use of the definite article to introduce a claim recitation. In addition, even if a specific quantity for introducing a claim recitation is explicitly recited, those skilled in the art will also recognize that such recitation should be interpreted as meaning at least the recited quantity (e.g., the mere recitation of "two recitations" without other modifiers means at least two recitations, or two or more of the recited recitations). In addition, in the case of using a convention similar to "at least one of A, B, and C, etc.", generally the meaning of such a structure is consistent with what those skilled in the art understand (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, A and B simultaneously, A and C simultaneously, B and C simultaneously, and / or A, B, and C simultaneously, etc.). In the case of using a convention similar to "at least one of A, B, or C, etc.", generally the meaning of such a structure is consistent with what those skilled in the art understand (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, only B, only C, A and B simultaneously, A and C simultaneously, B and C simultaneously, and / or A, B, and C simultaneously, etc.). Those skilled in the art should also understand that in fact any alternative word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as contemplating the possibility of including one of the terms, any one of the terms, or both terms.

[0250] In addition, when a feature or aspect of the present disclosure is described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any single member or subgroup of members of the Markush group.

[0251] Those skilled in the art should understand that, for any purpose (e.g., in providing a written description), all ranges disclosed herein also cover any and all possible sub-ranges, and combinations of their sub-ranges. Any listed range can be readily understood as sufficient to describe the same range and to be divisible at least into halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily subdivided into lower thirds, middle thirds, upper thirds, etc. Those skilled in the art should also understand that all language such as "up to", "at least", "greater than", "less than", etc. includes the recited numbers and refers to ranges that can then be subdivided into sub-ranges as described above. Finally, those skilled in the art should understand that a range includes each individual member. Thus, for example, a group having 1 to 3 items means a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0252] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to be limiting, and their true scope and spirit are indicated by the following claims.

Claims

1. An in vitro method for generating mammalian synthetic embryos, the method comprising: Co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene under culture medium conditions that enable the ESCs to self-organize into post-implantation embryonic structures.

2. The in vitro method according to claim 1, wherein the first modified mammalian ESCs comprise an inducible GATA6 gene, an inducible SOX17 gene, or both.

3. The in vitro method according to claim 1 or 2, wherein the first modified mammalian ESCs comprise an inducible GATA6 gene and an inducible SOX17 gene.

4. The in vitro method according to any one of claims 1-3, wherein the second modified mammalian ESCs comprise an inducible GATA3 gene, an inducible TFAP2C gene, or both.

5. The in vitro method according to any one of claims 1-4, wherein the second modified mammalian ESCs comprise an inducible GATA3 gene and an inducible TFAP2C gene.

6. The in vitro method according to any one of claims 2-5, further comprising contacting the first modified mammalian ESCs and / or the second modified mammalian ESCs with an inducer.

7. The in vitro method according to claim 6, wherein the inducer is doxycycline.

8. The in vitro method according to claim 6 or 7, wherein the inducer is supplied to the culture medium, optionally for about 1 to 7 days.

9. The in vitro method according to any one of claims 2-8, further comprising adjusting the induction intensity, optionally by increasing or decreasing the concentration of the inducer, or increasing or decreasing the duration of the inducer in the culture medium.

10. The in vitro method according to any one of claims 6-9, wherein the inducer is supplied to the culture medium throughout the co-culture process.

11. The in vitro method according to any one of claims 1-10, wherein the wild-type mammalian ESCs and / or the modified mammalian ESCs are naive ESCs or primed ESCs.

12. The in vitro method according to claim 11, wherein the wild-type mammalian ESCs and / or the modified mammalian ESCs are pre-implantation naive hESCs, peri-implantation-like pluripotent naive hESCs, or post-implantation primed hESCs.

13. The in vitro method according to claim 12, wherein the pre-implantation naive hESCs are cultured in PXGL medium before co-culture, the peri-implantation-like pluripotent hESCs are cultured in RSeT medium before co-culture, and the post-implantation-like primed hESCs are cultured in mTeSR1 medium before co-culture.

14. The in vitro method according to any one of claims 1-13, wherein the wild-type mammalian ESCs and the modified mammalian ESCs are peri-implantation-like pluripotent hESCs, optionally cultured in RSeT medium before co-culture.

15. The in vitro method according to any one of claims 1-14, wherein the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 and / or SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 and / or TFAP2C gene are provided in a ratio of about 1:1:1 to 1:1:5, optionally in a ratio of about 1:1:1 to 1:1:

2.

