Methods and compositions for in vitro embryonic development from pluripotent stem cells

Abstract

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

Description

Related Applications

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63 / 403,684, filed September 2, 2022, and U.S. Provisional Patent Application No. 63 / 457,670, filed April 6, 2023, the contents of which related applications are incorporated herein by reference in their entirety for all purposes.

[0002] Array list reference This application is submitted in electronic format with a Sequence Listing. The Sequence Listing is provided as file entitled 30KJ-302455-WO-SeqList, created on August 24, 2023, and is 33 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. background Field

[0003] The present disclosure relates generally to the field of cell culture, and more particularly to embryonic and stem cell culture. 2. Description of Related Art

[0004] Mammalian embryos, particularly human embryos, undergo morphogenetic changes after implantation in the uterus, but our knowledge of this critical stage is limited by the inability to observe embryos in vivo. Embryonic stem cell-derived models are important tools for investigating developmental events and tissue interactions during these stages. Summary of the Invention

[0005] Disclosed herein are in vitro methods for generating mammalian synthetic embryos. The methods, in some embodiments, include co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising a GATA6 gene and / or a SOX17 gene, and second modified mammalian ESCs comprising a GATA3 gene and / or a TFAP2C gene in a culture medium under conditions that allow 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.

[0006] In some embodiments, the method can further include contacting the first modified mammalian ESCs and / or the second modified mammalian ESCs with an inducer. The inducer can be, for example, doxycycline. In some embodiments, the inducer is provided in the culture medium, optionally for a period of about 1-7 days. In some embodiments, the method can further include adjusting the intensity of induction by, optionally, 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 provided in the culture medium throughout the entire co-culture process. In some embodiments, the wild-type mammalian ESCs and / or modified mammalian ESCs are naive ESCs or primed ESCs. In some embodiments, the wild-type mammalian ESCs and / or modified mammalian ESCs are pre-implantation naive hESCs, peri-implantation-like pluripotent naive hESCs, or post-implantation primed hESCs. In some embodiments, pre-implantation naive hESCs are cultured in PXGL medium prior to co-culture, pre- and post-implantation-like pluripotent hESCs are cultured in RSeT medium prior to co-culture, and post-implantation-like primed hESCs are cultured in mTeSRl medium prior to co-culture. In some embodiments, the wild-type mammalian ESCs and modified mammalian ESCs are pre- and post-implantation-like pluripotent hESCs, optionally cultured in RSeT medium prior to co-culture. In some embodiments, 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 at a ratio of about 1:1:1 to 1:1:5, optionally at a ratio of about 1:1:1 to 1:1:2. In some embodiments, the ESCs are cultured on a substrate, optionally comprising a dish, U-plate, flask, or microwell plate. In some embodiments, ESCs are cultured in inverted pyramidal microwells. In some embodiments, one or more, or each, of the inverted pyramidal microwells is about 400 μm or about 800 μm in size, optionally about 400 μm or about 800 μm in diameter.In some embodiments, the co-culturing comprises co-culturing the ESCs in stem cell growth medium for about 5 days, optionally passaged at least twice in the stem cell growth medium. In some embodiments, the stem cell growth medium is serum-free. In some embodiments, the stem cell growth medium comprises Dulbecco's Modified Eagle Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), neurobasal, N2, B27, L-glutamine or an analog thereof, a reducing agent, an antibiotic, or a combination thereof. The reducing agent can be or include beta-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof.

[0007] In some embodiments, the stem cell growth medium is N2B27 medium. In some embodiments, N2B27 medium comprises DMEM / F12, Neurobasal, B27, N2, GlutaMax, β-mercaptoethanol, penicillin / streptomycin, or a combination thereof. In some embodiments, 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.

[0008] In some embodiments, the ESCs aggregate after co-culturing in stem cell expansion medium for up to 24 hours. In some embodiments, the aggregated ESCs exhibit differentiation between inner and outer cell domains. In some embodiments, the co-culturing comprises co-culturing the ESCs in post-transplantation medium for at least two days following co-culturing in stem cell expansion medium. In some embodiments, co-culturing the ESCs in post-transplantation medium begins about two days after aggregation of the ESCs. In some embodiments, the post-implantation culture medium comprises Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or serum substitute, antibiotic, antimicrobial, L-glutamine or analog thereof, insulin, insulin analog, or insulin receptor agonist, estrogen analog, or estrogen receptor agonist, progesterone, progesterone analog, or 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 (knock out, TM (represents a trademark designation in the U.S. and elsewhere) Serum Replacement, or any combination thereof. In some embodiments, the antibiotic includes penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tyrosine, or any combination thereof. The estrogen receptor agonist can be or include, for example, β-estradiol, estrone, estriol, and estetrol, or any analog thereof.

[0009] In some embodiments, the insulin receptor agonist is selected from the group consisting of IGF-I, IGF-II, analogs thereof, or any combination thereof. In some embodiments, the post-implantation culture medium comprises an antimicrobial agent, and optionally the antimicrobial agent is sodium lactate. In some embodiments, the post-implantation culture medium comprises transferrin, sodium selenium, ethanolamine, or any analog thereof. In some embodiments, the post-implantation culture 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-estrodiol, progesterone, or any combination thereof.

[0010] In some embodiments, 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-estrodiol, about 200 ng / mL progesterone, or any combination thereof. In some embodiments, the co-culturing comprises transferring ESCs from one substrate to another. In some embodiments, the post-implantation embryo structure comprises an inner epiblast-like domain, a single outer layer of trophoblast-like cells, and an intermediate hypoblast-like domain between the epiblast-like domain and the single outer layer of trophoblast-like cells. In some embodiments, the inner epiblast-like domain is SOX2-positive and contains a central lumen, a single outer layer of trophoblast-like cells is GATA3-positive, and the intermediate hypoblast-like domain is GATA6-positive. In some embodiments, the postimplantation embryo 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 human embryos.

[0011] In some embodiments, the post-implantation embryonic structure comprises cell clusters resembling embryonic late-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 combinations thereof. In some embodiments, the post-implantation embryonic structure produces amnion and primordial germ cells. In some embodiments, the efficiency of forming post-implantation embryos from wild-type mammalian ESCs, first modified mammalian ESCs comprising a GATA6 gene and / or a SOX17 gene, and second modified mammalian ESCs comprising a GATA3 gene and / or a TFAP2C gene is 5%, 10%, 15%, 20%, 25%, 30%, 35% or higher.

[0012] In some embodiments, the method does not include any in vivo steps. In some embodiments, none of the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or TFAP2C gene are present in an in vivo environment during co-culture, and optionally the in vivo environment includes a tissue, organ, organism, or combination thereof. In some embodiments, the method does not include culturing trophoblast stem cells, hypoblast stem cells, or both, alone or in combination with ESCs. In some embodiments, the wild-type mammalian ESCs, the first modified mammalian ESCs comprising the GATA6 gene and / or SOX17 gene, and the second modified mammalian ESCs comprising the GATA3 gene and / or TFAP2C gene are human ESCs. In some embodiments, the post-implantation embryo structure is a human embryo structure. In some embodiments, the post-implantation embryo structure resembles a post-implantation human embryo at about 8-9 days post-fertilization. In some embodiments, the methods do not include the use of exogenous signaling pathway factors, and optionally the culture medium does not include exogenous signaling pathway factors or is provided with exogenous signaling pathway factors, hi some embodiments, the exogenous signaling pathway factors include a WNT signaling pathway activator, a TGFβ superfamily member, or both.

[0013] Disclosed herein is a synthetic embryo obtained by any of the methods disclosed herein. In some embodiments, the synthetic embryo is a human embryo, and optionally, the synthetic embryo resembles a post-implantation human embryo at about day 8-9 post-fertilization.

[0014] Also disclosed herein is a method for investigating mechanisms involved in embryogenesis, comprising any of the methods disclosed herein. Disclosed herein is a method for identifying a compound useful for treating disease, comprising contacting a synthetic embryo obtained by any of the in vitro methods disclosed herein with the compound.

[0015] Also disclosed herein are methods of diagnosing or treating a disease or disorder in a subject. The methods, in some embodiments, include generating a synthetic embryo according to any of the methods disclosed herein; and implanting the synthetic embryo into a subject. In some embodiments, the wild-type mammalian ESCs and modified mammalian ESCs are obtained from the subject or derived from ESCs obtained from the subject.

[0016] Also disclosed herein is a method for elucidating the role of a candidate gene in embryonic development, comprising obtaining wild-type mammalian ESCs, first modified mammalian ESCs comprising a GATA6 gene and / or a SOX17 gene, and second modified mammalian ESCs comprising a GATA3 gene and / or a TFAP2C gene, wherein the candidate gene is modified or knocked out; and culturing the mammalian ESCs using any of the in vitro methods disclosed herein. [Brief explanation of the drawings]

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with this drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] [Figure 1]Figures 1A-1F depict non-limiting exemplary embodiments and data related to validation of extraembryonic-like induction. Figure 1A depicts the generation of inducible GATA6 (iG6) and / or SOX17 (iS17) hESCs and validation 24 hours after doxycycline addition in basal N2B27. iG6 (n=551), iS17 (n=550), and iG6-S17 (n=707) cells from three independent experiments. Figure 1B depicts the generation of inducible GATA3 (iG3) and / or AP2Y (iAP2Y) hESCs and validation 24 hours after doxycycline addition in basal N2B27. iG3 (n=1456), iAP2Y (n=1456), and iG3-AP2Y (n=782) cells from three independent experiments. Figure 1C depicts Uniform Manifold Approximation and Projection (UMAP)-based dimensionality reduction of sequenced wild-type (RSeT WT), inducible GATA6-SOX17 (day 3 iG6-S17), and inducible GATA3-AP2Y (day 3 iG3-AP2Y) RSeT hESCs 3 days after doxycycline induction. Figure 1D depicts logistic regression analysis and comparison of the cells with a human postimplantation embryonic population. Human embryo data from a previous report were used as training data, and cell line data were used as testing data. Figure 1E depicts selected differentially expressed genes from RNA sequencing (left) and predicted differential motif accessibility (right) from ATAC sequencing scored by chromVAR for wild-type, inducible GATA6-SOX17, and inducible GATA3-AP2Y RSeT hESCs 3 days after doxycycline induction. Figure 1F depicts validation of 2D coculture of wild-type, inducible GATA6-SOX17, and inducible GATA3-AP2Y RSeT hESCs (N = 3 independent experiments). Scale bars in Figure 1A, Figure 1B, and Figure 1F = 100 μm.In Figures 1A-1B, the mean ± SEM is plotted. [Figure 2]Figures 2A-2H depict non-limiting exemplary embodiments and data related to the generation of induced post-implantation human embryoid bodies. Figure 2A outlines the protocol for generating induced human embryoid bodies by combining wild-type RSeT hESCs with inducible GATA6-SOX17 (iG6-S17) and GATA3-AP2Y (iG3-AP2Y) cells. Extraembryonic-like cells were induced for 3 days prior to aggregation on day 0. Figure 2B shows that well-defined self-organization was demonstrated 96 hours after aggregation. Figure 2C depicts the size of cell aggregates between days 1 and 3 after aggregation. Structures at day 1 (n=175), day 2 (n=171), and day 3 (n=91) were from five independent experiments. The lengths of all individual embryoid bodies are plotted. Each symbol (orange cross, orange triangle, green triangle, blue circle, purple square) represents an independent experiment. Figure 2D depicts quantification of embryoid body formation across the initial pluripotency state. RSeT (n = 952), mTeSR (n = 30), and PXGL (n = 207) constructs from five independent experiments. Statistical analysis using one-way ANOVA with Holm-Sidak's multiple comparison test revealed a significant difference between RSeT and mTeSR (p = 0.0347) and between RSeT and PXGL (p = 0.0283). Unmarked pairwise comparisons were not significant (ns; > 0.05). Figure 2E depicts quantification of the proportion of cell types in correctly organized embryoid bodies. N = 16 embryoid bodies from three independent experiments. Figure 2F depicts a representative image of in vitro cultured human embryos at day 9 post-fertilization, showing a distinct lumenalized SOX2 domain surrounded by a layer of GATA6-positive cells. A subset of GATA6-positive cells expressed the anterior hypoblast marker CER1. Images are representative of three independent experiments. Figure 2G depicts a hypoblast-like domain expressing N-cadherin, SOX17, and GATA4, and an epiblast-like domain that maintained expression of the pluripotency factors SOX2, OCT4, and NANOG. GFP-expressing cells derived from inducible GATA3-AP2Y showed clear lateral localization. Images are representative of two experiments.Figure 2H depicts induced human embryoid bodies demonstrating clear apical-basal polarity, along with quantification of induced human organization. In the top right panel of Figure 2H, 1 = cavity, 2 = ECM, and 3 = SOX2 + cavity + ECM. n = 506 structures were from three independent experiments for cavity and ECM efficiency. n = 27 embryoid bodies were from two independent experiments for cavity number. In Figures 2A, 2B, and 2F-H, scale bars = 100 μM. * indicates P < 0.05. In Figure 2C, the lengths of all individual embryoid bodies are plotted. For Figures 2D-E and 2H, mean ± SEM is plotted. The inner domain of the embryoid body is outlined by a dashed line. [Figure 3]Figures 3A-3I depict non-limiting exemplary embodiments and data related to the differentiation of extraembryonic mesenchyme, amnion, and primordial germ cells. Figure 3A depicts a schematic of the inducible human embryoid body expansion culture protocol and sampling for combined single-cell RNA and single-cell ATAC sequencing using a 10x platform. Twelve embryoid bodies were sequenced at 4, 6, and 8 days post-aggregation, respectively. Figure 3B depicts cell annotation based on transcriptional projection onto diverse human and non-human primate embryo datasets using scmap in combination with RNA and chromatin velocity. Figure 3C depicts differentially expressed genes selected in RNA sequencing data (top) and predicted differentially accessible motifs scored by chromVAR on cluster-wide ATAC sequencing data (bottom). Figure 3D depicts induced human embryoid bodies in which SOX2 was downregulated at day 6 and CDX2, ISL1, and VTCN1 were upregulated at day 8, indicating robust amniotic differentiation and maturation. In some rare cases, dorsoventral and / or anterior-posterior patterns were observed. Images are representative of three experiments. Figure 3E depicts module scoring for primordial germ cell marker genes. Figure 3F depicts a Nebulosa plot visualizing the joint expression density of key primordial germ cell genes in induced human embryoid bodies. Figure 3G depicts a heat map of selected primordial germ cell gene expression across clusters. Figure 3H depicts quantification of SOX17 / NANOG / AP2Y triple-positive (+) cells at day 4 (n = 10 embryoid bodies) and day 6 (n = 10 embryoid bodies). n = 2 independent experiments were performed. Figure 3I depicts immunofluorescence identification of SOX17 / NANOG / AP2Y triple-positive primordial germ cell-like cells in induced human embryoid bodies, highlighted by arrowheads. Mean ± SEM is plotted. Scale bar = 100 μm. The inner domain of the embryoid body is enclosed by a dashed line. [Figure 4]Figures 4A-4H depict non-limiting exemplary embodiments and data related to BMP signaling driving amnion specification in induced human embryoid bodies. Figure 4A depicts expression of ID1-4, downstream targets of BMP signaling, in embryoid bodies. Figure 4B depicts 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- and GATA6-positive cells from representative induced human embryoid bodies at day 4 (n=60 cells each, <0.0001) and day 6 (n=40 cells each, <0.0001) from N=3 independent experiments. Figure 4D depicts representative images and quantification of SMAD2.3 in OCT4- and GATA6-positive cells from representative induced human embryoid bodies at 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 demonstrates that inhibition of BMP signaling blocks the transition from pluripotency and the upregulation of amniotic markers AP2a and CDX2. Figure 4F depicts quantification of the percentage of the medial domain at day 4 expressing SOX2 and CDX2 (control: n = 147; LDN-treated: n = 126; BMP4-treated: n = 57; and Act-A-treated: n = 60 embryoid bodies from five independent experiments). For the SOX2+ / CDX2 domain, control vs. LDN P = 0.0002, control vs. Act-A P = 0.0433, and control vs. BMP4 P = 0.1753. Figure 4G demonstrates that BMP suppression reduces the number of primordial germ cell-like cells in embryoid bodies. Figure 4H depicts quantification of the number of SOX17 / NANOG / AP2Y triple-positive primordial germ cell-like cells (PGCLCs) at day 4 (control: n = 45; LDN-treated: n = 30; BMP4-treated: n = 48; and Act-A-treated: n = 36 embryoid bodies from six independent experiments). Control vs. LDN P = 0.0011. Control vs. BMP4 P > 0.99. Control vs. Act-A P = 0.98. Comparisons with controls are not marked and are therefore ns.Statistics used in Figures 4C-4D were two-tailed Mann-Whitney. In Figure 4F, it was RM two-way ANOVA with Holm-Sidak's multiple comparison test. In Figure 4H, it was Kruskal-Wallis with Dunn's multiple comparison test. Scale bars = 100 μm. Mean ± SEM is plotted in Figures 4C-4D and 4F. For Figure 4H, boxes enclose the 25th-75th quartiles with whiskers for minimum and maximum values. The center line indicates the median, and + symbols indicate the mean. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The inner domain of the embryoid bodies is enclosed by a dashed line. [Figure 5]Figures 5A-5H depict non-limiting exemplary embodiments and data related to the antagonistic effects 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 regulon activity as scored by SCENIC, and the co-expression of SOX17 and CER1 in human hypoblasts after implantation (days 9-11 post-fertilization). Data were from a previous report. Figure 5C shows a representative example of an embryoid body exhibiting CER1-positive cells generated using induced GATA6 (iG6), but not induced GATA6-SOX17 (iG6-S17) dual induction of hypoblast-like cells. CER1 expression was suppressed when doxycycline was discontinued on day 3 post-aggregation. Figure 5D depicts representative images from day 4, demonstrating a decrease in pSMAD1.5 in the epiblast-like domains of structures with CER1-positive cell populations. Figure 5E depicts the percentage of embryoid bodies from Figure 5C expressing CER1. iG6-S17: n = 78; iG6: n = 25; iS17: n = 26; iG6-S17-Dox. Day 1: n = 54; and iG6-S17-Dox. Day 3: n = 65. Embryoid bodies were from seven independent experiments. iG6-S17 vs. iG6 P < 0.0001. iG6-S17 vs. iG6-S17-Dox. Day 3 P < 0.001. iG6-S17 vs. iS17 P = 0.87. iG6-S17 vs. iG6-S17-Dox. Day 1 P = 0.87. Figure 5F depicts quantification of pSMAD1.5 levels in SOX2-positive cells in CER1-negative (CER1-) versus CER1-positive (CER1+) iG6-S17 embryoid bodies at day 4. CER1-: n = 108 cells; and CER1+: n = 123 cells from eight embryoid bodies each from two independent experiments. P < 0.0001 in Figure 5F. Figure 5G depicts quantification of Brachyury expression in Figure 5D. Figure 5H depicts representative images of BRY / TBXT expression in induced human embryoid bodies generated by iG6, iS17, or iG6-S17 cells (doxycycline maintained and 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; and iG6-S17-Dox Day 3: n = 20 embryoid bodies from six independent experiments. iG6-S17 vs. iG6 = 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. Statistics used in Figure 5E and Figure 5G were RM two-way ANOVA with Holm-Sidak multiple comparisons test; in Figure 5F, two-way Mann-Whitney test. Scale bar = 100 μm. In Figures 5E, 5F and 5G, mean ± SEM is plotted. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The inner domain of the embryoid bodies is circled by a dashed line. [Figure 6]Figures 6A-6H depict non-limiting exemplary embodiments and data related to the selection of transgenes to drive extraembryonic-like cells. Figure 6A depicts a uniform manifold approximation projection (UMAP) showing the combined human pre- and post-implantation datasets, color-coded according to the original publication. Figure 6B depicts the UMAP of the combined human datasets, color-coded according to embryonic stage (dpf stands for days post-fertilization). Figure 6C depicts the UMAP of the combined human datasets, color-coded according to cell type. Figure 6D depicts key cell type gene expression on the UMAP of the human datasets. Figure 6E depicts a plot from single-cell RNA sequencing of key marker gene expression in the human datasets separated by cell type (n=10,223 cells). Figure 6F depicts the inferred epiblast, hypoblast, and trophoblast gene regulatory networks generated by SCENIC during pre- and post-implantation human embryo development. Candidate factors are marked with boxes (TFAP2C, GATA3, GATA6, and SOX17). Figure 6G depicts regulon activity scored by SCENIC for the hypoblast markers GATA6 and SOX17, and the TrB markers GATA3 and TFAP2C (n = 10,223 cells). Figure 6H depicts qRT-PCR analysis of individual inducible cell lines. Doxycycline-inducible constructs were inserted in Shef6 hESCs using piggybac transposase (inducible GATA6, SOX17, GATA6-SOX17, GATA3, AP2Y, GATA3-AP2Y). After single-cell plating, colonies were manually isolated and expanded. Appropriate transgene expression was verified by RT-qPCR 72 hours after the addition of 1 μg / mL doxycycline in basal N2B27 conditions. N = 3 technical replicates were included. Clones selected for further analysis were marked with a box. For box plots, the box encloses the 25th-75th quartiles with whiskers for the minimum and maximum values, and the middle line marks the median. [Figure 7]Figures 7A-7F depict non-limiting exemplary embodiments and data relating to immunofluorescence analysis of key marker genes in the hypoblast and trophoblast after doxycycline induction across pluripotent states. Figure 7A depicts qRT-PCR analysis of induced GATA6 (iG6), induced SOX17 (iS17), or induced GATA6-SOX17 (iG6-S17) from three pluripotent states, both alone and simultaneously, after 3 days of doxycycline induction. Figure 7B depicts qRT-PCR analysis of induced GATA3 (iG3), induced AP2Y (iAY), or induced GATA3-AP2Y (iG3-AY) from various pluripotent starting states, 3 days after DOX induction. For Figures 7A-7B, n=3 technical replicates were from three independent experiments. Figure 7C depicts immunofluorescence analysis of iG6, iS17, or iG6-S17 cells after 3 days of induction from various pluripotent states. Figure 7D depicts quantification of the immunofluorescence levels of Figure 7C. Figure 7E depicts immunofluorescence analysis of iG3, iAY, or iG3-AY after 3 days of induction from various pluripotent states. Figure 7F depicts quantification of the immunofluorescence levels of Figure 7E. For Figures 7C-7F, n=3 technical replicates were from two independent experiments. Cells were initially cultured in either mTeSR, RSeT, or PXGL conditions and subsequently cultured under the same conditions for 3 days, or alternatively, transferred to either basal N2B27 medium or basal N2B27 medium supplemented with doxycycline. Induced transgenes are marked with boxes. Scale bar = 100 μm. [Figure 8]Figures 8A-8D depict non-limiting exemplary embodiments and data relating to a comparison of transcription factor-mediated induction with published directed differentiation methods. Figure 8A depicts the comparison and quantification of GATA6, SOX17, and SOX2 following yolk sac-like cell (activin-A, CHIR99021, and LIF) directed differentiation, doxycycline-mediated induction in inducible GATA6-SOX17 cells, or both. Cells were differentiated from RSeT conditions. The figure legend in the bottom panel of Figure 8A is the same as in Figure 8B. Figure 8B depicts the comparison and quantification of EOMES, N-cadherin, and OTX2 following yolk sac-like cell (activin-A, CHIR99021, and LIF) directed differentiation, doxycycline-mediated induction in inducible GATA6-SOX17 cells, or both. In Figure 8A-8B, N2B27: n = 717; ACL: n = 1211; N2B27+Dox: n = 522; and ACL+Dox: n = 544 cells were from three fields obtained from two independent experiments. Figure 8C depicts the comparison and quantification of GATA3, AP2a, and SOX2 expression after PA (PD0325901 and A83-01) or PAL (PD0325901, A83-01, and LPA) directed differentiation, doxycycline-mediated induction in inducible GATA3-AP2Y cells, or both. The figure legend for the bottom panel of Figure 8C is the same as for Figure 8D. Figure 8D depicts the comparison and quantification of GATA2, KRT7, and AP2Y following PA (PD0325901 and A83-01) or PAL (PD0325901, A83-01, and LPA) directed differentiation, induction with doxycycline in inducible GATA3-AP2Y RseT cells, or both. In Figures 8C-D, N2B27: n = 443; PA: n = 487; PAL: n = 371; N2B27+Dox.: n = 357; PA+Dox.: n = 412; and PAL+Dox.: n = 287 cells were from three fields obtained from two independent experiments. Scale bar = 100 μm. In Figures 8A-D, mean ± SEM is plotted. Differentiation was performed on hESCs in RSeT conditions. [Figure 9]Figures 9A-9G depict non-limiting exemplary embodiments and data related to the evaluation of extraembryonic-like induction from RseT cells. Figure 9A depicts a quality control plot of cell line 10x multi-ome sequencing data (n=5328 cells). The violin plot is from minimum to maximum. Figure 9B depicts a logistic regression framework for assessing similarity between clusters, which was applied to cell line RNA sequencing data using published in vitro blastoid and directed differentiation protocols as training data. Figure 9C depicts a uniform manifold projection and approximation (UMAP) visualization of gene expression of selected genes 3 days after doxycycline induction from sequencing of wild-type, inducible GATA6-SOX17 (iG6-S17), and inducible GATA3-AP2Y (iG3-AP2Y) RSeT hESC populations. A visualization of sample distribution in the UMAP is shown in Figure 1C. Figure 9D depicts an immunofluorescence image of a late blastocyst-stage human cell-mouse embryo chimera, showing that iG6-S17 induction shifts human cells marked by human nuclear antigen (HuNAg) contributing to the SOX2-positive epiblast to SOX17-positive primitive endoderm. Figure 9E depicts quantification of the contribution of HuNAg-positive cells stained for SOX2 and SOX17. Control: n = 12 and iG6-S17: n = 30 embryos from three independent experiments. Figure 9F depicts an immunofluorescence image of a late blastocyst-stage human cell-mouse embryo chimera, showing that iG3-AP2Y induction shifts human cells from the SOX2-positive epiblast to GATA3-positive trophectoderm. Figure 9G depicts quantification of the contribution of HuNAg-positive cells stained for SOX2 and GATA3. Control: n = 10 and iG6-AP2Y: n = 27 embryos from three independent experiments. Derivation was performed from hESCs in RSeT conditions. For box plots, the 25th-75th quartiles are surrounded by whiskers for the minimum and maximum values ​​and a center line marking the median. Scale bar = 100 μm. [Figure 10]Figures 10A-10F depict non-limiting exemplary embodiments and data related to the identification of human embryoid model clusters after transplantation. Figure 10A depicts day 4 embryoid bodies generated from a second hESC line, RUES2. N=371 structures were from two independent experiments. Figure 10B depicts brightfield images of induced human embryoid bodies selected for sequencing at days 4, 6, and 8 (n=12 at each stage). Note the presence of an inner domain surrounded by two concentric domains. Figure 10C depicts quality control plots for embryoid body sequencing data at days 4, 6, and 8 after aggregation (n=5217 cells). Violin plots are from minimum to maximum. Figure 10D depicts scmap projections of induced human embryoid cells into cynomolgus macaques (M. fascicularis) and human datasets (H. sapien) spanning from around implantation through gastrulation. Figure 10E depicts key marker gene expression for epiblast, endoderm, and mesoderm, as well as trophoblast and amnion within the stem cell-derived model. Figure 10F depicts an alluvial plot showing the contribution of day 4, 6, or 8 embryoid bodies to assigned cell types. Scale bar = 100 μm. The inner domain of the embryoid body is outlined by a dashed line. [Figure 11]Figures 11A-11D depict non-limiting exemplary embodiments and data relating to a comparison of embryoid body clusters to human and cynomolgus monkey datasets. Figure 11A depicts a logistic regression analysis comparing annotated clusters (training data) from cynomolgus macaque (M. fasicularis) and human (H. sapiens) datasets spanning pre- and post-implantation through gastrulation to post-implantation human embryoid model clusters (test data). The cynomolgus monkey data is from a previous report. Figure 11B depicts a logistic regression analysis comparing an in vitro human embryoid model and directed differentiation dataset (training data) to induced human embryoid bodies (test data). Figure 11C depicts an SCMAP projection of the human induced embryoid body dataset onto an in vitro dataset. The in vitro datasets were from three previous reports. Figure 11D depicts a violin plot of gene expression in GFP-negative and GFP-positive cells from induced GATA3-AP2Y (iG3-AP2Y) cells from an induced human embryoid body sequencing dataset (n = 5217 cells). The violin plot is from minimum to maximum. [Figure 12]Figures 12A-12J depict non-limiting exemplary embodiments and data regarding the trajectory of extraembryonic mesenchyme and the differentiation potential of wild-type cells. Figure 12A depicts immunofluorescence of HAND1, demonstrating expression in GATA6-positive cells (putative extraembryonic mesenchyme) and upregulation in putative amniotes (AP2Y-positive) between days 4 and 6. Images are representative of two experiments. Figure 12B depicts HAND1 expression in an induced human embryoid body single-cell sequencing dataset. Figure 12C depicts immunofluorescence of TBX20, demonstrating high expression in a subset of GATA6-positive cells (putative extraembryonic mesenchyme). Images are representative of five experiments. Figure 12D depicts TBX20 expression in an induced human embryoid body single-cell sequencing dataset, demonstrating enrichment of extraembryonic mesenchyme clusters. Figure 12E depicts the differentiation of ISL1-positive amniotic membranes and GATA6 / TBX20-positive extraembryonic mesenchyme in structures derived from a second cell background, RUES2. Images are representative of two experiments. Figure 12F depicts the differentiation of primordial germ cell-like cells in embryoid bodies derived from a second cell background, RUES2. Images are representative of two experiments. Figure 12G depicts examples and quantification of day 4 embryoid bodies. Embryoid bodies displayed 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 composed primarily of unlabeled induced GATA6-SOX17 (iG6-S17) cells. N = 9 embryoid bodies were from two independent experiments. Figure 12H depicts ISL1-positive amniotic membranes overlapping with mKate2-positive wild-type cells. Images are representative of three experiments. Figure 12I depicts the expression of GATA6- and TBX20-positive extraembryonic mesenchyme-like cells overlapping with mKate2-positive wild-type cells. Images are representative of three experiments. Figure 12J depicts the expression of AP2Y-, SOX17-, and NANOG-triple-positive primordial germ cell-like cells overlapping with mKate2-positive wild-type cells. Images are representative of three experiments. Scale bar = 100 μm. In Figure 12G, mean ± SEM is plotted. The inner domain of the embryoid body is surrounded by a dashed line. [Figure 13]Figures 13A-13H depict non-limiting exemplary embodiments and data related to the role of BMPs and inducible GATA3-AP2Y cells in the generation of induced human embryoid bodies. Figure 13A depicts the expression of ID1-4 fits over incubation time, color-coded by cell type assignment. Figure 13B depicts motif accessibility scored by chromVAR for SMAD5 and SMAD2::SMAD3::SMAD4 fits over incubation time, color-coded by cell type assignment. Figure 13C depicts predicted ligand-receptor pairing in induced human embryoid bodies generated by CellPhoneDB. Figure 13D depicts the predicted interaction of inducible GATA6-SOX17 (G6-S17) cells and inducible GATA3-AP2Y (G3-AP2Y) cells 3 days after induction with wild-type RSeT hESCs, which are the cell types aggregated to generate induced human embryoid bodies. Figure 13E depicts the failure of induced human embryoid bodies to form when induced GATA3-AP2Y cells were omitted or when the BMP signaling antagonist LDN193189 (LDN) was added between days 0 and 2. Figure 13F depicts a quantification of the embryoid body formation efficiency in Figure 13E. N = 535 ESC + iG6-S17 and 500 LDN-treated constructs were from four independent experiments. Figure 13G depicts a quantification of embryoid body size after the addition of LDN193189 between days 0 and 2. N = 105 constructs per condition on each day were from three independent experiments. The statistical test used was a two-tailed Mann-Whitney test between control and LDN at each time point (day 1 P < 0.0001, day 2 P = 0.0019, day 3 P < 0.0001). Figure 13H depicts an overview of whole Aggrewells demonstrating the effects of BMP inhibition, BMP4 addition, and NODAL activation. Images are representative of five experiments. A significant increase in highly organized structures expressing SOX2 is noted after BMP inhibition. Scale bar = 100 μm. **P<0.01. ****P<0.0001. In Figure 13F, mean ± SEM is plotted. In Figure 13G, all individual data points are plotted. [Figure 14] Figures 14A-14C depict non-limiting exemplary embodiments and data related to the downregulation of CER1 expression during long-term culture of induced human embryoid bodies. Figure 14A depicts the formation efficiency of embryoid bodies generated under different conditions. Note that the highest efficiency was achieved using the standard conditions described here, consistent addition of doxycycline, and the use of GATA6-SOX17-induced cells (iG6-S17). iG6-S17: n = 224; induced GATA6 (iG6): n = 276; induced SOX17 (iS17): n = 247; iG6-S17 with doxycycline removed on day 1: n = 370; iG6-S17 with doxycycline removed on day 3: n = 410. Structures were from two independent experiments. Figure 14B depicts immunofluorescence of CER1 and SOX2 at day 6 after aggregation, demonstrating downregulation of both SOX2 and CER1 at this stage in structures generated by both wild-type ESCs and inducible GATA3-AP2Y (iG3-AP2Y) cells, along with inducible iG6 or iG6-S17 hypoblast-like cells (with consistent addition or early removal of doxycycline at day 3 after aggregation). Images are representative of three experiments. Figure 14C depicts embryoid bodies generated by Shef6-mKate2 ESCs, demonstrating differentiation of both ISL1- and BRY-positive cell populations from wild-type cells in structures generated by either iG6 or iG6-S17 hypoblast-like cells with early removal of doxycycline at day 3 after aggregation. In Figure 14C, the images depict, from top to bottom, DAPI, mKate2 (WT ESC), ISL1, BRY, and a merged view of mKate2, ISL1, BRY. Images are representative of three experiments. Scale bar = 100 μm. The inner domain of the embryoid body is enclosed by a dashed line. Detailed Description

[0019] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically clearly identify like components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as broadly described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and made a part of this disclosure.

[0020] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art. definition

[0021] Unless defined otherwise, 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 belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined as follows:

[0022] As used herein, the term "about" when referring to a measurable value such as the amount of a compound, dosage, time, temperature, etc., is meant to encompass variations of 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0023] The term "stem cells" as used herein can refer to cells that can retain a certain degree of 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; somatic stem cells and pluripotent stem cells present in tissues of living organs, such as liver stem cells, dermal stem cells, and germline stem cells that form the basis of each tissue; pluripotent stem cells derived from germline stem cells; pluripotent stem cells obtained by reprogramming the nucleus of somatic cells; totipotent stem cells and non-totipotent stem cells, and the like.

[0024] As used herein, the term "pluripotent stem cells" (PSCs) refers to stem cells that can be cultured in vitro and have the potential to differentiate into all cells except the placenta. Pluripotent stem cells have the potential to differentiate into any of three germ layers: endoderm (which forms structures such as the digestive tract and respiratory system), mesoderm (which forms structures such as the musculoskeletal system, vascular system, urogenital system, etc.), or ectoderm (which forms epidermal tissue and the nervous system).

[0025] As used herein, the term "embryonic stem cells" (ES cells or ESCs) refers to pluripotent stem cells derived from the inner cell mass of a blastocyst, an early preimplantation embryo. Such cells are expected to express genes involved in the naive pluripotency network (e.g., Oct4 / Nanog, Sox2, Klf4, etc.). Such cells may also possess Oct4 proximal enhancer activity. They may contribute to all embryonic tissues of a chimera. ES cells can be derived from mammalian embryos, derived from iPS cells, or derived from appropriate cell lines. Non-limiting examples of such stem cells include embryonic stem cells of mammals or other species established by culturing early preimplantation embryos, embryonic stem cells established by culturing early embryos prepared by nuclear transfer of somatic cell nuclei, induced pluripotent stem cells (iPS cells) established by transferring several different transcription factors into somatic cells, and pluripotent stem cells prepared by modifying genes on the chromosomes of embryonic stem cells or iPS cells using genetic engineering techniques. More specifically, embryonic stem cells include embryonic stem cells established from the inner cell mass that constitutes the early embryo, embryonic stem cells established from primordial germ cells, cells isolated from a pluripotent cell population (e.g., primitive ectoderm) of the early embryo before implantation, and cells obtained by culturing these cells.

[0026] As used herein, the term "trophoblast stem cells" refers to stem cells derived from the trophoblast lineage of the embryo. Trophoblast stem cells are preferably extraembryonic cells derived from two types of cells that are precursors of the human placenta: the cytotrophoblast and the syncytiotrophoblast. While these cells can be derived from late preimplantation or early postimplantation stages, the resulting cell lineage is equivalent to the stem cell compartment present in the extraembryonic ectoderm of the mouse egg cylinder after implantation. Transcription factors such as Cdx2, Tead4, Gata3, Elf5, Eomes, and Tfap2C characterize this lineage. TS cells are also thought to be precursors of differentiated cells of the placenta. In mice, TS cells can be derived from either the polar trophectoderm of the blastocyst or the extraembryonic ectoderm that develops from the polar trophectoderm after implantation.

[0027] As used herein, the term "extraembryonic endodermal stem cells" (XEN stem cells) refers to stem cells derived from the extraembryonic endoderm of an embryo. Extraembryonic endoderm is typically a derivative of hypoblast cells that migrate into the blastocyst cavity (starting on day 8 of human embryonic development), lining the cavity and giving rise to the primary and definitive yolk sac. The extraembryonic endoderm fills the remaining cavity of the blastocyst.

[0028] As used herein, the term "differentiation" can refer to the process by which unspecified ("uncommitted") or less-specialized cells acquire the characteristics of specialized cells, such as neurons. A differentiated cell is one that has become more specialized ("committed") within a cellular lineage. The term "committed," when applied to the process of differentiation, refers to a cell that, under normal circumstances, would continue to differentiate into a specific cell type or subset of cell types, but has progressed along the differentiation pathway to a point where, under normal circumstances, it is no longer able to differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the lineage of a cell defines the inheritance of the cell, i.e., what cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a genetic scheme of development and differentiation. As used herein, a "lineage-specific marker" refers to a characteristic that is specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of uncommitted cells into a lineage of interest.

[0029] As used herein, the terms "marker," "lineage marker," or "lineage-specific marker" can refer to a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest. Differential expression can mean that the level of a positive marker is increased and the level of a negative marker is decreased compared to undifferentiated cells. The detectable level of the marker nucleic acid or polypeptide is sufficiently high or low in the cell of interest compared to other cells so that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art. In some embodiments, the marker can be enriched. The term "enriched" as used herein should 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) in one condition compared to another condition (e.g., one cell layer compared to another cell layer).

[0030] As used herein, the term "concentration" has its ordinary meaning and can refer to (a) mass concentration, molar concentration, volume concentration, mass fraction, mole 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, concentration can refer to the ratio of activity units per volume (e.g., U / ml).

[0031] As used herein, the term "analog" refers to a compound that may be structurally related to the related molecule. As used herein, the term "agonist" can refer to a compound that may not be structurally related to the related molecule. For example, an agonist may activate the related receptor by altering the receptor's structure. In either case, however, the term is used herein to refer to a compound or molecule that can mimic, reproduce, or otherwise generally replace a specific biological activity of the related molecule.

[0032] As used herein, the phrase "culture medium" refers to a liquid substance used to promote the growth and development of stem cells and embryos. The culture medium used in accordance with some embodiments of the present invention can be an aqueous medium containing a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, and / or proteins necessary for cell growth and embryo development, such as cytokines, growth factors, hormones, and the like.