16. The method according to any one of claims 1-15, wherein the ESCs are cultured on a substrate, optionally wherein the substrate comprises a culture dish, a U-bottom plate, a flask, or a microplate.

17. The method according to claim 16, wherein the ESCs are cultured in inverted pyramid-shaped microwells.

18. The method according to claim 17, wherein each of the inverted pyramid-shaped microwells has a size of about 400 μm or about 800 μm, optionally a diameter of about 400 μm or about 800 μm.

19. The in vitro method according to any one of claims 1-18, wherein the co-culture comprises co-culturing the ESCs in a stem cell proliferation medium for about 5 days, optionally passaging the ESCs in the stem cell proliferation medium at least twice.

20. The in vitro method according to claim 19, wherein the stem cell proliferation medium is a serum-free medium.

21. The in vitro method according to claim 19 or 20, wherein the stem cell proliferation medium comprises Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F-12 (DMEM / F12), Neurobasal, N2, B27, L-glutamine or analogs thereof, a reducing agent, an antibiotic, or a combination thereof.

22. The in vitro method according to claim 21, wherein the reducing agent comprises β-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof.

23. The in vitro method according to any one of claims 19-22, wherein the stem cell proliferation medium is N2B27 medium.

24. The in vitro method according to claim 23, wherein the N2B27 medium comprises DMEM / F12, Neurobasal, B27, N2, GlutaMax, β-mercaptoethanol, penicillin / streptomycin, or a combination thereof.

25. The in vitro method according to claim 24, wherein the N2B27 medium comprises 1:1 DMEM / F12 and Neurobasal A, 0.5×B27, 0.5×N2, 100 μM β-mercaptoethanol, 1×GlutaMAX, and 1×penicillin-streptomycin.

26. The in vitro method according to any one of claims 19 - 25, wherein the ESC aggregates after co - culturing in the stem cell proliferation medium for up to 24 hours.

27. The in vitro method according to claim 26, wherein the aggregated ESC exhibits a distinction between an inner cell mass and an outer cell mass.

28. The in vitro method according to any one of claims 19 - 27, wherein the co - culturing comprises co - culturing the ESC in the post - implantation medium for at least 2 days after co - culturing in the stem cell proliferation medium.

29. The in vitro method according to claim 28, wherein the ESC is co - cultured in the post - implantation medium starting about 2 days after the ESC aggregates.

30. The in vitro method according to claim 28 or 29, wherein the post - implantation medium comprises Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture F - 12 (DMEM / F12), non - human serum or a serum substitute thereof, an antibiotic, an antimicrobial agent, L - glutamine or an analogue thereof, insulin, an insulin analogue or an insulin receptor agonist, an estrogen analogue or an estrogen receptor agonist, progesterone, a progesterone analogue or a progesterone receptor agonist, or any combination thereof.

31. The in vitro method according to claim 30, wherein the non-human serum or serum substitute comprises fetal bovine serum, bovine serum albumin, KnockOut TM serum substitute or any combination thereof.

32. The in vitro method according to claim 30 or 31, wherein the antibiotic comprises penicillin - streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof.

33. The in vitro method according to any one of claims 30 - 32, wherein the estrogen receptor agonist is selected from β - estradiol, estrone, estriol, estetrol, or any analogue thereof.

34. The in vitro method according to any one of claims 30 - 33, wherein the insulin receptor agonist is selected from IGF - I, IGF - II, an analogue thereof, or any combination thereof.

35. The in vitro method according to any one of claims 30 - 34, wherein the post - implantation medium comprises an antimicrobial agent, optionally the antimicrobial agent is sodium lactate.

36. The in vitro method according to any one of claims 30 - 35, wherein the post - implantation medium comprises transferrin, sodium selenite, ethanolamine, or any analogue thereof.

37. The in vitro method according to any one of claims 30 - 36, wherein the post - implantation medium comprises DMEM / F12, fetal bovine serum, GlutaMax, non - essential amino acids, essential amino acids, insulin - transferrin - selenium - ethanolamine (ITS - X), penicillin and / or streptomycin, glucose, sodium lactate, β - estradiol, progesterone, or any combination thereof.

38. The in vitro method according to any one of claims 30-36, wherein the post-implantation culture medium comprises DMEM / F12, about 20% fetal bovine serum, about 1× GlutaMax, about 1× non-essential amino acids, about 1× essential amino acids, about 1× ITS-X, about 25 U / mL penicillin and / or streptomycin, about 1.8 nM glucose, about 0.22% sodium lactate, about 8 nM β-estradiol, about 200 ng / ml progesterone, or any combination thereof.