[0033] Provided herein are methods, compositions, and culture media for generating mammalian (e.g., human) postimplantation embryo models composed of embryonic and extraembryonic tissues. Two types of extraembryonic cell-like cells, generated by overexpression of transcription factors, are combined with wild-type embryonic stem cells and self-organize into structures that mimic aspects of postimplantation embryos, such as postimplantation human embryos. The self-organized structures contain pluripotent epiblast-like domains surrounded by hypoblast-like and trophoblast-like tissues.

[0034] Disclosed herein are in vitro methods for generating synthetic embryos from embryonic stem cells. In some embodiments, the methods 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 in a culture medium under conditions that allow the ESCs to self-organize into post-implantation embryonic structures. Disclosed herein are also synthetic embryonic structures obtained by the disclosed methods.

[0035] Disclosed herein are methods for investigating mechanisms involved in embryogenesis according to the in vitro methods disclosed herein. Also disclosed herein are methods for identifying compounds useful for treating disease, comprising contacting a synthetic embryo obtained by the in vitro methods disclosed herein with a compound. Also disclosed herein are methods for diagnosing or treating a disease or disorder in a subject. The method can include generating a synthetic embryo according to the methods disclosed herein and implanting the synthetic embryo into a subject. Also disclosed herein are methods for elucidating the role of a candidate gene in embryonic development. The method can include obtaining wild-type mammalian ESCs, first modified mammalian ESCs comprising a GATA6 gene and / or a SOX17 gene, and second modified mammalian ESCs comprising a GATA3 gene and / or a TFAP2C gene in which the candidate gene has been modified or knocked out, and culturing the mammalian ESCs using the in vitro methods disclosed herein. Generating synthetic embryonic structures from pluripotent stem cells

[0036] Human reproduction is highly inefficient, with an estimated 60% of pregnancies failing during the first two weeks after fertilization. Since the advent of in vitro fertilization, human embryos have been studied throughout their first week of development. However, the second week, which includes preparation for uterine implantation and gastrulation, remains a "black box." The human blastocyst, 5-6 days after fertilization, consists of the outermost layer, the trophectoderm, which is the precursor to the placenta, and an inner cell mass that gives rise to both the embryonic epiblast and the hypoblast, the precursors to the yolk sac. Between 7-8 days after fertilization, the blastocyst implants into the uterine lining, and the epiblast polarizes and transitions from a naive to a primed state of pluripotency. The central amniotic cavity forms within the blastoderm epithelium, separating the dorsal and ventral amniotic epithelium, which maintains pluripotency and gives rise to the embryo itself. After implantation, the trophectoderm develops into several trophoblast subtypes, and the hypoblast forms the primary and then secondary yolk sac. A subset of cells in the hypoblast maintains expression of NODAL, BMP, and WNT inhibitors and protects the future anterior epiblast from posteriorizing signals during primitive streak formation, characterized by upregulation of BRY / TBXT. An additional extraembryonic tissue, the extraembryonic mesenchyme, is located between the inner cell mass-derived tissue and the trophoblast, but the origin of these cells remains unknown.

[0037] Recent mouse embryo studies have established conditions amenable to in vitro culture of human embryos by implantation, unlocking for the first time this developmental black box. This system has been used to characterize key developmental events, including the formation of the anterior hypoblast domain, trophoblast subtype specification, and the transition from the epiblast to the pluripotent state. However, mechanistic studies in human embryos remain challenging. Thus, stem cell-derived human embryo models serve as important and complementary tools for understanding this critical developmental stage. Several groups have reported the generation of blastocyst-like structures derived from human embryonic stem cells (hESCs). These blastocyst-like structures resemble preimplantation embryos but develop poorly to the postimplantation stage. Other models, including gastruloids, 2D micropatterns, and embryoid bodies, are capable of modeling aspects of postimplantation development. However, these models are derived entirely from hESCs, lack extraembryonic tissues, and do not recapitulate embryonic morphology. Recent studies combining epiblast-like spheroids with BMP4-treated hESCs expressing a mixture of extraembryonic markers mark a step toward a model of postimplantation embryonic integration. However, this model does not show self-organization of the epiblast-like compartment in the context of extraembryonic tissues until after lumen formation, and BMP4-treated hESCs do not correlate with the targeted extraembryonic lineage.

[0038] Several protocols have been developed to induce trophoblast and hypoblast cells from hESCs. Importantly, the pluripotent state influences the developmental trajectory of differentiated cells. Derivation of lineage-specific cell lines provides the opportunity to model these tissues in vitro. However, generating a modular, integrated model system that includes both embryonic and extraembryonic tissues has proven challenging. This is likely due to opposing signaling pathway modulators required for hESC culture, hypoblast-like cell differentiation, and trophoblast-like cell differentiation. Furthermore, while tissue-directed crosstalk is an advantage of an integrated model system, generating embryoid bodies in media containing exogenous factors can impair tissue-driven self-organization.

[0039] 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, such as pluripotent embryonic stem cells ("ESCs"), based on an approach of expressing (e.g., overexpressing) transcription factors that can promote the generation of extraembryonic-like cells, including trophoblast-like and hypoblast-like cells, from pluripotent embryonic stem cells. The pluripotent stem cell-based in vitro embryo model described herein uses exclusively pluripotent embryonic stem cells (e.g., human ESCs or "hESCs") to generate embryonic and extraembryonic lineages. Extraembryonic trophoblast-like and hypoblast-like cells can be generated by overexpression of transcription factors by wild-type embryonic stem cells. This 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 postimplantation development, including lumenogenesis, amniogenesis, primordial germ cell formation, and anterior hypoblast specification. These induced embryoid bodies are modular, independent of exogenous signaling factors, and amenable to genetic perturbations. In some embodiments, the induced embryoid bodies generated herein are human embryoid bodies. 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, which is incorporated herein by reference in its entirety.

[0040] Provided herein are methods for generating synthetic embryos in vitro from mammalian pluripotent stem cells, such as pluripotent embryonic stem cells or ESCs. The methods 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 in a culture medium under conditions that allow 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, pre-implantation-like pluripotent ESCs, or post-implantation primed ESCs. In some embodiments, the ESCs used herein are pre- and post-implantation-like pluripotent ESCs, e.g., pre- and post-implantation-like pluripotent hESCs (e.g., RSeT hESCs). In some embodiments, the ESCs used herein express lower levels of amnion-specific genes during trophoblast-like cell induction compared to ESCs in other pluripotent states (e.g., primed cells). ESCs in different pluripotent states can be pre-cultured using culture media / conditions distinguishable to those skilled in the art. For example, pre-implantation naive hESCs can be generated by culturing in PXGL medium before co-culture. Pre- and post-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 mTeSRl medium before co-culture.

[0041] Modified mammalian pluripotent stem cells, such as ESCs, can contain one or more genes encoding one or more transcription factors capable of driving the generation of extraembryonic 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. Inducible ESCs can express one or more inducible genes upon induction. For example, modified mammalian ESCs can contain only an inducible GATA6 gene. Alternatively or additionally, modified mammalian ESCs can contain only an inducible SOX17 gene. In some embodiments, modified mammalian ESCs contain an inducible GATA6 gene and an inducible SOX17 gene. Pluripotent stem cells containing an inducible GATA6 gene and / or an inducible SOX17 gene can attain any pluripotent state. For example, modified mammalian ESCs containing only an inducible GATA6 gene can attain a pre-implantation naive state or an intermediate pre- or post-implantation-like state. Modified mammalian ESCs containing only the inducible SOX17 gene can be in a naive state before transplantation, an intermediate pre- and post-transplantation-like state, or a primed state after transplantation.Modified mammalian ESCs containing the inducible GATA6 gene and the inducible SOX17 gene can be in a naive state before transplantation, an intermediate pre- and post-transplantation-like state, or a primed state after transplantation.

[0042] Modified mammalian ESCs can contain only an inducible GATA3 gene. Alternatively or additionally, modified mammalian ESCs can contain only an inducible TFAP2C gene. In some embodiments, modified mammalian ESCs contain an inducible GATA3 gene and an inducible TFAP2C gene. Pluripotent stem cells containing an inducible GATA3 gene and / or an inducible TFAP2C gene can be in any pluripotent state. For example, modified mammalian ESCs containing only an inducible TFAP2C gene can exist as pre-implantation naive pluripotent stem cells, such as PXGL cells, or pre- and post-implantation pluripotent stem cells, such as RSeT cells. Modified mammalian ESCs containing only an inducible GATA3 gene can be in a pre-implantation naive state, an intermediate pre- and post-implantation-like state, or a post-implantation primed state. Modified mammalian ESCs containing an inducible GATA3 gene and an inducible TFAP2C gene can be in a pre-implantation naive state, an intermediate pre- and post-implantation-like state, or a post-implantation primed state.

[0043] In some embodiments, GATA6 and / or SOX17 can drive pluripotent stem cells (e.g., ESCs) to develop into hypoblast-like cells. In some embodiments, GATA3 and / or TFAP2C can drive pluripotent stem cells (e.g., ESCs) to develop into trophoblast-like cells.

[0044] 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 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 demonstrate at least a 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 increase in GATA6 and / or SOX17 mRNA expression compared to wild-type ESCs. In some embodiments, modified ESCs comprising a GATA3 gene and / or a TFAP2C gene demonstrate at least a 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 increase in GATA3 and / or TFAP2C mRNA expression compared to wild-type ESCs.

[0045] The method can further include contacting the inducible mammalian ESCs with an inducer (e.g., doxycycline) to generate induced mammalian ESCs. Induction can occur during or prior to co-culture of the modified ESCs with wild-type ESCs. Contacting can be performed for any duration suitable for increasing mRNA expression of a transcription factor (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 administered to the culture medium containing the wild-type ESCs and the modified inducible ESCs for a duration of about, at least, at least about, up to, or up to 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 during the entire co-culture process. Alternatively or additionally, modified mammalian ESCs can be induced before co-culture. For example, inducible ESCs can be induced (e.g., in the presence of an inducer) before co-culture with wild-type ESCs to generate induced ESCs that express or overexpress one or more transcription factors (e.g., GATA6, SOX17, GATA3, and TFAP2C). The induced ESCs are then co-cultured with wild-type ESCs under conditions that allow the ESCs to self-organize into aggregated structures. 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 present in the culture medium. In some embodiments, induction can be terminated by removing the inducer from the culture medium, for example, by replacing or adding fresh culture medium without the inducer.

[0046] The number or amount of wild-type ESCs and modified ESCs can be any suitable ratio, which can vary depending on different embodiments. In some embodiments, ESCs comprising the GATA6 gene and / or SOX17 gene and / or ESCs comprising the GATA3 gene and / or TFAP2C gene are provided in greater amounts than wild-type ESCs. In some embodiments, ESCs comprising the GATA3 gene and / or TFAP2C gene are provided in greater amounts than ESCs comprising the GATA6 gene and / or SOX17 gene. ESCs comprising the GATA3 gene and / or TFAP2C gene can be provided in an amount or cell number that is at least 2, 3, 4, 5, 6, 7, or 8 times greater than the amount or cell number of wild-type ESCs and / or ESCs comprising the GATA6 gene and / or SOX17 gene. In some embodiments, ESCs comprising the GATA6 gene and / or SOX17 gene and wild-type ESCs are provided in approximately the same amount (e.g., a 1:1 ratio). In some embodiments, the ratio between wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene is from about 1:1:2 to about 1:1:10, e.g., 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 between wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene is about 1:1:2. In some embodiments, the ratio between wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene is about 1:1:1.

[0047] In some embodiments, co-culture of wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene is carried out for a duration of about, at least, at least about, up to, or up to about 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the co-culture comprises co-culturing wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene in a culture medium suitable for the growth of pluripotent stem cells (e.g., N2B27 medium). In some embodiments, the culture in the pluripotent stem cell growth medium lasts for about 5 days, and optionally, the ESCs are passaged at least twice in the stem cell growth medium. During culture in the stem cell growth medium, the ESCs aggregate and can form an aggregate structure showing a distinction between an inner cell domain and an outer cell domain, for example, after 3 days of culture in the stem cell growth medium. The ESCs can be cultured in pluripotent stem cell expansion medium for about two more days after aggregation, and then transferred to a post-transplantation culture medium. The method further includes co-culturing the ESCs in a post-transplantation culture medium (e.g., a post-transplantation culture medium). After co-culturing in the pluripotent stem cell expansion medium, the ESCs are cultured in a human embryo transfer medium (e.g., hIVC1). Culturing in the post-transplantation culture medium can be carried out for at least about 1 day, 2 days, 3 days, 4 days, 5 days, or longer. Co-culturing the ESCs in the post-transplantation culture medium can begin about 2 days after aggregation of the ESCs. In some embodiments, co-culturing the ESCs includes transferring the ESCs from one substrate to another.

[0048] Thus, 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 SOX17 gene, and second modified mammalian ESCs comprising the GATA3 gene and / or TFAP2C gene in a culture medium suitable for the growth of pluripotent stem cells (e.g., N2B27 medium) under conditions that allow the ESCs to form aggregate structures. The method can further include culturing the aggregate structures in a post-implantation culture medium under conditions that allow the aggregate structures to develop into synthetic embryos that mimic post-implantation embryo structures. Cell aggregates formed by the wild-type ESCs, the ESCs comprising the GATA6 gene and / or SOX17 gene, and the ESCs comprising the GATA3 gene and / or TFAP2C gene can self-organize into synthetic embryo structures that mimic post-implantation embryo structures. The post-implantation embryo structures can be human embryo structures. In some embodiments, the post-implantation embryo structures resemble post-implantation human embryos at approximately 8-9 days post-fertilization.

[0049] The synthetic embryonic structures produced using the methods and culture media described herein contain both embryonic and extraembryonic tissues and recapitulate embryonic morphology. The post-implantation embryonic structures described herein (e.g., human post-implantation embryonic structures) can contain a pluripotent epiblast-like domain surrounded by hypoblast-like tissue and trophoblast-like tissue. In some embodiments, the post-implantation embryonic structures contain a SOX2-positive epiblast-like domain containing a central lumen; an outer single layer of GATA3-positive presumptive trophoblast-like cells; and an intermediate presumptive hypoblast-like domain of GATA6-positive cells between the inner lumen-forming domain and the outer layer.

[0050] The efficiency of co-culturing wild-type and modified ESCs to form post-implantation embryo structures can vary in various embodiments depending on factors such as the pluripotent state of the ESCs, the expression level of one or more transcription factors described herein, the time in cell culture, the time and intensity of induction, and / or other factors discernible by a person of skill in the art upon reviewing this disclosure. For example, in some embodiments, the use of ESCs in an intermediate pluripotent state, such as pre- or post-implantation pluripotent ESCs (e.g., RSeT hESCs), can more efficiently generate post-implantation embryos compared to the use of ESCs in other pluripotent states (e.g., naive ESCs before implantation or primed ESCs after implantation). In some embodiments, the efficiency of co-culturing wild-type ESCs, ESCs comprising the GATA6 gene and / or SOX17 gene, and ESCs comprising the GATA3 gene and / or TFAP2C gene to form post-implantation embryos is 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).

[0051] The synthetic embryo structure described here is an ESC-derived embryo model capable of forming a pluripotent epiblast-like domain surrounded by hypoblast and trophoblast-like tissue. Specifically, the trophoblast and hypoblast cells are derived or programmed from ESCs (e.g., hESCs). Thus, the method does not involve culturing trophoblast cells, hypoblast cells, or both. Notably, the method does not involve culturing trophoblast and / or hypoblast cells in combination with pluripotent embryonic stem cells.

[0052] In some embodiments, the methods disclosed herein do not include any in vivo steps. In some embodiments, neither wild-type ESCs comprising the GATA6 gene and / or SOX17 gene, nor ESCs comprising the GATA3 gene and / or TFAP2C gene, nor synthetic embryos are present in an in vivo environment during any of the culture steps disclosed herein. The in vivo environment can include a tissue, an organ, an organism, or a combination thereof.

[0053] The methods, compositions, and culture media described herein also eliminate the need for exogenous signaling pathways that may otherwise impair tissue-driven self-organization. In some embodiments, the methods, compositions, and culture media described herein for generating synthetic embryos (e.g., human synthetic embryos) do not include the use of exogenous signaling pathway factors (e.g., WNT signaling pathway activators and / or TGFβ superfamily members). For example, none of the culture media used herein contain exogenous signaling pathway factors or have exogenous signaling pathway factors added. Pluripotent embryonic stem cells and embryonic development

[0054] Disclosed herein are methods, compositions, and culture media for in vitro culturing of synthetic embryos from mammalian pluripotent stem cells (e.g., ESCs). In some embodiments, the mammalian pluripotent ESCs are hESCs. In some embodiments, the methods involve 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 culture medium under conditions that allow the ESCs to self-organize into a synthetic embryo that mimics a post-implantation embryo structure. In some embodiments, the generated post-implantation embryo structure represents a multi-lineage stem cell-derived model of the human post-implantation embryo, which undergoes lumen formation in its epiblast-like domain and differentiation events that reflect interactions between extraembryonic and embryonic-like tissues.

[0055] Although mammalian embryonic development shares some characteristics with all species, it is recognized that different mammalian species develop in different ways and at different rates. However, generally, the fertilized egg undergoes several division stages (passing through the 2-cell, 4-cell, and 8-cell stages) before compacting to form a solid ball of cells called a morula, within which cells continue to divide. Eventually, the inner cells of the morula give rise to the inner cell mass, and the outer cells give rise to the trophectoderm. The morula develops into a blastocyst, which is surrounded by trophectoderm and contains a fluid-filled vesicle with an inner cell mass at one end.

[0056] As used herein, the term "embryo" refers to a mammalian organism at the single-cell stage. Embryos as described herein are produced from the culture of embryonic stem cells in vitro under appropriate conditions, and resemble or mimic a natural embryo produced in vivo at the corresponding stage, such as having similar morphology, length, weight, cell type composition, and expression of developmental marker genes.

[0057] Embryonic developmental stages can be defined by the development of specific structures and can be used to define equivalent stages in the development of other species. In some embodiments, embryonic developmental stages can be defined according to "Carnegie stages," a standardized system used to provide a unified developmental chronology of vertebrate embryos. The earliest Carnegie stages are listed in Table 1. [Table 1]

[0058] In some embodiments, the methods, compositions, and culture media described herein may enable culture to post-implantation stages corresponding to Carnegie stages (a), 5(b), 5(c), 6, 7, 8, 9, and beyond, and to corresponding stages in other species.

[0059] The methods, compositions, and culture media described herein can be applied to embryos from any suitable mammalian species, including humans and non-humans, such as: primates, including humans, great apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (including rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or domesticated mammal. The methods, compositions, and culture media described herein can be applied to embryos from humans. Thus, any of the culture medium embodiments provided herein can support the in vitro development of human embryos on a substrate from pre-implantation to post-implantation stages of development.

[0060] The term "preimplantation stage" as used herein can be used to refer to a stage of development that corresponds to Carnegie stage 5(a) and that precedes corresponding stages in other species. The term "postimplantation stage" as used herein can refer to a stage of development that corresponds to Carnegie stage 5(a) and that precedes corresponding stages in other species. The "postimplantation stage" can be determined by detecting the upregulation of one or more genes by the embryo. For example, such a stage can be determined by detecting one or more of the following changes: the epiblast upregulates Fgf5; the primitive endoderm differentiates into visceral endoderm, which upregulates Cer1 in a subpopulation of cells (anterior visceral endoderm); the visceral endoderm upregulates Eomes; and the trophectoderm upregulates Handl.

[0061] Stem cells (e.g., mammalian pluripotent stem cells, such as embryonic stem cells) can be cultured using the 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 stem cells in tissues. Also included are cells that have been artificially conferred pluripotency similar to embryonic stem cells by transferring multiple different genes into somatic cells (also called 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. All 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, great apes (e.g., gorillas, chimpanzees, and orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, and hamsters); cats; dogs; lagomorphs (including rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or domestic mammal. The methods of the present disclosure may be applied to stem cells from any non-human mammal, including, but not limited to, those 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).

[0062] As used herein, pluripotent stem cells (e.g., ESCs) can be in different pluripotent states. Pluripotent embryonic stem cells can be naive ESCs, primed ESCs, or intermediate pluripotent states between naive and primed pluripotent stem cells, such as formative ESCs. Pluripotent stem cells can be pre-implantation naive stem cells, pre-implantation pluripotent stem cells, or post-implantation primed stem cells. Naive ESCs include cells that exhibit naive characteristics, such as global DNA hypomethylation, expression of naive pluripotency markers such as Klf4, Tfcp2l1, Esrrb, Klf2, Tbx3, Prdm14, and Dppa3, active mitochondria, reduced glucose dependence, and the ability to proliferate by enzymatic degradation of single cells, as would be understood by one skilled in the art. Markers highly expressed in naive ESCs can be silenced in formative and primed ESCs. Formative ESCs can possess upregulated Otx2, Dnmt3b, Fgf5, Zic2 / 5, Etvl / 4, Oct6, and Grhl2, which have been reported to play important roles in the transition from the naive to the primed state. For example, when cells exit naive pluripotency, increased Otx2 binds to many enhancer domains of formative genes, which are pre-bound or not bound by Oct4, activating correlated genes. Thus, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape for the transition from naive pluripotency to the formative state. Primed pluripotent stem cells possess upregulated Soxll, Zic23, Dusp6, and Cd24 and downregulated or silenced naive markers (e.g., silenced Klf4). Additional information related to the pluripotent state in mammalian cells, such as mouse and human cells, can be found, for example, in Genes (Basel). 2022 Aug;13(8):1459, doi: 10.3390 / genesl3081459, the contents of which are incorporated by reference in their entirety.

[0063] 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 peri- or early-stage pluripotent intermediate stem cells. In some embodiments, the pluripotent stem cells used herein are more readily differentiated into yolk sac-like endoderm cells before and after implantation compared to naive cells such as PXGL cells. In some embodiments, the ESCs used herein can be naive hESCs before implantation, pluripotent hESCs before and after implantation, or primed hESCs after implantation. In some embodiments, the ESCs are pluripotent hESCs before and after implantation. Pre- and post-implantation pluripotent hESCs exhibit naive-like features such as tightly packed, dome-shaped colonies with curved edges and can exhibit increased expression of gene markers such as Klf2, Klf4, and Tfcp2L1. As used here, ESCs can express amnion-specific genes at lower levels during trophoblast-like cell induction compared to ESCs in other pluripotent states (e.g., primed cells).

[0064] Pluripotent stem cells (e.g., ESCs) in different pluripotent states can be pre-cultured using appropriate culture media / conditions identifiable by one skilled in the art. For example, pre-implantation naive hESCs can be generated by culturing them in PXGL medium before co-culture. Pre- and post-implantation-like pluripotent hESCs can be generated by culturing them in RSeT medium before co-culture. Post-implantation-like primed hESCs can be generated by culturing them in mTeSRl medium before co-culture.

[0065] Conversion between distinct pluripotent states can be achieved in vitro. In some embodiments, primed pluripotent stem cells (e.g., hESCs) can be converted into formative or naive cells under appropriate culture conditions, as will be understood by those skilled in the art. For example, to convert primed pluripotent stem cells (e.g., primed hESCs) into pre- or post-implantation pluripotent stem cells (e.g., RSeT cells), primed hESCs can be passaged onto mitomycin C-inactivated CF-1 MEFs in a medium containing DMEM / F12 with knockout serum replacement, a reducing agent, non-essential amino acids, antibiotics, L-glutamine or an analog thereof, a fibroblast growth factor family (FGF) member, and a ROCK inhibitor. To culture pre-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 defined cell culture medium used to convert primed human pluripotent stem cells (hPSCs) to a naive-like state and to maintain naive-like hPSCs under feeder-dependent and hypoxic conditions. In some embodiments, RSeT medium does not contain bFGF or TGFβ. RSeT medium is compatible with human embryonic stem cells and human induced pluripotent stem cells. Additional information regarding culture conditions for naive cells (e.g., PXGL cells) is provided in the Examples section and in "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 contents of which are incorporated herein by reference in their entirety.

[0066] As will be appreciated by those skilled in the art, pluripotent stem cells such as ESCs may be obtained from stem cell banks, such as the UK stem cell bank, which provide human stem cell lines for research, or from participating fertility centers. Preferably, the ESCs are obtained or available by methods that do not involve the destruction of human or non-human animal embryos. synthetic embryo structure

[0067] Provided herein are methods, compositions, and culture media for modeling mammalian embryonic development by culturing pluripotent stem cells (e.g., embryonic stem cells). The disclosed methods, compositions, and culture media can generate synthetic embryonic structures through co-culturing wild-type embryonic stem cells and extraembryonic-like cells generated by overexpression of transcription factors with wild-type embryonic stem cells. Accordingly, provided herein are 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 postimplantation embryo model structures, such as human embryoid bodies. In some embodiments, the synthetic embryonic structures represent self-organized assemblies comprising pluripotent epiblast-like domains surrounded by extraembryonic-like tissues, where the epiblast-like domains can differentiate into amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP signaling.

[0068] In some embodiments, wild-type and modified pluripotent stem cells used herein can form cell aggregates within 24 hours of culturing in culture medium (e.g., stem cell growth medium). Approximately four days after aggregation, the cell aggregates can self-organize into a structure with a SOX2-positive epiblast-like domain containing a central lumen, 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 lumenalized domain and the outer layer. Synthetic embryonic structures can be composed of aggregates containing organized SOX2-positive domains surrounded by concentric layers of GATA6- and GATA3-positive cells. In some embodiments, the aggregates do not undergo a blastocyst-like morphology before forming postimplantation-like structures.

[0069] In some embodiments, the synthetic embryo structures produced using the methods and compositions described herein can reach a post-implantation stage (e.g., post-implantation gastrulation). In some embodiments, the synthetic embryo structures produced herein can reach an early gastrulation stage. In some embodiments, the synthetic embryos produced herein can reach a late gastrulation stage. As used herein, the term "gastrulation" in the context of an embryo refers to an embryo that follows the expanded blastocyst stage and precedes the somitogenesis stage, and is characterized by the formation of three germ layers through the formation of a primitive streak and the transition from epithelium to mesenchyme. The gastrulation process generally involves the transformation of a bilayered embryonic disc into a three-layered disc, as intraembryonic mesoderm appears between the ectoderm and endoderm. The gastrulation stage can be early gastrulation, mid-gastrulation, late gastrulation, or advanced gastrulation.

[0070] In some embodiments, embryonic structures produced using the methods and culture media described herein include post-implantation embryos, e.g., post-implantation pre-gastrulation embryonic structures. As used herein, the term "post-implantation pre-gastrulation," as used with respect to mammalian embryos (e.g., human embryos), refers to embryos after the implantation blastocyst stage and before the early gastrulation stage, and is characterized by an egg cylindrical shape before symmetry is broken.

[0071] The embryonic stage of synthetic embryo structures generated using the methods and culture media disclosed herein can be assessed by comparing them with their in vivo natural embryonic counterparts at the same developmental stage by a variety of methods, including, but not limited to, morphology, length, weight, cell type composition, chromatin accessibility patterns, expression of developmental marker genes (e.g., Oct4, Nanog, Sox2, Klf4, Cdx2, Gata4, Gata6, Brachyury, Otx2, Fgf5, and other genes described herein and known in the art) using specific antibodies or primers, or by transcriptional profiling, single-cell RNA sequencing, and other methods further described in this Examples section.

[0072] In some embodiments, the postimplantation embryo structure generated herein comprises an inner epiblast-like domain, a single outer layer of trophoblast-like cells, and an intermediate hypoblast-like domain between the epiblast-like domain and the single outer layer of trophoblast-like cells. The inner epiblast-like domain can be SOX2-positive and contain a central cavity, the single outer layer of trophoblast-like cells can be GATA3-positive, and the intermediate hypoblast-like domain can be GATA6-positive. In some embodiments, the postimplantation embryo 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 inner epiblast-like domain can exhibit pluripotency and epithelial identity similar to human embryos.

[0073] In some embodiments, the post-implantation embryonic structures produced herein comprise cell clusters resembling late embryonic epiblast, amnion, mesoderm, extraembryonic mesenchyme, and / or hypoblast / visceral endoderm. In some embodiments, the post-implantation embryonic structures express 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.

[0074] In some embodiments, the post-implantation embryonic structure can generate amniotic membranes and primordial germ cells. The post-implantation embryonic structure can generate primordial germ cell-like cells that express the pluripotency marker NANOG and the primordial germ cell markers PRDM1 (BLMP1) and NANOS3.

[0075] BMP signaling can play a role during differentiation of epiblast-like domains. In some embodiments, the human embryonic structures generated herein can express one or more downstream BMP-responsive genes ID1, ID2, ID3, or ID4 (see, e.g., Example 4). In some embodiments, phosphorylated (p)SMAD1.5 expression in OCT4-positive epiblast-like domains at days 4 and 6 after aggregation indicates active BMP signaling.

[0076] One or more of the genetic markers described herein can be upregulated or downregulated in the generated synthetic embryonic structures by one or more inducible genes introduced into pluripotent stem cells. For example, induction of SOX17 alone or in combination with GATA6 can result in a reduced ability to upregulate CER1 compared to GATA6 overexpression alone (see, e.g., Example 5). Alternatively, or in addition, synthetic embryonic structures generated with single GATA6 induction, or reduced induction strength and / or duration (e.g., withdrawal of doxycycline after a period of time, e.g., day 3), show increased expression of the primitive streak marker BRY / TBXT at day 6 post-aggregation compared to structures with consistent GATA6-SOX17 or SOX17 induction. Thus, in some embodiments, the in vitro synthetic embryonic structures generated herein can be used as modular embryoid body models to study the regulation of genetic markers and interactions between embryonic and extraembryonic tissues, to examine the roles of specific tissues and tissue-specific gene requirements, and to investigate mechanisms involved in embryogenesis.

[0077] In some embodiments, the synthetic embryonic structures produced 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 embryoid bodies. The human embryonic-like structures produced herein can exhibit organization reminiscent of a human embryo at about 8-9 days post-fertilization.

[0078] In vitro culturing of embryonic cells from stem cells (e.g., pluripotent ESCs) can be accomplished after implantation or until any developmental stage in between is reached. In some embodiments, synthetic embryos generated using the methods and culture media described herein do not mimic stages beyond primitive streak formation. In some embodiments, synthetic embryos generated using the methods and culture media described herein may not contain all cell types of a gastrulation-stage embryo. In some embodiments, synthetic embryos generated using the methods and culture media described herein may not further develop to form a viable human embryo.

[0079] The culture conditions for producing synthetic embryos, including substrates, culture media, and the like, are described in the following sections and in the specific embodiment of human embryos in the Examples section.

[0080] The embryonic stage of the synthetic embryos described herein can be assessed in comparison with in vivo or natural embryonic counterparts at the same developmental stage by a variety of methods, including but not limited to, morphology, length, weight, expression of developmental marker genes using specific antibodies or primers, transcriptional profiling, and the like, as described in more detail below and in the Examples section.

[0081] Morphological assessment of embryonic development can be performed by previously established morphological features as described in the Carnegie developmental stages (see, e.g., Table 1; Developmental stages in human embryos. R. O'Rahilly and F. Muller (eds.), Carnegie Institution of Washington, Washington, DC, 1987) or according to embryonic age.

[0082] In some embodiments, one or more developmental markers described herein can be used to assess the developmental stage of a synthetic embryonic structure. Numerous methods exist in the art for detecting the presence, absence, or quantity of marker gene products (e.g., mRNA and / or protein), as well as their localization or subcellular location (e.g., nuclear and / or cytoplasmic) in an embryonic structure. Marker expression can be assessed by any of a variety of well-known methods for detecting the expression of transcribed molecules or proteins. Non-limiting examples of such methods include immunological methods for detecting secreted, cell surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification and sequencing methods.

[0083] In some embodiments, the activity of a particular gene is characterized by measuring the gene transcript (e.g., mRNA), by measuring the amount of translated protein, or by measuring the activity of the gene product. Marker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can include quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or alternatively, can include qualitative assessment of the gene expression level, particularly by comparison with a control level. The type of level detected will be clear from the context.

[0084] In another embodiment, detecting or determining the expression levels of a marker and its functionally similar homologs, fragments thereof, or those containing genetic mutations (e.g., in its regulatory or promoter regions) comprises detecting or determining RNA levels for the marker of interest. In some embodiments, one or more cells can be obtained from the synthetic embryonic structure, and RNA is isolated from the 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). This technique can be used to isolate cells from tissue sections, including stained tissue sections, thereby ensuring that the desired cells are isolated. It is also possible to obtain cells from, for example, synthetic embryonic structures, and culture them in vitro, e.g., to obtain a larger population of cells from which RNA can be extracted. Methods for establishing cultures of non-transformed cells, i.e., primary cell cultures, are known in the art. In some embodiments, 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.).

[0085] For example, when isolating RNA from synthetic embryonic structures at various developmental stages and / or cells comprising said synthetic embryonic structures, it may be important to prevent any further changes in gene expression after the tissue or cells are removed from the subject. Changes in expression levels are known to change rapidly after perturbations, such as heat shock or activation by lipopolysaccharide (LPS) or other reagents. In addition, RNA in tissues and cells can undergo rapid degradation. Therefore, in a preferred embodiment, tissues or cells obtained from a subject are flash-frozen as soon as possible.

[0086] RNA can be extracted from cells by various methods, such as guanidinium thiocyanate lysis followed by CsCl centrifugation. Methods for obtaining RNA from single cells are also known in the art. The RNA sample can then be enriched for specific species. In some embodiments, poly(A)+ RNA is isolated from the RNA sample. Often, such purification utilizes the poly(A) tail on mRNA. In particular, and as mentioned above, poly(T) oligonucleotides can be immobilized on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, such as the MessageMaker kit (Life Technologies, Grand Island, NY). In some embodiments, the RNA population is enriched for marker sequences. Enrichment can be achieved, for example, by primer-specific cDNA synthesis or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription.

[0087] A population of RNA, whether enriched in a particular species or sequence, can be further amplified. As defined herein, an "amplification process" increases the copy number of a polynucleotide (e.g., RNA). For example, if the RNA is mRNA, an amplification process such as RT-PCR can be used to amplify the mRNA, making the signal detectable or enhancing detection. Such an amplification process is particularly useful when the size or volume of a biological, tissue, or tumor sample is small.

[0088] Various amplification and detection methods can be used. For example, within the scope of the disclosed method, mRNA can be reverse transcribed into cDNA followed by polymerase chain reaction (RT-PCR); or a single enzyme can be used for both steps as described in U.S. Patent No. 5,322,770, or mRNA can be reverse transcribed into cDNA followed by symmetric gap ligase chain reaction (RT-AGLC) as described by R.L. Marshall et al., PCR Methods and Applications, 4:80-84 (1994). Real-time PCR can also be used.Other known amplification methods that can be utilized in the present invention include, but are not limited to, the so-called "NASBA" or "3SR" technique described in PNAS USA (Proceedings of the National Academy of Sciences of the United States) 87: 1874-1878 (1990) and also in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification, described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (G.T. Walker et al., Clin. Chem. 42:9-13 (1996) and European Patent Application No. 684315); target-mediated amplification, as described in PCT International Publication No. WO 9322461; PCR; ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4, 560 (1989); Landegren et al., Science 241, 1077 (1988)); self-sustained sequence replication (SSR) (see, e.g., Guatelli et al., Proc. Nat. Acad. Sci. USA 87, 1874 (1990)); and transcriptional amplification (see, e.g., Kwoh et al., Proc. Nat. Acad. Sci. USA 86, 1173 (1989). Numerous techniques for determining absolute and relative levels of gene expression are currently known, and commonly used techniques suitable for use in the disclosed methods include Northern analysis, RNase protection assays (RPA), microarrays, and PCR-based techniques such as quantitative PCR and differential display PCR.For example, Northern blotting involves running a preparation of RNA on a denaturing agarose gel and transferring it to a suitable support, such as activated cellulose, nitrocellulose, or a glass or nylon membrane, etc. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed, and analyzed by autoradiography.

[0089] In situ hybridization visualization can also be employed, in which radiolabeled antisense RNA probes are hybridized with thin sections of samples, washed, cleaved with RNase, and exposed to a sensitive emulsion for autoradiography.The samples are stained with hematoxylin to reveal the histological composition of the samples, and the developed emulsion is shown by dark-field imaging using appropriate light filters.Non-radioactive labels such as digoxigenin can also be used.In some embodiments, the probe is labeled with a fluorescent moiety.

[0090] Alternatively, mRNA expression can be detected on a DNA array, chip, or microarray. The labeled nucleic acid of a test sample collected from a subject can be hybridized to a solid surface containing marker DNA. A sample containing marker transcripts will obtain a positive hybridization signal. The method of preparing DNA arrays and their use are well known in the art (see, for example, U.S. Patent No. 66,186,796; No. 6,379,897; No. 6,664,377; No. 6,451,536; No. 548,257; U.S. Patent Application Publication No. 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 the total mRNA expression from one (e.g., single-cell RNA-seq) or more cells. Nucleic acid target molecules labeled with barcodes (e.g., origin-specific barcodes) can be sequenced using the barcodes to generate single reads and / or contigs containing the sequences or portions thereof of both the target molecule and the barcode. Exemplary next-generation sequencing technologies 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.

[0091] In some embodiments of the present disclosure, the single-cell sequencing is high-throughput single-cell RNA sequencing. In certain embodiments, the single-cell sequencing is low-cost high-throughput single-cell RNA sequencing. Without being bound by any particular theory, single-cell RNA sequencing is capable of efficiently and cost-effectively sequencing thousands to tens of thousands of single cells. In certain embodiments, the single-cell RNA sequencing comprises pairing a single cell in a droplet with an oligonucleotide for reverse transcription, wherein the oligonucleotide is configured to provide a cell-origin-specific barcode that uniquely identifies the transcript from each cell and a unique molecular identifier (UMI) that uniquely identifies each transcript. In certain embodiments, the single-cell RNA sequencing comprises pairing a single cell in a droplet with a single oligonucleotide-coated microparticle bead for reverse transcription, wherein the oligonucleotide comprises a bead-specific barcode that uniquely identifies each bead and a unique molecular identifier (UMI) that uniquely identifies each primer. In some embodiments of the present disclosure, unbiased classification of cells in a biological sample involves sequencing the transcriptomes of thousands of cells, preferably tens of thousands of cells (e.g., more than 1,000 cells, or more than 10,000 cells).

[0092] 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 several means well known to those skilled in the art. Any method known in the art for detecting a polypeptide can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, Western blotting, binder-ligand assays, immunohistochemistry techniques, agglutination, complement assays, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, and the like.

[0093] Described below are non-limiting examples of techniques that may be used to detect marker proteins according to the practitioner's preference based on the present disclosure. One such technique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)), in which an appropriately treated sample is scanned on an SDS-PAGE gel before being transferred to a solid support such as a nitrocellulose filter. An anti-marker protein antibody (unlabeled) is then contacted with the support, and labeled protein A or anti-immunoglobulin (suitably labeled) is added. 125 The antibody is assayed with secondary immunoreagents such as IgG, ...

[0094] Immunohistochemistry can be used to detect the expression of marker proteins. An appropriate antibody is, for example, contacted with a thin layer of cells, washed, and then contacted with a secondary, labeled antibody. The label can be a fluorescent marker, an enzyme such as peroxidase, avidin, or a radioactive label. The assay is scored visually using a microscope.

[0095] Anti-marker protein antibodies, such as intrabodies, can also be used for imaging purposes, e.g., to detect 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.

[0096] Antibodies that can be used to detect marker proteins include any antibody, natural or synthetic, full-length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the marker protein to be detected. Antibodies can be up to about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 K of M d The phrase "specifically binds" refers, for example, to an antibody binding to an epitope or antigen or antigenic determinant in such a way that the binding can be displaced or competed for by a second preparation of the same or similar epitope, antigen or antigenic determinant. An antibody may preferentially bind to a marker protein over other proteins, such as related proteins.