39. The in vitro method according to any one of claims 1-37, wherein the co-culture comprises transferring the ESCs from one substrate to another substrate.

40. The in vitro method according to any one of claims 1-38, wherein the post-implantation embryonic structure comprises an inner epiblast-like domain, an outer single-layer trophoblast-like cell, and an intermediate hypoblast-like domain between the epiblast-like domain and the outer single-layer trophoblast-like cell.

41. The in vitro method according to claim 39, wherein the inner epiblast-like domain is SOX2 positive and contains a central cavity, the outer single-layer trophoblast-like cell is GATA3 positive, and the intermediate hypoblast-like domain is GATA6 positive.

42. The in vitro method according to any one of claims 1-39, wherein the post-implantation embryonic structure expresses N-cadherin and SOX17 in the hypoblast-like domain, CDX2 in the trophoblast-like cell, and / or SOX2, NANOG, and E-cadherin in the epiblast-like domain.

43. The in vitro method according to any one of claims 39-41, wherein the inner epiblast-like domain exhibits pluripotency and epithelial identity similar to that of a human embryo.

44. The in vitro method according to any one of claims 1-42, wherein the post-implantation embryonic structure comprises cell clusters similar to late embryonic epiblast, amnion, mesoderm, extraembryonic mesenchyme, and / or hypoblast / visceral endoderm.

45. The in vitro method according to any one of claims 1-43, wherein the post-implantation embryonic structure expresses TDGF1, SOX2, NANOG, TFAP2A, ID1, ISL1, TFAP2C, VTCN1, GRHL1, MEIS1, TBXT, MESP1, MIXL1, CER1, SNAI1, EOMES, POSTN, COL6A3, IGF2, TBX20, BMP6, CDH2, HNF1B, FOXA2, or a combination thereof.

46. The in vitro method according to any one of claims 1-44, wherein the post-implantation embryonic structure generates amnion and primordial germ cells.

47. The in vitro method according to any one of claims 1-45, wherein the efficiency of the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene in forming post-implantation embryos is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35% or higher.

48. The in vitro method according to any one of claims 1-46, wherein the method does not include any in vivo steps.

49. The in vitro method according to any one of claims 1-47, wherein during co-culture, the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene are all not present in an in vivo environment, and optionally wherein, The in vivo environment includes tissues, organs, organisms or combinations thereof.

50. The in vitro method according to any one of claims 1-48, wherein the method does not include culturing trophoblast stem cells, hypoblast stem cells or both alone or in combination with the ESCs.

51. The in vitro method according to any one of claims 1-49, wherein the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene are human ESCs.

52. The in vitro method according to any one of claims 1-50, wherein the post-implantation embryo structure is a human embryo structure.

53. The in vitro method according to any one of claims 1-51, wherein the post-implantation embryo structure is similar to a post-implantation human embryo about 8-9 days after fertilization.

54. The in vitro method according to any one of claims 1-52, wherein the method does not include using exogenous signal transduction pathway factors, and optionally the culture medium does not contain or provide exogenous signal transduction pathway factors.

55. The in vitro method according to claim 53, wherein the exogenous signal factors include a WNT signal transduction pathway activator, a TGFβ superfamily member or both.

56. A synthetic embryo obtained by the method according to any one of claims 1-55.

57. The synthetic embryo according to claim 56, wherein the synthetic embryo is a human embryo, and optionally the synthetic embryo is similar to a post-implantation human embryo about 8-9 days after fertilization.

58. A method for studying mechanisms involved in embryogenesis, comprising the method according to any one of claims 1 to 55.

59. A method for identifying a compound useful for treating a disease, comprising contacting a synthetic embryo obtained by the in vitro method according to any one of claims 1-55 with the compound.

60. A method for diagnosing or treating a disease or disorder of a subject, comprising: generating a synthetic embryo by the method according to any one of claims 1-55; and transplanting the synthetic embryo into the subject.

61. The method according to claim 59, wherein the wild-type mammalian ESCs and the modified mammalian ESCs are obtained from the subject or are derived from ESCs obtained from the subject.

62. A method for elucidating the role of a candidate gene in embryonic development, comprising: Obtaining wild-type mammalian ESCs, first-modified mammalian ESCs comprising the GATA6 gene and / or the SOX17 gene, and second-modified mammalian ESCs comprising the GATA3 gene and / or the TFAP2C gene, wherein the candidate gene has been modified or knocked out; And culturing the mammalian ESCs using the in vitro method according to any one of claims 1-55.

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