[0097] 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), veneered antibodies, or single-chain antibodies, as well as functional fragments of antibodies, i.e., marker protein-binding fragments. For example, antibody fragments capable of binding to a marker protein or a portion thereof, including but not limited to Fv, Fab, Fab', and F(ab')2 fragments, can be used. Such fragments can be produced by enzymatic cleavage or recombinant technology. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the required substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain, and hinge region of the heavy chain. In some embodiments, agents that specifically bind to marker proteins other than antibodies, such as peptides, are used. Peptides that specifically bind to marker proteins can be identified by any means known in the art. For example, specific peptide binders of marker proteins can be screened using peptide phage display libraries. Substrate for synthetic embryo culture

[0098] In some embodiments, wild-type and modified pluripotent stem cells (e.g., ESCs), cell aggregates, post-implantation embryos, and / or synthetic embryos described herein are cultured on a substrate. In some embodiments, the methods include transferring ESCs and / or embryos from one substrate to another. The substrates used in the methods disclosed herein can be the same or different. For example, mammalian ESCs (e.g., WT and modified ESCs) can be cultured on a first substrate to form aggregate structures. Cell aggregates can be transferred to a second substrate to develop into post-implantation embryos. The first and second substrates can be the same type or different types. In some embodiments, the first and second substrates are of different types. In some embodiments, the first and second substrates are cultured on AggreWell substrates. TM It can be a microwell plate containing inverted pyramidal microwells, such as a microplate.

[0099] As used herein, a substrate can include a dish, U-plate, flask, or microwell plate. The microwell plate can include an inverted pyramidal microwell. The size (e.g., depth and / or diameter) of each inverted microwell can vary. Each inverted pyramidal microwell can be about 400 μm or about 800 μm in size. Each inverted pyramidal microwell can be about 400 μm or about 800 μm in diameter. In some embodiments, each inverted pyramidal microwell can be about 100, 200, 300, 400, 500, 600, 700, 800, 900 μm, 1 μm in size and / or diameter, or a value or range between any two of these values. Each microwell (e.g., receptacle) can have a depth of about 250 μm to about 400 μm, for example, about 300 μm to about 350 μm. Additionally or alternatively, the plurality of receptacles may have an average depth of about 250 μm to about 400 μm, for example, about 300 μm to about 350 μm. In particular, when the receptacles are wells, they may be arranged on the substrate in an array, i.e., in a grid pattern with regular spacing in substantially orthogonal directions. Regardless of the substrate's configuration, the substrate may accommodate one or more embryos. When the substrate includes one or more receptacles, each receptacle may independently contain one or more embryos, for example, 2, 3, 4, 5, 6, 7, or 8 or more embryos. In some embodiments, each embryo structure is disposed in a different well. In alternative embodiments, each receptacle contains multiple embryos, for example, 2, 3, 4, 5, 6, 7, or 8 embryos, or more.

[0100] The methods disclosed herein can be applied to any suitable size culture volume. For example, the culture volume per embryo can be from about 50 μl to about 10 ml, optionally from about 100 μl to about 5 ml, optionally from about 250 μl to about 5 ml, and optionally from about 1 ml to about 5 ml. The culture volume per embryo can be about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000 μl, or more. Culture medium

[0101] In some embodiments, the method includes co-culturing wild-type mammalian embryonic stem cells (ESCs), first modified mammalian ESCs comprising a GATA6 gene and / or a SOX17 gene, and second modified mammalian ESCs comprising a GATA3 gene and / or a TFAP2C gene in a culture medium suitable for the growth of pluripotent stem cells (e.g., N2B27 medium) under conditions that allow the ESCs to form an aggregate structure. The method can further include culturing the aggregate structure in a post-implantation culture medium under conditions that allow the aggregate structure to self-organize into a synthetic embryonic structure that mimics the post-implantation embryonic structure.

[0102] In some embodiments, the method includes co-culturing mammalian pluripotent stem cells (e.g., ESCs) in stem cell growth medium, and optionally passaged the ESCs at least twice (e.g., 2, 3, 4, or more) in the stem cell growth medium. The mammalian pluripotent stem cells can be cultured in the stem cell growth medium for 1, 2, 3, 4, or 5 days. In some embodiments, the pluripotent stem cells aggregate after about 3 days of co-culturing in the stem cell growth medium.

[0103] The method can also include co-culturing the pluripotent stem cells in post-transplantation culture medium for at least two days (e.g., 2, 3, 4, 5, 6, or more days) following co-culturing in stem cell growth medium. In some embodiments, the pluripotent stem cells are cultured in the post-transplantation culture medium for at least two days after being cultured in the stem cell growth medium for about five days.

[0104] In some embodiments, the method includes partially replacing a portion of the stem cell growth medium (e.g., at least half of the medium) with refreshed stem cell growth medium or post-transplantation culture medium. The replacement can occur every 20-28 hours (e.g., every 24 hours) of culture. In some embodiments, the method includes partially replacing a portion of the post-transplantation culture medium (e.g., at least half of the medium) with refreshed post-transplantation culture medium.

[0105] The culture media disclosed herein, including stem cell growth media and post-implantation culture media, can include basal culture media. Basal media are composed of water, salts, amino acids, a carbon source, vitamins, lipids, and buffers. Suitable carbon sources can be determined by those skilled in the art from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. Basal media are commercially available, for example, under the trade names Advanced DMEM / F12 (Gibco, 12634-010) and CMRL-1066 (Invitrogen or Sigma). The basal culture media were Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Roswell Park Memorial Institute (RPMI) Medium 1640, Neurobasal (R) (Neurobasal, (R) indicates registered trademarks in the United States and other countries), Neurobasal (R)A, Connaught Medical Research Laboratory 1066 (CMRL-1066), or any combination thereof.

[0106] Basal culture media include Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or its serum substitute, antibiotics, L-glutamine or its analogs (e.g., GlutaMAX TM ), or any combination thereof.

[0107] Non-human serum or serum substitutes include fetal bovine serum, bovine serum albumin, rat serum, and KnockOut. TM Antibiotics can include penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tyrosine, or any combination thereof.

[0108] The concentration or amount of one or more components in the solution or medium can be varied, e.g., non-human serum or serum substitutes, antibiotics, reducing agents, and / or L-glutamine (e.g., GlutaMax TMThe amount of ) can vary and in some embodiments can be adjusted as needed by one skilled in the art. In some embodiments, the amount of non-human serum or serum substitute can comprise from about 0.01% to about 40% (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 number or range between any two of these values) volume per volume (% v / v), weight per volume (% w / v), or weight per weight (% w / w) of medium. In some embodiments, the amount of antibiotic can comprise from about 0.01% to about 10% (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) volume per volume (% v / v), weight per volume (% w / v), or weight per weight (% w / w) of medium. 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 from about 0.1 μM to about 1 mM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900 μM, 1 mM, or a value or range between any two of these values). TM The amount of L-glutamine can vary. For example, in some embodiments, the concentration of L-glutamine in the culture 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, e.g., about 2 mM. When percentages are provided for agents, ingredients, and compounds, they can be % w / w, % w / v, or % v / v of the formulation as a whole, unless otherwise specified.

[0109] Each component of the culture medium described herein may be present in an amount suitable for the culture medium to support the self-organization of stem cells (e.g., ESCs) into post-implantation embryonic structures and / or the further development of post-implantation embryonic structures.

[0110] In the embodiments described herein, the culture medium and compositions used herein do not contain or do not contain exogenous signal transduction pathway factors.In some embodiments, the culture medium and compositions used herein do not contain WNT signal transduction pathway activators (e.g., WNT agonists or WNT signal transduction agonists). Exemplary WNT signaling pathway agonists include, but are not limited to, CHIR99021, derivatives of CHIR99021, e.g., salts of CHIR99021, e.g., the trihydrochloride salt, the hydrochloride salt of CHIR99021, Wnt3a recombinant protein, glycogen synthase kinase 3 (GSK3) inhibitors, e.g., 3F8, A 1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, 10Z-Hymenialdisine, indirubin-3'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and analogs or derivatives thereof.

[0111] In some embodiments, the culture medium or composition used herein does not contain a member of the TGF-β superfamily. The term "TGF-β superfamily" refers to proteins with structural and functional characteristics of known TGF-β family members. Proteins in the TGF-β family are well characterized from both structural and functional aspects. They include the TGF-β series of proteins, inhibins (including inhibin A and inhibin B), activins (including activin A, activin B, and activin AB), MIS (Müllerian inhibiting substance), BMPs (bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF (monoclonal nonspecific suppressor factor), and others. The activity of this protein family is based on specific binding to certain receptors on various cell types. Members of this family share regions of sequence identity, particularly in the C-terminus, that correlate with function. The TGF-β family includes over 100 distinct 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 TGF-β superfamily members include, but are not limited to, growth differentiation factor 8 (GDF8) (GenBank accession EAX10880), growth differentiation factor 11 (GDF11) (GenBank accession AAF21630), activin A, Nodal, activin A, activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and any functional fragments thereof.

[0112] The mammalian pluripotent stem cells described herein, e.g., wild-type ESCs and modified ESCs with transcription factor overexpression, can be cultured separately in an appropriate culture medium suitable for the growth 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 culture medium. The culture medium may comprise a serum replacement medium. Such a serum replacement medium may be KSR (KnockOut) or other suitable medium. TM Serum replacement media are commercially available under the trade names N2B27 (e.g., Invitrogen, 10828-010) and N2B27 (e.g., Invitrogen, ME100137L1). Serum replacement media may comprise from about 5% to about 60%, from about 10% to about 50%, from about 15% to about 45%, or from about 20% to about 40% of the culture medium. Exemplary culture media include, but are not limited to, RSeT medium, PXGL medium, cRM-1 medium, mTeSRl medium, and others identifiable by one of skill in the art.

[0113] 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-pyridinyl)phenyl]-urea (AS 1892802), fasudil hydrochloride (also known as HA 1077), -[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 (GSK 429286), (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-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 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-Benzodioxin-2-carboxamide dihydrochloride (SR 3677 dihydrochloride), and trans-4-[(R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (thiazovivin), a Rock inhibitor, isoquinoline sulfonamide compounds (Rho kinase inhibitors), 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; commercially available Rock antibodies from Santa Cruz Biotechnology, selected from the group consisting of 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), and Rock-2(30-J); ROCK CRISPR / Cas9 knockout plasmids, selected from the group consisting of 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 plasmid and / or shRNA lentiviral particle gene silencer, including those selected from the group consisting of 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 comprises 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.

[0114] The culture media described herein may contain other components or analogs thereof. As used herein, the term "analog" can refer to a biologically active analog of any of the components of the culture medium. Such analogs may be natural or synthetic.

[0115] The specific biologically active ligands and compounds used in the media defined herein, such as insulin, progesterone, etc., are used for illustrative purposes. However, those skilled in the art will readily recognize that analogs of such ligands and compounds may equally be used as substitutes, so long as they retain the relevant biological activity. Those skilled in the art will be able to routinely identify other biologically active compounds suitable for use as substitutes. By way of example, these may be naturally occurring compounds or compounds that can be made by synthetic or semi-synthetic methods. Stem Cell Growth Medium

[0116] In some embodiments, the pluripotent stem cell growth medium used herein is serum-free or substantially serum-free. Alternatively, the stem cell growth medium may be supplemented with KSR, optionally at about 5%-15% KSR. In some embodiments, the stem cell growth medium is a serum-free or substantially serum-free defined in vitro culture medium comprising a basal medium containing water, salts, amino acids, a carbon source, vitamins, lipids, and a buffer. In some embodiments, the stem cell growth medium can further comprise sodium pyruvate. Sodium pyruvate may be included in the culture 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 growth medium comprises a neurobasal medium (e.g., Neurobasal or Neurobasal A from Thermo Fisher Scientific). The stem cell growth medium can further comprise or be supplemented with B-27 supplement and N-2 supplement. As will be appreciated by those skilled in the art, B-27 supplement is a defined mixture of antioxidant enzymes, proteins, vitamins, and fatty acids combined in optimized ratios to support neuronal survival in culture. N2 supplement is a chemically defined, serum-free supplement that can be used for the growth and expression of neuroblastoma and postmitotic neurons in primary culture from both the peripheral and central nervous systems.

[0117] Stem cell growth medium can contain an effective amount of L-glutamine or its analogue.L-glutamine can be contained in the culture medium at a concentration of 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, for example, about 2 mM.In some embodiments, L-glutamine is contained in the stem cell growth medium at a concentration of about 2 mM.

[0118] The stem cell growth medium further includes or is supplemented with an effective amount of a reducing agent. The reducing agent can include beta-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 growth medium can be from about 0.1 μM to about 1 mM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900 μM, 1 mM, or a value or range between any two of these values). In some embodiments, the reducing agent is included in the stem cell growth medium at a concentration of about 0.1 mM. In some embodiments, the stem cell growth medium includes beta-mercaptoethanol (BME) at a concentration of about 0.1 mM.

[0119] In some embodiments, the stem cell growth medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Neurobasal (R) A, N2, B27, L-glutamine or analogs thereof, a reducing agent, an antibiotic, or a combination thereof, wherein these components are provided in amounts such that the medium is capable of supporting the growth of pluripotent stem cells on the substrate. In some embodiments, the stem cell growth medium is a medium such as DMEM / F12, Neurobasal (R) A, B-27, N-2, GlutaMax TM , β-mercaptoethanol, penicillin / streptomycin, or a combination thereof. In some embodiments, the stem cell growth medium is N2B27 medium. N2B27 medium can include 1:1 DMEM / F12 and Neurobasal A, 0.5x B-27, 0.5x N-2, 100 μM β-mercaptoethanol, 1x GlutaMAX, and IX penicillin-streptomycin. Post-transplant culture medium

[0120] The methods described herein also include co-culturing pluripotent stem cells (e.g., cell aggregates formed by ESCs) in a post-implantation culture medium following co-culturing in stem cell expansion medium. In some embodiments, the post-implantation culture medium is a post-implantation human embryo culture medium (e.g., hIVC1).

[0121] The post-implantation medium can include non-human serum. The non-human serum in the post-implantation culture medium can vary. In some embodiments, the post-implantation medium can include 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) volume per volume (% v / v), weight per volume (% w / v), or weight per weight (% w / w) of non-human serum (e.g., fetal bovine serum) of the medium. In some embodiments, the post-implantation medium comprises from about 15% to about 30% non-human serum (e.g., fetal bovine serum). In some embodiments, the post-implantation medium comprises about 20% fetal bovine serum. In some embodiments, the fetal bovine serum is inactivated.

[0122] The post-implantation medium can further comprise: (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.

[0123] The amount of insulin, estrogen, progesterone, or analogs or receptor agonists thereof present in the post-implantation medium can vary. For example, in some embodiments, the post-implantation medium contains from about 1 ng / ml to about 100 mg / ml (e.g., about 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml, 1 μg / ml, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / ml, 1 mg / ml, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / ml, or any value or range between any two of these values) of one or more hormones (e.g., progesterone) and / or one or more growth factors (e.g., insulin or insulin-like growth factor).In some embodiments, the post-transplant medium contains from about 0.5 nM to about 1 mM (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900 nM, 0.5 mM, 1 mM, or a number or range between any two of these values) of a hormone (e.g., estrogen) and / or insulin or insulin-like growth factor.

[0124] In some embodiments, the insulin receptor agonist is selected from the group consisting of IGF-I, IGF-II, analogs thereof, or any combination thereof. The estrogen receptor agonist can be selected from the group consisting of β-estradiol, estrone, estriol, and estetrol, or any analogs thereof. The post-transplantation medium can include transferrin, sodium selenium, ethanolamine, or analogs thereof. The post-transplantation medium can include insulin-transferrin-selenium-ethanolamine (ITS-X). In some embodiments, the post-transplantation medium further includes an agonist of the activin type 1 or type 2 receptor. In some embodiments, the post-transplantation medium does not include a reducing agent.

[0125] In some embodiments, the post-implantation culture medium may comprise the basal medium defined above (e.g., Advanced DMEM / F12) 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 (e.g., 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).

[0126] The post-transplantation medium may also contain an effective amount of a non-essential amino acid selected from the group including L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The post-transplantation medium may also contain an effective amount of an essential amino acid selected from the group including L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine. The non-essential and / or essential amino acids can have an effective amount of, for example, from 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) volumes per volume (% v / v), weight per volume (% w / v), or weight per weight (% w / w). In some embodiments, the post-transplant medium includes about 1% non-essential and / or essential amino acids.

[0127] The post-transplantation medium may contain L-glutamine. L-glutamine may be contained in the culture medium at a concentration of 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, for example, about 2 mM. In some embodiments, L-glutamine is contained in the culture medium at a concentration of about 2 mM.

[0128] Penicillin may be included in the post-implantation culture medium at a concentration of about 1 to about 500 units / ml, about 2 to about 250 units / ml, about 5 to about 100 units / ml, about 10 to about 50 units / ml, or about 20 to about 30 units / ml, e.g., about 25 units / ml. Streptomycin may be included in the culture medium at a concentration of about 1 to about 500 μg / ml, about 2 to about 250 μg / ml, about 5 to about 100 μg / ml, about 10 to about 50 μg / ml, or 25 or about 20 to about 30 μg / ml, e.g., about 25 μg / ml. The culture medium may include 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 culture medium may further include an antibacterial agent, such as sodium lactate.

[0129] The post-transplant culture medium may also contain an effective amount of glucose. Glucose may be present in the culture medium at a concentration of about 0.5 mM to about 5 mM, about 1 mM to about 4 mM, about 1.5 mM to about 3 mM, for example, about 2 mM. In some embodiments, glucose is present in the culture medium at a concentration of about 1.8 mM.

[0130] In some embodiments, the post-implantation culture medium comprises DMEM / F12, fetal bovine serum, GlutaMax, essential and non-essential amino acids, ITS-X, β-estrodiol, progesterone, glucose, sodium lactate, penicillin and / or streptomycin, or any combination thereof. In some embodiments, 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-estrodiol, about 200 ng / mL progesterone, or any combination thereof. application

[0131] Provided herein are synthetic embryo models obtained by the in vitro methods described herein for use in patients in need thereof in methods for diagnosing, preventing, or treating disease. For example, embryonic cells obtained from the present invention can be used in stem cell therapies, such as for the treatment of cancer, tissue replacement, reconstructive surgery, tissue repair, wound healing, bone marrow transplantation, stroke, alopecia, blindness, hearing loss, diabetes, heart disease, bowel disease, arthritis, skeletal injuries, tooth replacement, neurological disorders, and other conditions in which replacement cells or tissues may be beneficial. As will be appreciated by those skilled in the art, the cells can also be used to screen therapeutic compounds for efficacy and safety.

[0132] In some embodiments, synthetic embryo structures for use in a patient in need thereof in methods for diagnosing, preventing, or treating disease, as described herein, may be used for transplantation into the patient. In certain embodiments, the pluripotent stem cells used to obtain the embryo may originally have been obtained from the patient, thus reducing the likelihood of rejection by the patient's immune system. Thus, pluripotent stem cells, e.g., embryonic stem cells, obtained from a patient may be cultured using the methods described herein to provide material for transplantation into the patient to prevent or treat a disease condition. For example, embryos may be used to grow replacement organs or tissues to restore function to a patient whose function has been lost through degeneration, aging, and / or disease.

[0133] Also disclosed herein are methods for providing transgenic non-human animals, including gestating embryos derived from cells cultured using the in vitro methods described herein. Such transgenic non-human animals can be useful in drug screening or disease research. For example, model animals can be produced to study specific conditions. It is envisioned that the methods provided herein could be used to make developing transgenic and chimeric embryos (which currently rely on the labor-intensive process of, for example, harvesting blastocysts and manually replacing the inner cell mass) even more efficient.

[0134] Disclosed herein are methods for investigating the effect of a test agent on embryonic development. In some embodiments, the method includes: a) generating a synthetic embryo model using the methods described herein; b) contacting the synthetic embryo model with a test agent; and c) determining the effect of the test agent on the synthetic embryo model. In some embodiments, the determining includes comparing the phenotype or genotype of the synthetic embryo in the presence of the test agent to the phenotype or genotype of the synthetic embryo in the absence of the test agent. The method can include contacting mammalian pluripotent stem cells (e.g., wild-type and modified ESCs) with the test agent during or after step (a), and before, during, or after step (b), and before, during, or after step (c).

[0135] The method can include determining the subsequent effect on the formation of a synthetic embryo at various developmental stages. The determining can be performed using any method known in the art. For example, the method can include recording one or more images of the embryonic structure.

[0136] Disclosed herein are methods for investigating mechanisms involved in embryogenesis. In some embodiments, the methods include any of the in vitro methods for generating synthetic embryonic structures at various developmental stages described herein. Investigating mechanisms involved in embryogenesis can include any method known in the art. For example, the investigation can include investigating the effects of test agents on embryonic development, as described above. In some embodiments, investigating mechanisms involved in embryogenesis can include determining the effects of genetic perturbations on embryonic structures.

[0137] The method may include recording multiple images of the composite embryonic structure. The multiple images may be recorded over a predetermined period of time, thus illustrating the development of the embryonic structure over time. The imaging device may include a microscope device, a suitable recording device, and optionally an image processing device.

[0138] Typically, fluorescent markers, such as fluorescent dyes or fluorescent marker proteins, are used in imaging embryonic development. Such markers can be added to the culture system. For example, fluorescent dyes 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 embryonic structure may endogenously produce such fluorescent markers; for example, it may contain one or more cells that express a fluorescent marker protein. Such cells may be genetically engineered to confer the ability to express such marker proteins. Thus, fluorescent imaging devices may be particularly suitable for the described methods. Imaging devices may include fluorescent microscopes, such as confocal microscopes, including, but not limited to, wide-field microscopes, scanning and spinning-disk confocal microscopes, and light-sheet microscopes.

[0139] Confocal microscopes image a single point in a specimen at a given time, but can generate two- or three-dimensional images by scanning various points within the specimen in a regular raster to provide image data that can be assembled into two- or three-dimensional images. For example, scanning a specimen in a single plane can generate a two-dimensional image of a slice through the specimen. Multiple such two-dimensional images, or "stacks," are combined to provide a three-dimensional image. Spinning disk confocal microscopes offer additional benefits over confocal laser scanning microscopes. Additionally, light sheet microscopes can also provide better images of embryonic development.

[0140] Also disclosed herein are methods for elucidating the role of genes in embryonic development, comprising obtaining pluripotent stem cells in which a gene has been modified or knocked out, and culturing the pluripotent stem cells (e.g., ESCs) and extraembryonic-like cells generated by overexpression of a transcription factor with the ESCs using the in vitro methods described herein. Thus, the methods may be useful in developing treatments for conditions related to embryonic development, such as infertility treatments.

[0141] Also disclosed herein is a method of imaging a developing embryo, comprising culturing mammalian pluripotent stem cells (e.g., ESCs) and mammalian extraembryonic-like cells generated by overexpression of transcription factors with ESCs or mammalian synthetic embryonic structures using the methods described herein, and recording an image of the embryo using an imaging device. The image may be a two-dimensional image or a three-dimensional image. Multiple images may be recorded of the same embryo. The imaging device may further include a microscope and a suitable recording device. The imaging device may additionally include an image processing device. Additionally, the imaging device may further include a fluorescence microscope. Additionally or alternatively, the imaging device may further include a confocal microscope. [Example]

[0142] Certain aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the invention in any way. material and method

[0143] The following experimental materials and methods were used for Examples 1-5 below. ethics statement

[0144] Work with human embryonic stem cells (Shef6) was performed under license SCSC21-38 with approval from the UK Human Stem Cell Bank Steering Committee and in accordance with the UK Code of Practice for Use of Human Stem Cell Lines. Research on human embryos was regulated by the Human Fertility and Embryology Authority (HFEA) and conducted under license R0193. Ethical approval was obtained from the Human Biology Research Ethics Committee at the University of Cambridge. Patients undergoing IVF (in vitro fertilization) at CARE Fertility, Bourn Hall Fertility Clinic, Herts & Essex Fertility Clinic, and King's Fertility were given the option of continuing to bank their embryos at the end of treatment, discarding them, or donating them for research (including project-specific information) or training. Patients were provided with counseling, received no financial benefits, and could withdraw from participation at any time until the embryos were used in research. Informed research consent for donated embryos was obtained from both gamete donors. Only blastocysts displaying appropriate morphology (i.e., an expanded blastocyst cavity and a healthy inner cell mass) were used for subsequent experiments. Embryos were not cultured until 14 days post-fertilization (dpf) or the first appearance of the primitive streak. Mice were housed in animal enclosures with a 12:12-h light / dark cycle and had free access to food and water.Experiments with mice were conducted under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and in accordance with ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). Experiments were approved by the Home Office. This study used CD1 and Fl wild-type males between 6 and 45 weeks of age and CD1 and Fl wild-type females between 6 and 18 weeks of age. Animals were examined daily. Animals exhibiting health concerns were killed by cervical dislocation. All embryo and embryoid body work was performed in the UK and in accordance with the 2021 ISSCR guidelines. hESCs culture

[0145] Human ESCs from Shef6 (from the UK Stem Cell Bank) or RUES2 (kindly provided by Ali Brivanlou, Rockefeller University) were cultured on Matrigel-coated plates in mTESR medium (05825, STEMCELL Technologies) at 37°C, 20% O2, and 5% CO2. Plates were coated with 1.6% growth factor-reduced Matrigel (356230, BD Biosciences) in DMEM / F12 (21331-020, Life Technologies) and incubated at 37°C for 1 hour. hESCs were subcultured using TrypLE (12604013, ThermoFisher Scientific). During the first 24 hours after passage, 10 μM of ROCK inhibitor Y-27632 (72304, STEMCELL Technologies) was added. The medium was changed every 24 hours. Cells were routinely tested for mycoplasma contamination by PCR (6601, Takara Bio) and authenticated by short tandem repeat analysis.To convert primed hESCs to RSeT or PXGL culture conditions, cells were cultured on mitomycin C-inactivated CF-1 MEFs (3 × 10 cells / mL) in medium consisting of DMEM / F12 supplemented with 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 (University of Cambridge, Department of Biochemistry), and 10 μM ROCK inhibitor Y-27632 (72304, STEMCELL Technologies). 3 cells / cm 2GSC-6101G, Amsbio). For RSeT cells, the medium was switched to RSeT medium after 24 hours (05978, STEMCELL Technologies). Cells were maintained in RSeT and passaged every 4–5 days as described above. For PXGL cells, transformation was performed as previously described. Briefly, cells were cultured at 5% O2 and 7% CO2. The medium was switched to chemical resetting media 1 (cRM-1), consisting of N2B27 medium supplemented with 1 μM PD0325901 (Stem Cell Institute, University of Cambridge), 10 ng / mL human recombinant LIF (300-05, PeproTech), and 1 mM valproic acid. N2B27 medium contained 1:1 DMEM / F12 and Neurobasal A (10888-0222, Thermo Fisher Scientific) supplemented with 0.5x B27 (10889-038, Thermo Fisher Scientific), 0.5x N2 (in-house), 100 μM β-mercaptoethanol, 1x GlutaMAX, and 1x penicillin-streptomycin. cRM-1 medium was changed every 48 h for 4 days, after which the medium was replaced with PXGL medium. PXGL medium consists of N2B27 supplemented with 1 μM PD0325901, 10 ng / mL human recombinant LIF, 2 μM Go6983 (2285, Tocris), and 2 μM XAV939 (X3004, Merck). PXGL cells were passaged every 4–6 days using TrypLE (12604013, Thermo Fisher Scientific) for 3 min. At the time of passage, 10 μM of ROCK inhibitor Y-27632 and 1 μL / cm 2The cells were cultured for 24 hours with 100 μg of Geltrex (A1413201, Thermo Fisher Scientific). For differentiation into yolk sac-like cells or trophoblasts, RSeT cells were passaged onto Matrigel-coated IBIDI chamber slides. After 24 hours, the medium was switched 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 "PA" (1 μM PD0325901 and 1 μM A83-01, 72022, STEMCELL Technologies) with or without 500 nM lysophosphatidic acid-LPA (3854, Tocris). Generation of inducible hESC cell lines

[0146] The full-length coding sequence was amplified from human cell line cDNA containing AttB overhangs using Phusion High-Fidelity DNA polymerase (M0530S, New England BioLabs) according to the manufacturer's instructions to generate the piggyBac plasmid. The amplicon was transferred to the pDONR221 entry plasmid using BP clonase (11789100, ThermoFisher Scientific) and then transferred to the destination plasmid using LR clonase (11791020, ThermoFisher Scientific) according to the manufacturer's instructions. hESCs were electroporated with pBase plasmids expressing PB-CAG-rTTA3-Bsd or PB-CAG-rTTA3-Zeo and PiggyBac transposase, plus GATA6-3XFLAG-TetOn-Zeo (entry plasmid 72922, Addgene) and / or SOX17-TetOn-Hygro or GATA3-EGFP-TetO-Hygro and / or TFAP2C-TetOn-G418, using the Neon transfection system with the following settings: 1200V, 20ms, and two pulses. Two days after transfection, antibiotics were applied at a quarter dose and increased to a final concentration of 100 μg / mL zeocin (ant-zn-1, Invitrogen), 20 μg / mL blasticidin (Al 13903, 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. Clones were generated by manually picking single colonies under a dissecting microscope.Transgene activation was induced by the addition of 1 μg / mL doxycycline hydrochloride (D9891, Sigma). To select clones for downstream experiments, isolated colonies that survived manual picking were induced for 72 hours, and cell pellets were collected for qPCR or stained for immunofluorescence analysis. Immunofluorescence analysis was performed on primed hESCs. Transgene expression and other key lineage markers were assessed for changes in expression compared to uninduced controls. Clones with robust transgene upregulation and downstream upregulation of uninduced lineage markers were selected for subsequent experiments (e.g., 1-2 clones per transgenic line). Note that AP2Y-induced cells could not be reset in PXGL-naive conditions. qRT-PCR analysis

[0147] Cell pellets were harvested and RNA was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions. Reverse transcriptase reactions were performed with 1 μg of RNA using random primers (C1181, Promega), dNTPs (N0447S, New England Biolabs), RNAse inhibitor (M0314L, New England Biolabs), and M-MuLV reverse transcriptase (M0253L, New England Biolabs). RT-qPCR was performed on a Step One Plus Real-Time PCR machine (Applied Biosystems) using Power SYBR Green PCR Master Mix (4368708, ThermoFisher Scientific). The following program was used: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A single melting curve was observed for all primers used in this study. The oligonucleotides used in this study are provided in Table 2 below. [Table 2] Generation of hPSC-mouse embryo chimeras

[0148] For human cell-mouse embryo chimeras, oviducts and uterine horns were collected and flushed with M2 medium (in-house preparation) supplemented with 4 mg / mL BSA (A9418, Sigma) on E2.5. The collected, uncompacted 8-cell embryos were then treated with Acidic Tyrode's Solution to remove the zona pellucida. Human cells (wild-type, 3-day-induced GATA6-SOX17 cells, and 3-day-induced GATA3-AP2Y RSeT cells) were prepared by dissociating cells with TrypLE and washing as described above. Cells were resuspended in either RSeT or N2B27 medium supplemented with 5% KSR and 1 μg / mL doxycycline. The resulting small cell clumps were allowed to accumulate in these medium wells with 8-cell mouse embryos for 24 hours, then transferred to KSOM ± 1 μg / mL doxycycline and cultured for an additional 24 hours until E4.5. For negative controls, embryos were cultured under these conditions without the addition of human cells. 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 either the SOX2, SOX17, and / or GATA3 populations was quantified. Generation of inducible human embryoid bodies

[0149] To generate 3D stem cell-derived models of postimplantation embryos, RSeT cells were routinely subcultured between passages 2 and 6 after transfer to RSeT medium. The following day (day -3), the medium for extraembryonic-like cells (induced GATA6, induced GATA6-SOX17, or induced GATA3-AP2Y) was changed to N2B27 containing 5% Knockout Serum Replacement and 1 μg / mL DOX. This medium was refreshed every 24 h for 3 days. On day 0 (the day of aggregation), Aggrewell dishes (34415, STEMCELL Technologies) were precoated with anti-adhesion solution (07010, STEMCELL Technologies) and centrifuged at 2000 g for 5 min. Before adding experimental medium, wells were washed twice with PBS. This medium consisted of N2B27 with 5% knockout serum replacement, 1 μg / mL doxycycline, and 10 μM Y-27632. Induced cells and wild-type ESCs were enzymatically detached 1 hour after adding 10 μM Y-27632 to wells containing cells for inducible human embryoid body generation. Dissociated cells were pelleted and resuspended in experimental medium and placed in gelatin-coated wells for MEF depletion. After 15–30 minutes, cells were counted, mixed, and plated in Aggrewell dishes. The final count was 8 wild-type ESCs, 8 hypoblast-like cells, and 16 trophoblast-like cells per microwell in Aggrewell. In vitro cultured human embryos had a 32:24:228 epiblast:hypoblast:trophoblast ratio at day 8 of gestation. Importantly, however, many of the trophoblast cells were not in contact with tissue derived from the inner cell mass or had terminally differentiated. Additionally, inducible GATA6-SOX17 cells in culture proliferated slower than the other two cell populations after the addition of doxycycline. Therefore, we utilized (1) a total cell number similar to that used in the mouse model to ensure successful cell sorting; (2) a cell ratio reflecting the embryo before and after implantation; and (3) an initial seeding density with a reduced number of inducible GATA3-AP2Y cells and an increased number of inducible GATA6-SOX17 cells.

[0150] On day 1, the medium was replaced twice, two-thirds N2B27 with 5% knockout serum replacement and 1 μg / mL doxycycline. On day 2, half of the Aggrewell was replaced with hIVCl medium containing 25 ng / mL hIGFl (78022.1 STEMCELL Technologies) and 1 μg / mL doxycycline. hIVCl medium consisted of Advanced DMEM / F12 (12634-010 Thermo Fisher Scientific) supplemented with 20% inactivated FBS (10270106 Thermo Fisher Scientific), 1× Glutamax, 1× NEAA, 1× Essential AA, 1× ITS-X, 25 U / mL Pen / Strep, 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). Half of this medium was replaced daily from day 3 onward. On day 4, aggregates were manually harvested using a mouth pipette under a dissecting microscope and transferred into individual wells of an ultra-low attachment 96-well plate (CLS7007, Corning) in hIVCl medium with IGF1 and doxycycline as described above for subsequent culture. Immunostaining and image analysis

[0151] Samples were washed with phosphate-buffered saline (PBS) and fixed in 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 incubated in PBS with 0.3% (vol / vol) Triton X-100 (T8787, Sigma-Aldrich) containing 0.1 mM glycine (BP381-1, Thermofisher Scientific) for 30 minutes at room temperature. Samples were blocked in blocking buffer (PBST with 5% (w / vol) BSA, A9418, Sigma) and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. A list of primary antibodies is provided in Table 3 below. Samples were washed three times with PBST and incubated with fluorescently conjugated AlexaFlour secondary antibody (ThermoFisher Scientific, 1:500) and DAPI (D3571, ThermoFisher Scientific, 1 μg / mL) diluted in blocking buffer for 2 hours at room temperature. For pSMAD1.5 quantification, OCT4- or GATA6-positive nuclei were isolated, and the fluorescence intensity of pSMAD1.5 was quantified. For SMAD2.3 quantification, the fluorescence intensity of nuclei and cytoplasm (excluding the outermost GFP+ cell layer) was quantified. Data are presented as the ratio of nuclear to cytoplasmic fluorescence intensity. Immunofluorescence images were analyzed using FIJI. The Spot tool with manual curation in Imaris software (version 9.1.2, Oxford Instruments) was used to quantify total vesicle number in day 4 embryoid bodies and generate spot renders. [Table 3-1] [Table 3-2] Thawing and culturing human embryos

[0152] Human embryos were thawed and cultured as previously described. Briefly, cryopreserved human blastocysts (5 or 6 dpf) were thawed using the Kitazato Thawing Kit (VT8202-2, Hunter Scientific) according to the manufacturer's instructions. The day before thawing, thawing solution (TS) was placed at 37°C overnight. The following day, in vitro fertilization (IVF) straws were immersed in 1 mL of prewarmed TS for 1 minute. The embryos were then transferred to diluent solution (DS) for 3 minutes, wash solution 1 (WS1) for 5 minutes, and wash solution 2 (WS2) for 1 minute. These steps were performed in a Reproplate (Reproplate, Hunter Scientific) using a Stripper micropipette (Origio). Embryos were cultured for 1-4 hours in pre-equilibrated human IVC1 supplemented with 50 ng / mL insulin growth factor-1 (IGF1) (Product Code: 78078, Stemcell Technologies) under mineral oil for recovery at 37°C and 5% CO2 in normoxia. After thawing, blastocysts were briefly treated with acidic Tyrode's solution (Product Code: T1788, Sigma) to remove the zona pellucida and placed in pre-equilibrated human post-implantation medium (hIVC1) in an 8-well μ-Slide tissue culture plate (Product Code: 80826, Ibidi) at a volume of approximately 400 μL per well per embryo. Half-medium changes were performed every 24 hours. statistical analysis

[0153] Statistical analyses were performed using Graphpad Prism v9.4. Sample sizes were not predetermined, and researchers were blinded to the conditions. All experiments were performed at least twice independently. Data were tested for normality using the Shapiro-Wilk test. 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 indicated in the figure legends. Sample numbers are indicated in the figure legends. All statistical tests were two-sided. For each test, independent samples were used. Replicates are biological unless otherwise stated. Within plots, all data are presented as mean ± SEM. For box plots, boxes represent 25 th -75 th The figures represent quartiles, and the whiskers represent minimum and maximum values, the center line represents the median, and the + symbol represents the mean. In multiple comparison tests, comparisons were made only with the control condition. Unmarked pairwise comparisons were not significant (p>0.05). Collection, generation, and sequencing of single-nucleus ATAC / RNA10x libraries

[0154] To collect postimplantation embryoid-like models for single-cell sequencing, properly organized embryoid bodies were visually collected on days 4, 6, and 8 and washed twice with PBS in a 4-well dish before being transferred to TrypLE. Samples were mixed by pipetting every 5 minutes for 10–20 minutes until dissociated. Enzyme activity was inactivated by adding 2 volumes of 20% fetal bovine serum (FBS) in PBS. Cells were collected in Falcon tubes, pelleted, and resuspended in a freezing buffer consisting of 50 mM Tris (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), 1x protease inhibitor cocktail (P8340, Sigma-Aldrich), and a 1:2500 dilution of superasin (AM2694, Invitrogen) at pH 8.0. For cell lines, 10,000 cells were counted, pelleted, and resuspended in the above freezing medium before slow freezing at -80°C.

[0155] Nuclei were isolated and libraries were constructed using the 10x Genomics low-input nuclei isolation protocol. Briefly, frozen cell pellets were thawed in a 37°C water bath for 30 seconds and centrifuged (500xg for 5 minutes at 4°C) to pellet the cells. The supernatant was then aspirated. The cell pellets were washed twice with 200 μL of 1x PBS containing 0.04% BSA and centrifuged. The supernatant was aspirated between washes. Next, chilled lysis buffer (45 μL per sample) was added to the washed cell pellets. The lysis buffer-added cell pellets were placed on ice for 3 minutes. Wash buffer (50 μL per sample) was then added. The washed, isolated nuclei were resuspended in diluted nuclei buffer. The isolated nuclei were resuspended in 5 μL of diluted nuclei buffer and added directly to the transposition reaction. All following steps followed the 10x Genomics Single Cell Multiome ATAC and Gene Expression protocols according to the manufacturer's specifications and guidelines. The final libraries were loaded onto a NextSeq 2000 using the P2 100-cycle kit at a loading concentration of 650 μM and paired-end sequenced according to the 10x Genomics recommended sequencing reads (28 / 10 / 10 / 90 cycles for gene expression libraries and 50 / 8 / 24 / 49 cycles for ATAC libraries). Single-cell sequencing analysis Processing and Quality Control

[0156] Raw reads were analyzed using the CellRangerARC pipeline to generate ATAC and RNAfastq files for each sample, and then genome and transcriptome reads were aligned. The matrices were then imported into Seurat48 and Signac49 using the Read10X_h5 command. For ATAC-seq data, peaks from standard chromosomes were used, and peaks were called using macs2, with the addition of an additional Signac assay. Cells with RNA UMI counts >500, mitochondrial reads <20%, ATAC reads >500, TSS enrichment >1, and called as singlets using scDblFinder50 were retained for downstream analysis. For UMAP projections, SCTransform was used to regress RNA counts on mitochondrial percentage and cell cycle score. PCA and LSI graphs were used to generate weighted nearest neighbor (wnn) embeddings that considered both modalities. ChromVAR51 was used to calculate motif accessibility scores for peak analysis. Data visualization was performed using Seurat's DimPlot, FeaturePlot, VlnPlot, TSSPlot, and FragmentHist functions, as well as SCpubr's do_Alluvialplot and do_Nebulosaplot functions. Comparison with published datasets

[0157] We used the analysis tool scmap to project cell labels from other single-cell datasets onto the transcriptional data of the postimplantation embryo-like model. All reference data used were publicly available with published cell type annotations. Cynomolgus macaque gene names were converted to hgnc gene symbols using biomaRt. For data generated by smart-seq2 or other non-UMI-based single-cell sequencing methods, we used the scmapCluster method with a similarity threshold of 0.5. For UMI-based methods, we used the scmapCell followed by the scmapCell2Cluster method with w=2. We used diverse datasets to draw conclusions using scmap. Due to limited cell assignments in certain datasets, transcriptionally similar clusters (e.g., trophoblasts or amniotes) may be mismapped if both clusters are absent. For cell type assignment and processing of sequenced cell lines, a previously reported and validated logistic regression framework was applied to project cell line data onto published single-cell data and published cluster annotations (examples shown in training data) onto postimplantation embryo-like model clusters (examples shown in test data), resulting in quantitative measures of predicted similarity. Here, only differentially expressed genes (generated using Seurat's FindAllMarkers function on course cell assignments collapsed across amniote and mesoderm clusters) were used. Multivelo (Multivelo) RNA / chromatin velocity

[0158] The recently published velocity calculation method "Multivelo" was applied to the data, considering both single-cell ATAC and RNA data. Multivelo was run on all cells and passed the QC and processing described above. Analysis was based on available vignettes and "grid" methods with 1000 highly variable genes. Gene expression and chromvar were plotted over incubation time using the switchde package. CellPhoneDB analysis

[0159] Potential tissue signaling crosstalk was assessed using CellPhoneDB 2.0 with default settings. For simplicity, course cell assignments were used that grouped the isolated amniotic (AM-1, AM-2, AM-3) and mesodermal (MESO-1, MESO-2) clusters. Selected significant interactions were plotted as dot plots. Reanalysis of the human in vitro cultured embryo dataset

[0160] Previously published data were realigned to the hg38 human genome using kallisto or kb-bustools. Datasets not sequenced with UMI-based technologies were normalized using quminorm to quasiumis. Datasets were combined using SCTransform-based integration to generate single-cell RNA-seq datasets of human embryos spanning zygotes up to day 14 post-fertilization. Cells were clustered and assigned identity based on previous annotations and expression of reference markers. The datasets showed good overlap with datasets with cell type separation and some temporal resolution. SCENIC was used with default settings in R, and the AUC-regulon table was used to generate new assays in the Seurat object. Using this assay, we compared epiblast, hypoblast, and trophoblast lineages using the FindMarkers function in Seurat and performed a Wilcoxon rank test with Bonferroni correction to identify pairwise predicted differentially active regulons. Regulons enriched across both relevant comparisons (e.g., hypoblast vs. epiblast; hypoblast vs. trophoblast) were used as enriched active transcription factors for subsequent analysis (e.g., in the hypoblast). These factors were plotted relative to each other in Cytoscape. Data and code availability

[0161] GRCh38 (www.ncbi.nlm.nih.gov / assembly / GCF_000001405.26 / ) and GRCm38 (www.ncbi.nlm.nih.gov / assembly / GCF_000001635.20 / ) were used to align the sequencing data. The code used to analyze the data mentioned here is available at http: / / github.com / bweatherbee / human_model. Example 1 Induction of extraembryonic lineages

[0162] For the first time, factors capable of similarly upregulating extraembryonic gene programs in human ESCs were identified. We integrated published single-cell RNA sequencing data from human embryos cultured to gastrulation (Figure 6A-6E). The computational tool, SCENIC, was used to assess the predicted activity of transcription factors enriched in the epiblast, trophoblast, or hypoblast (Figure 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). Overexpression of GATA6 or SOX17 was shown to drive the endodermal gene program from primed hESCs. Therefore, GATA6 or SOX17 were selected as candidates for programming hESCs to become hypoblast-like. Similarly, GATA3 and TFAP2C were reported to share high chromatin co-occupancy during hESC differentiation into trophoblast stem cells. GATA3 and TFAP2C also demonstrated high predictive activity in trophoblasts. Thus, GATA3 and TFAP2C were selected as candidates for driving hESCs to become trophoblast-like cells. hESCs were generated and validated with doxycycline-inducible individual or combinatorial transgenes for transcription factors of interest (Figure 1A-B and Figure 6H).

[0163] Notably, the pluripotent state partially determines the differentiation potential of ESCs. Therefore, to assess the ability of selected candidate transcription factors to induce hESCs toward an extraembryonic-like expression profile, we overexpressed these factors—alone and in combination—in cells across the naive-to-primed pluripotency spectrum. Cells were cultured using three established starting conditions: PXGL, which supports preimplantation-like cells; RSeT, which generates preimplantation-like intermediate cells; and conventional mTeSRl, which maintains postimplantation-like cells. Significant differences in extraembryonic gene induction, at both protein and mRNA levels, were observed when using individual or combination transgenes and starting from different pluripotent states (Figure 7A-F). During hypoblast-like induction, GATA6 overexpression did not drive SOX17 expression from RSeT or PXGL conditions, whereas SOX17 overexpression resulted in robust GATA6 upregulation across the initial pluripotent state conditions (Figure 7A and Figure 7C-D). FOXA2 expression was consistently upregulated after combinatorial induction of GATA6 and SOX17 from primed and RSeT, but not from PXGL conditions (Figure 7C-D). These data indicated that GATA6 and SOX17 could indeed drive the endoderm gene program, but the regulation of specific downstream targets differed depending on the initial pluripotent state.

[0164] The AP2Y transgene appears to be particularly effective at upregulating GATA2 and CK7 expression when driving a trophoblast-like gene program. However, induction of AP2Y alone led to cell death and loss of transgene expression in primed cells, but not in RSeT or PXGL cells (Figure 7E-F). Combinatorial induction of GATA6 and SOX17 or GATA3 and AP2Y resulted in a consistent downregulation of pluripotency markers, including NANOG, SOX2, and OCT4 (Figure 7A-F).

[0165] RSeT hESCs may be the best starting cell type for generating the disclosed human postimplantation embryo model because they: (1) represent the pre- and post-implantation developmental stage; (2) express lower levels of amnion-specific genes after induction of GATA3 and AP2Y compared to primed cells (Figure 7B, 7E-F); and (3) are known to differentiate more easily into yolk sac-like endoderm cells pre- and post-implantation compared to PXGL cells. For these reasons, and due to the synergistic effect of dual induction of candidate transcription factors, inducible GATA6-SOX17 and inducible GATA3-AP2Y RSeT hESCs were used for hypoblast-like and trophoblast-like cell induction, respectively, in subsequent experiments. Dual induction of GATA6 and SOX17 from RSeT cells in basal medium induced endoderm gene expression comparable to that of the directed differentiation protocol in yolk sac-like cell differentiation conditions (Figure 8A-B). Dual induction of GATA3 and AP2Y from RSeT cells in basal medium induced trophoblast gene expression, albeit at different levels, when compared to the directed trophoblast differentiation protocol (Figures 8C-8D).

[0166] To further characterize RSeT hESCs induced to express GATA6-SOX17 or GATA3-AP2Y, we performed single-cell 10x multi-ome sequencing. Transcriptome and chromatin accessibility were simultaneously assessed. Cells were clustered based on their sample origin (Figure 1C, Figure 9A). Applying a logistic regression framework, wild-type, inducible GATA6-SOX17, and inducible GATA3-AP2Y RSeT hESCs showed the highest similarity to the epiblast, hypoblast, and cytotrophoblast of postimplantation embryos, respectively (Figure 1D). Furthermore, when compared to in vitro blastoid and directed differentiation models, RSeT hESCs showed similarity to pluripotent populations, and induced GATA6-SOX17 cells resembled blastoid-derived hypoblasts, while induced GATA3-AP2Y cells resembled postimplantation-like trophoblast stem cells but not blastoid-derived trophectoderm-like cells (Figure 9B). Analysis of differentially expressed genes and differentially accessible motifs revealed similar embryonic and extraembryonic dynamics (Figure 1E and Figure 9C). Specifically, we detected enriched expression and motif accessibility scores for pluripotency and epiblast markers in RSeT hESCs, hypoblast markers in GATA6-SOX17-induced cells, and trophoblast markers in GATA3-AP2Y-induced cells (Figure 1E). Taken together, these data demonstrate that transcription factor-mediated induction of extraembryonic cell fates from RSeT hESCs drove a hypoblast- or trophoblast-like gene program without the need for exogenous factors, despite heterogeneity and minor defects in marker gene expression (Figure 9C). The ability of induced cells to grow as stable cell lines in culture has not been tested. When aggregated with 8-cell mouse embryos, induced GATA6-SOX17 and GATA3-AP2Y cells shifted human cell contributions toward SOX17-positive primitive endoderm and GATA3-positive trophectoderm, respectively, relative to wild-type controls (Figure 9D-G).If this relative shift toward extraembryonic identity is sufficient to enable self-organization, it overcomes the challenges to successful co-culture of embryonic and extraembryonic-like cells caused by their competing culture medium requirements. Indeed, 1:1:1 co-culture of wild-type RSeT hESCs with inducible GATA6-SOX17 and inducible GATA3-AP2Y RSeT hESCs demonstrated good viability and mixed identity (Figure 1F). Example 2 Assembly of the 3D post-implantation model

[0167] Three RSeT hESC-derived cell types—wild-type, GATA6-SOX17-inducible, and GATA3-AP2Y-inducible—co-cultured in N2B27 medium, and expression of selected transcription factors was induced with doxycycline for 3 days. The cell mixture was then aggregated in Aggrewell dishes (Figure 2A). Cells aggregate within 24 hours. By 48 hours after aggregation, clear distinction between inner and outer cell domains was observed using bright field imaging (Figure 2A). At 48 hours after aggregation, the medium was changed to human embryonic post-implantation medium (hIVCl). Incubation with doxycycline was continued throughout the culture period, and proliferation was consistent across experiments (Figure 2A and Figure 2C). On day 4 after aggregation, the cell aggregates self-organized into a structure with a SOX2-positive, epiblast-like domain containing a central lumen; an outer monolayer of GATA3-positive putative trophoblast-like cells; and an intermediate, presumptive hypoblast-like domain GATA6-positive cells located between the inner lumenal domain and the outer layer (Figure 2B and Figure S10A).

[0168] Similar to the postimplantation mouse embryo model, the aggregates did not undergo a blastocyst-like morphology before forming postimplantation-like structures. The efficiency of inducible human embryoid body formation (defined as aggregates containing organized SOX2-positive domains surrounded by concentric layers of GATA6- and GATA3-positive cells) was approximately 23% (Figure 2D). In contrast, when primed mTeSRl or naive PXGL hESCs were used as the starting pluripotent state for constitutive wild-type, GATA6-SOX17-inducible, and GATA3-AP2Y-inducible cells, the efficiency of organized, multilineage structure formation was less than 5% (Figure 2D-E). The organized embryoid-like structures displayed organization reminiscent of human embryos at 8-9 days postfertilization (PFS) (Figure 2F).

[0169] The induced human embryoid bodies described herein expressed several other lineage markers in an organized manner, including N-cadherin, SOX17, and GATA4 in the presumed hypoblast-like compartment (Figure 2G). Structures with SOX17 and / or GATA6 expression were also observed within the outer GATA3-AP2Y-induced cells (marked with eGFP), which may reflect the reported tendency of peripheral cells to adopt an endodermal identity within the embryoid body proper. The epiblast-like inner compartment expressed SOX2, NANOG, and E-cadherin and maintained pluripotency and epithelial identity similar to human embryos (Figure 2G). Furthermore, this inner domain displayed apical-basal polarity with basal deposition of laminin and apical expression of PODLX, PARD6, and ZO-1 (Figure 2H). These data demonstrated that embryoid-like structures derived from RSeT hESCs can self-organize in minimal medium conditions. Example 3 Differentiation within embryoid bodies

[0170] To gain insight into whether our human embryo-like model developed gene expression and chromatin accessibility patterns reflective of native human embryos, we performed single-cell multiome RNA and transposase-accessible chromatin sequencing (ATAC-seq) at 4, 6, and 8 dpf (Figure 3A). Individual structures were selected for sequencing based on the development of three tissues: (1) the inner epithelial domain; (2) the intermediate domain surrounding the central epithelium; and (3) the outer GFP-positive cell layer (Figures 10B-10C). To unbiasedly assign clusters, we used scmap to project our dataset onto human and cynomolgus monkey datasets spanning the pre- and post-implantation stages through the gastrula stage (Figures 3B and 10D). This analysis allowed us to project gene expression signatures from previously annotated cynomolgus monkey cell type clusters onto our human embryo model. Using multiome-based rate estimation (multivelo), we found that estimated differentiation times correlated well with structural transitions from day 4 to day 8 post-aggregation. These data, combined with canonical marker expression, enabled annotation of cell types in our human embryonic-like structures (Figure 3B, Figures S10E-S10F). Clusters resembling late embryonic epiblast (L-EPI), amniote (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 and Figures S10E-S10F). These assigned clusters showed differences in their composition based on the date of sample collection, with the L-EPI cluster consisting exclusively of day 4 and day 6 structures, which progressively shifted over time from AM-1 to AM-2 and AM-3, and similarly from mesoderm to EXMC (Figure 10F).

[0171] Finally, the clusters described here were directly compared with previously annotated pre-implantation and pre-gastrulation cynomolgus monkey and human embryo datasets. This analysis demonstrated similarities between induced human embryoid body clusters and primate embryos (Figure 11A-11B). Similarly, induced human embryoid body clusters were aligned with in vitro hESC-derived models, including post-implantation amniotic sac embryoid bodies (PASEs), blastoid cells, and recently identified extraembryonic mesenchyme-like cells generated during TSC-like directed differentiation (Figure 11B-11C). When compared with embryoid-like structures, striking similarities were observed between in vitro amnion, hypoblast, and extraembryonic mesenchyme populations. However, distinct trophoblast-like clusters derived from GFP-positive induced GATA3-AP2Y cells were not identified, despite their presence as an outer layer within induced human embryoid bodies (Figure 3A and Figure 11D). Given the aberrant upregulation of endoderm markers after aggregation, it was unlikely that the induced GATA3-AP2Y-derived cells represented bona fide trophoblasts. Nevertheless, when human embryos were cultured in vitro at postimplantation stages, including those containing amnion and extraembryonic mesenchyme, the induced human embryoid bodies generated several cell types that failed to differentiate robustly. Indeed, immunofluorescence analysis demonstrated that the inner SOX2-positive domain upregulated amniotic markers, including CDX2 and ISL1, by day 6 after aggregation. By day 8 after aggregation, the inner domain expressed mature amniotic markers VTCN1 and HAND1 (Figure 3D and Figures 11A-11B), correlating with the transition between AM-1, AM-2, and AM-3. The GATA6-positive domain also expressed HAND1, confirming the presence of extraembryonic mesenchyme (Figures 12A-12B). A subset of GATA6-positive cells showed high coexpression of TBX20, further highlighting the presence of extraembryonic mesenchyme in this intermediate domain (Figures 12C-12D). In the majority of human embryoid bodies described herein, the entire epiblast-like domain differentiated toward an amniotic fate. However, in rare cases of embryoid-like structures at days 6-8 postaggregation, ISL1, SOX2, and BRACHYURY expression was regionally concentrated, disrupting dorsoventral and / or anterior-posterior symmetry (Figure 3D).

[0172] Recent reports have hypothesized that both amniocytes and primordial germ cells (PGCs) are precursors to gametes and are generated, at least in part, from bipotent progenitors. Therefore, we assessed whether such progenitors or their descendants are specified in the disclosed human embryo-like model. A PGC module score was first assigned based on the expression of genes identified in in vitro differentiated human PGC-like cells. Cells with transcriptomes resembling those of PGCs were identified (Figures 3E-3F). Cells at the 98th percentile of the PGC gene expression module score were labeled as putative PGC-like cells ("PGCs"). PGCs expressed TFAP2A (AP2a), a key marker for bipotent amniotic and PGC-like progenitors. In contrast to other cells in the AM-1 and AM-2 clusters, PGC-like cells expressed the pluripotency marker NANOG and the PGC markers PRDM1 (also known as BLIMP1) and NANOS3 (Figure 3G). Immunofluorescence analysis of a canonical set of human PGC markers confirmed that AP2Y / SOX17 / NANOG triple-positive PGC-like cells were observed by day 4 after aggregation and increased in number by day 6 (Figures 3H-I and ​and12F). These data demonstrate that robust PGC-like cell specification occurred simultaneously with amniotic cell formation and within the inner epiblast-like compartment, supporting the existence of bipotent progenitor cells for these two lineages.

[0173] Next, we used Shef6-mKate wild-type ESCs to confirm differentiation pathways within embryoid bodies. At day 4 after aggregation, the majority of wild-type cells contributed to the inner SOX2-positive epiblast-like domain, while some contributed to the GATA6-positive population (Figure 12G), consistent with the small proportion of early extraembryonic mesenchyme differentiation observed in sequencing analysis (Figure 10F). By day 6 after aggregation, wild-type cells contributed to the ISL1-positive amniotic domain, as well as GATA6- and TBX20-positive extraembryonic mesenchyme-like cells (Figures 12H and 12I). Finally, we confirmed the induction of AP2Y / SOX17 / NANOG triple-positive primordial germ cell-like cells from the mKate2-labeled wild-type population (Figure 12J). These data confirmed that the epiblast-like domain differentiated into several postimplantation lineages. Example 4 BMPs mediate epiblast differentiation

[0174] Amniotes, primordial germ cells, and extraembryonic mesenchyme are thought to differentiate in response to BMP signaling in primate embryos. To understand whether this is consistent in the human embryonic model disclosed herein, we examined the expression of downstream BMP-responsive genes ID1-4. ID1 and ID4 are upregulated during amniote formation, while ID2 and ID3 are enriched in both amniote and extraembryonic mesenchyme pathways, indicating that BMP signaling is likely activated (Figures 4A and 13A). In addition, the accessibility of the SMAD5 motif was high in both pathways, but the accessibility score of the SMAD2::SMAD3::SMAD4 motif, a downstream target of activin-NODAL signaling, was not (Figures 4B and 13B). Consistent with this observation, a high BMP and low NODAL signaling environment has recently been implicated in amniotic differentiation of marmoset ESCs and hESCs during extraembryonic mesenchymal differentiation, suggesting that similar dynamics may drive the differentiation of these populations within inductive human embryo-like structures.

[0175] To further understand potential tissue-tissue crosstalk in the disclosed human embryonic-like model, we used the computational tool CellPhoneDB to predict ligand-receptor pairings between clusters in the single-cell sequencing data (Figures 13C-13D). In this analysis, we used the expression of select receptor-ligand pairs across clusters to score potential tissue-tissue crosstalk. CellPhoneDB predicted that hypoblast cluster-derived BMP2 / 6 and extraembryonic mesenchyme-secreted BMP4 were likely mediators of tissue-tissue crosstalk. In contrast, predicted NODAL signaling between tissues was low, further supporting the existence of a high-BMP, low-NODAL signaling environment in the human embryonic-like model. When CellPhoneDB was applied to the single-cell sequencing data of three cell lines to generate the embryonic-like model, inducible GATA3-AP2Y cells were predicted to be the initial source of BMP (Figure 13D). Aggregation of inducible GATA6-SOX17 and wild-type RSeT hESCs alone (i.e., without inducible GATA3-AP2Y cells) or addition of the ALK1 / 2 / 3 / 6 (type I BMP receptor) inhibitor LDN193189 between days 0 and 2 blocked the formation of organized structures (Figures S13E-S13G), demonstrating the requirement for BMP secreted by inducible GATA3-AP2Y cells during embryoid body formation.

[0176] To confirm the role of BMP signaling during epiblast-like domain differentiation, we examined the expression of phosphorylated (p)SMAD1.5. The enrichment of pSMAD1.5 in the OCT4-positive epiblast-like domain at days 4 and 6 post-aggregation indicates active BMP signaling (Figure 4C). In contrast, these cells had a low nuclear / cytoplasmic ratio of total SMAD2.3, reflecting low NODAL signaling within the epiblast-like domain (Figure 4D), consistent with CellPhoneDB predictions. To functionally validate the role of BMP signaling in medial domain differentiation, human embryonic-like models were treated with LDN193189 between 48 and 96 hours post-aggregation. Treated structures showed increased maintenance of SOX2 expression and decreased upregulation of CDX2 and AP2a in the medial domain at days 4 and 6 post-aggregation compared to untreated controls or BMP4-treated structures. Addition of activin A, an agonist of SMAD2.3 signaling, resulted in a similar phenotype, albeit to a lesser extent (Figures 4E-F and S13H). Additionally, LDN193189 treatment reduced the number of primordial germ cell-like cells, whereas BMP4 or activin A treatment had little effect on the emergence of this population (Figures 4G-H). These data demonstrate that endogenous BMPs and NODAL are key drivers of amniotic and primordial germ cell-like cell differentiation from epiblast-like domains within inducible embryoid bodies. Example 5 SOX17 represses the anterior hypoblast

[0177] BMP signaling is localized to the posterior of the embryo through antagonistic activity in the anterior hypoblast, which secretes inhibitors of BMP, WNT, and NODAL, including CER1 and LEFTY1 (Figure 2F). Recently, these markers of the anterior hypoblast were demonstrated to be expressed in in vitro cultured pre- and postimplantation human embryos. Neither CER1 nor LEFTY1 were significantly expressed in the HYPO / VE single-cell sequencing cluster (Figure 5A). Reanalysis of previously published 10-fold single-cell RNA sequencing data from in vitro cultured postimplantation human embryos revealed that SOX17 regulon activity was significantly increased in the CER1-negative hypoblast subcluster (Figure 5B). These sequencing data are also listed in Supplementary Data Table 8 of Mole et al., "A single cell characterization of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast center," Nature Communications 2021;12(1):3679, the contents of which are incorporated by reference in their entirety. Indeed, induction of SOX17, alone or in combination with GATA6, reduced its ability to upregulate CER1 when compared with GATA6 overexpression alone (Figure 6H). To test whether induction of GATA6 alone alters the identity of the hypoblast-like cell subpopulation in our human embryonic-like model, we generated hypoblast-like cells with inducible expression of either GATA6 or SOX17, alone or in combination. We observed an increased proportion of CER1-positive cells in embryoid bodies induced from inducible GATA6 cells alone compared with embryoid bodies generated from inducible GATA6-SOX17 cells or SOX17 cells (Figures 5C and 5E and Figure S4A). To verify that SOX17 induction suppressed CER1 expression, doxycycline was discontinued on days 1 or 3 after aggregation.Removal of doxycycline on day 3 promoted CER1 expression in embryoid bodies, but not on day 1 (Figure 5C and Figure 14A). Co-staining of pSMAD1.5 with CER1 demonstrated 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). By day 6 after aggregation, CER1 expression decreased in all embryoid bodies, regardless of the initial hypoblast induction regime (Figure 14B). However, transient CER1 expression in anterior hypoblast-like cells affected the epiblast-like domain of embryoid bodies. Embryoid bodies generated by single GATA6 induction or doxycycline removal on day 3 showed increased expression of the primitive streak marker BRY / TBXT on day 6 after aggregation compared with structures with consistent GATA6-SOX17 or SOX17 induction (Figure 5G-Figure 5H and Figure 14C).

[0178] Together, these data demonstrate that functional differentiation of gene regulatory networks underlying the differentiation of hypoblast subpopulations is observed in embryoid bodies. They also demonstrate the repressive role of long-term SOX17 overexpression on CER1-positive pre-hypoblast identity. These experiments highlight the value of the modular embryoid body model for studying interactions between embryonic and extraembryonic tissues. Additional considerations

[0179] In this disclosure, we have generated a multilineage stem cell-derived model of human postimplantation embryos. This model reflects the developmental interactions between extraembryonic and embryonic-like tissues, undergoing lumen formation and differentiation of epiblast-like domains. In this stem cell-derived human embryo model, amniotic cells are generated in response to BMP signaling, which then undergo progressive maturation. Similarly, primordial germ cell-like cells differentiated smoothly in this human embryonic stem cell model. These cells were identified along an amniotic differentiation trajectory, providing evidence that they likely arise from a common AP2a-positive progenitor, as reported in other in vitro systems. Extraembryonic mesenchymal cells were also observed, closely resembling those in primate embryos. This analysis suggested a trajectory from a late epiblast-like population to a mesoderm intermediate, consistent with recently reported in vitro extraembryonic mesenchymal differentiation protocols, data from cynomolgus monkeys, and historical observations in rhesus monkey and human embryos.

[0180] Unexpectedly, modulation of the transgene used to drive hypoblast-like identity shifted the balance of hypoblast-like cell contribution from CER1-negative to CER1-positive hypoblast-like cells. This observation demonstrated that overexpression of SOX17 suppresses CER1-positive anterior hypoblast formation. NODAL signaling is required for anterior visceral endoderm formation in CER1-positive mice, and SOX17 can suppress excessive NODAL activity and antagonize its targets. The low NODAL activity observed in induced human embryoid bodies is further exacerbated by SOX17 overexpression, which may partially explain the low levels of anterior hypoblast formation in embryoid bodies. Additionally, the low NODAL environment, combined with the lack of anterior hypoblast, may contribute to the differentiation of an epiblast-like population over time.

[0181] In the disclosed human embryo model containing a CER1-positive hypoblast, a significant increase in primitive streak-like BRACHYUYRY / TBXT expression was observed despite the disappearance of CER1-positive cells by day 6. The transient presence of an anterior hypoblast-like population could protect the pluripotency of the epiblast-like domain for a long period of time, allowing cells to exit pluripotency at a later developmentally gastrulation-competent cell stage. These results contrast with embryoid bodies lacking an anterior hypoblast-like population, which primarily generate amnion. These data pointed to the possibility of the existence of a distinct intermediate pluripotent state capable of giving rise to both amnion and extraembryonic mesenchyme, but not to germ layer derivatives.

[0182] The modular generation of integrated embryoid bodies from their constituent parts is useful for interrogating the roles of specific tissues and tissue-specific gene requirements. However, using overexpression of transcription factors to generate extraembryonic tissues can also lead to differentiation defects. For example, GATA3-AP2Y induction drove a trophoblast-like gene program in 2D, but when aggregated in a human embryonic model, this cell population abnormally upregulated endoderm markers (including SOX17 and GATA6). Nevertheless, GATA3-AP2Y-induced cells were necessary for the successful organization of embryonic-like structures and likely served as an important source of BMPs. The initial pluripotent state was a key factor in inducing downstream gene regulatory networks. Evidence was provided that embryonic models can be efficiently formed using preimplantation-stage hESCs but not using more naive hESCs. However, because induction of trophoblast gene networks appears to be more robust in naive hESCs, the use of discordant pluripotency-generating embryo models better recapitulates the embryo. Similarly, lineage specification from different starting states may require different combinations of transcription factors. Thus, studies further investigating the epigenetic landscape and binding sites of these factors may help improve strategies for generating bona fide extraembryonic cells.

[0183] In summary, a modular model of human postimplantation development was presented, containing both embryonic-like and extraembryonic-like cells. The postimplantation embryo model self-organized and occasionally exhibited axis formation. Similar to other integrated human embryonic models, such as the blastoid model, further optimization was required to fully encompass all major lineages of the postimplantation embryo and maintain an embryonic-like morphology. Because this model was not transplantable, it lacked the ability to develop to the fetal stage and did not mimic stages beyond primitive streak formation. Furthermore, it did not include all cell types present in the gastrulation stage. However, the construction of these integrated models of postimplantation human embryos was an important step toward mechanistic studies of postimplantation development that were not feasible with in vivo human embryos. Terminology

[0184] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will recognize that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the present claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0185] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can interpret the plural to the singular and / or the singular to the plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a (indefinite article, one)," "an (indefinite article before a vowel)," and "the (definite article, the, this, the, etc.)" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless expressly stated otherwise.

[0186] It will be understood by those skilled in the art that the terms used generally herein, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to, including only," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to, including only," etc.). Furthermore, it will be understood by those skilled in the art that where a specific number of introduced claim recitations are intended, such intention will be expressly set forth in the claim, and that in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of "at least one" and "one or more" as introductory phrases to introduce claim recitations. However, the use of such phrases should not be construed to mean that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such an introduced claim recitation to embodiments containing only one of such recitations, even when the same claim includes the 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 to mean "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. Even when a specific number of specifically introduced claims is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations).Furthermore, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, such an interpretation is generally intended in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, such an interpretation is generally intended in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, or C, etc." includes, but is not limited to, systems including A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Furthermore, it should be understood by one skilled in the art that virtually any separating word and / or phrase, whether in the description, claims, or drawings, presenting two or more alternative terms, contemplates the possibility of including one of the terms, either term, or both terms.

[0187] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0188] As will be understood by those skilled in the art, for all purposes, such as in terms of providing a written description, all ranges disclosed herein include all possible subranges and combinations of subranges. Any range listed fully expresses, and one can readily recognize, that the same range can be divided into at least one half, third, quarter, fifth, tenth, etc. As a non-limiting example, each range described herein can be easily broken down into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," and the like, refer to ranges that are inclusive of the recited numbers and that can then be subdivided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 members refers to a group having 1, 2, or 3 members. Similarly, a group having 1-5 members refers to a group having 1, 2, 3, 4, or 5 members.

[0189] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. An in vitro method for generating a mammalian synthetic embryo, the following: Co-culturing wild-type mammalian ESCs, first modified mammalian ESCs containing the GATA6 gene and / or SOX17 gene, and second modified mammalian ESCs containing the GATA3 gene and / or TFAP2C gene under conditions in a culture medium that allows ESCs (embryonic stem cells) to self-organize into embryonic structures after transplantation. Methods that include...

2. The in vitro method of claim 1, wherein (1) the first modified mammalian ESC comprises an inducible GATA6 gene, an inducible SOX17 gene, or both, and / or (2) the second modified mammalian ESC comprises an inducible GATA3 gene, an inducible TFAP2C gene, or both.

3. The in vitro method of claim 1, wherein (1) the first modified mammalian ESC comprises an inducible GATA6 gene and an inducible SOX17 gene, and / or (2) the second modified mammalian ESC comprises an inducible GATA3 gene and an inducible TFAP2C gene.

4. (1) Contacting a first modified mammalian ESC and / or a second modified mammalian ESC with an inducer, and / or (2) Further comprising adjusting the intensity of induction by arbitrarily increasing or decreasing the concentration of the inducing factor, or by increasing or decreasing the duration of the inducing factor in the culture medium, The in vitro method according to any one of claims 1 to 3, optionally comprising: (i) the inducing factor being doxycycline; (ii) the inducing factor being supplied to the culture medium for a period of optionally about 1 to 7 days; and / or (iii) the inducing factor being supplied to the culture medium for the entire co-culture process.

5. (1) The wild-type mammalian ESCs and / or modified mammalian ESCs are naive ESCs or primed ESCs, and optionally, the wild-type mammalian ESCs and / or modified mammalian ESCs are pre-transplant naive hESCs, pre- and post-transplant-like pluripotent naive hESCs, or post-transplant primed hESCs, and optionally, the pre-transplant naive hESCs are cultured in PXGL medium prior to co-culture, the pre- and post-transplant-like pluripotent hESCs are cultured in RSeT medium prior to co-culture, and the post-transplant-like primed hESCs are cultured in mTeSR1 medium prior to co-culture. (2) The wild-type mammalian ESCs and the modified mammalian ESCs are pre- and post-transplant pluripotent hESCs, which may be cultured in RSeT medium before co-culturing and / or (3) The in vitro method according to any one of claims 1 to 3, wherein the wild-type mammalian ESC, the first modified mammalian ESC containing the GATA6 and / or SOX17 gene, and the second modified mammalian ESC containing the GATA3 and / or TFAP2C gene are provided in a ratio of approximately 1:1:1 to 1:1:5, and optionally in a ratio of approximately 1:1:1 to 1:1:

2.

6. ESCs are cultured on a substrate, which may optionally include dishes, U-plates, flasks, or microwell plates. The method according to any one of claims 1 to 3, wherein the ESCs are optionally cultured in inverted pyramidal microwells, and each inverted pyramidal microwell is optionally about 400 μm or about 800 μm in size and optionally about 400 μm or about 800 μm in diameter.

7. Co-culture involves co-culturing ESCs in stem cell growth medium for approximately 5 days, optional. (1) Passaging ESCs at least twice in stem cell growth medium, (2) The stem cell proliferation medium is a serum-free medium, (3) The stem cell growth medium comprises Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), neurobasal, N2, B27, L-glutamine or its analogues, reducing agents, antibiotics, or combinations thereof, and optionally, the reducing agents may include beta-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof. (4) The stem cell proliferation medium is N2B27 medium, which optionally contains DMEM / F12, Neurobasal, B27, N2, GlutaMax, β-mercaptoethanol, penicillin / streptomycin or a combination thereof, and optionally contains 1:1 DMEM / F12 and Neurobasal A, 0.5×B27, 0.5×N2, 100 μM β-mercaptoethanol, 1×GlutaMAX, and 1X penicillin-streptomycin. (5) ESCs aggregate after co-culture in stem cell proliferation medium for up to 24 hours, and optionally, aggregated ESCs show a distinction between the inner and outer cellular domains. An in vitro method according to any one of claims 1 to 3.

8. Co-culture includes co-culturing ESCs in post-transplant medium for at least two days following co-culture in stem cell proliferation medium, and optionally, (1) Co-culturing ESCs in the culture medium after transplantation is initiated approximately 2 days after the aggregation of ESCs, and / or (2) The post-transplant culture medium shall include Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or serum substitute, antibiotics, antibacterial agents, L-glutamine or its analogues, insulin, insulin analogues, or insulin receptor agonists, estrogen analogues, or estrogen receptor agonists, progesterone, progesterone analogues, or progesterone receptor agonists, or any combination thereof, and further optionally, (i) Non-human serum or serum substitutes include fetal bovine serum, bovine serum albumin, KnockOut™ Serum Replacement, or any combination thereof. (ii) Antibiotics include penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tyrosine, or any combination thereof. (iii) Estrogen receptor agonists are selected from the group including β-estradiol, estrone, estriol, and estetrol, or any analogue thereof. (iv) Insulin receptor agonists are selected from the group including IGF-I, IGF-II, their analogues, or any combination thereof. (v) The culture medium after transplantation contains an antibacterial agent, and optionally the antibacterial agent is sodium lactate. (vi) The post-transplant culture medium comprises transferrin, sodium selenium, ethanolamine, or any analogue thereof. (vii) The post-transplant culture medium shall consist of 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-estrodiol, progesterone, or any combination thereof. (viiii) The post-transplant culture medium shall consist of DMEM / F12, approximately 20% fetal bovine serum, approximately 1X GlutaMax, approximately 1X non-essential amino acids, approximately 1X essential amino acids, approximately 1X ITS-X, approximately 25 U / mL penicillin and / or streptomycin, approximately 1.8 nM glucose, approximately 0.22% sodium lactate, approximately 8 nM β-estrodiol, approximately 200 ng / mL progesterone, or any combination thereof. The in vitro method according to claim 7.

9. (1) The co-culture comprises transferring ESCs from one substrate to another substrate, (2) The post-transplant embryonic structure includes an inner epiblast-like domain, a single outer layer of trophoblast-like cells, and an intermediate hypoblast-like domain between the epiblast-like domain and the single outer layer of trophoblast-like cells, optionally the inner epiblast-like domain being SOX2-positive and containing a central lumen, the single outer layer of trophoblast-like cells being GATA3-positive, and the intermediate hypoblast-like domain being GATA6-positive, and optionally the inner epiblast-like domain exhibiting pluripotency and epithelial identity similar to that of a human embryo. (3) Post-transplant embryonic structures express N-cadherin and SOX17 in the hypoblastoid-like domain, CDX2 in the trophoblast-like cells, and / or SOX2, NANOG, and E-cadherin in the epiblast-like domain. (4) Post-transplant embryonic structures include late embryonic epiblast, amnion, mesoderm, extraembryonic mesenchyme, and / or cell clusters resembling the subblast / visceral endoderm. (5) Post-transplant embryo structures express 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 combinations thereof. (6) The embryonic structure after transplantation generates the amnion and primordial germ cells. An in vitro method according to any one of claims 1 to 3.

10. (1) The efficiency of forming post-transplant embryos from wild-type mammalian ESCs, first modified mammalian ESCs containing the GATA6 gene and / or SOX17 gene, and second modified mammalian ESCs containing the GATA3 gene and / or TFAP2C gene is 5%, 10%, 15%, 20%, 25%, 30%, 35%, or higher. (2) Without any in vivo steps, (3) None of the wild-type mammalian ESCs, the first modified mammalian ESCs containing the GATA6 gene and / or the SOX17 gene, and the second modified mammalian ESCs containing the GATA3 gene and / or the TFAP2C gene are present in the in vivo environment during co-culture, and optionally the in vivo environment includes tissues, organs, organisms, or combinations thereof, and / or (4) Not including culturing trophoblast stem cells, subblastoblast stem cells, or both alone or in combination with ESCs. An in vitro method according to any one of claims 1 to 3.

11. (1) A wild-type mammalian ESC, 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 are human ESCs, (2) The embryonic structure after transplantation is that of a human embryo. (3) The embryonic structure after transplantation resembles that of a post-implantation human embryo approximately 8-9 days after fertilization, and / or (4) The culture medium does not include the use of exogenous signaling pathway factors, optionally, the culture medium does not contain exogenous signaling pathway factors, or exogenous signaling pathway factors are supplied, optionally, the exogenous signaling pathway factors include WNT signaling pathway activators, TGFβ superfamily members, or both. An in vitro method according to any one of claims 1 to 3.

12. A synthetic embryo obtained by the method of any one of claims 1 to 3, The synthetic embryo is, optionally, a human embryo, and further optionally, a synthetic embryo that resembles a post-implantation human embryo approximately 8-9 days after fertilization.

13. A method for investigating mechanisms involved in embryonic development, comprising any method of claims 1 to 3.

14. A method for identifying compounds useful for treating a disease, comprising contacting a synthetic embryo obtained by an in vitro method according to any one of claims 1-3 with a compound.

15. A method for elucidating the role of candidate genes in embryonic development, comprising obtaining wild-type mammalian ESCs, first modified mammalian ESCs containing the GATA6 gene and / or the SOX17 gene, and second modified mammalian ESCs containing the GATA3 gene and / or the TFAP2C gene, wherein the candidate genes are modified or knocked out, and culturing the mammalian ESCs using the in vitro method of any one of claims 1 to 3.

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