Methods, culture media and devices for generating embryos in vitro from stem cells - Patents.com

Abstract

Disclosed herein are methods and compositions for culture media for the in vitro culture of synthetic embryos derived from mammalian pluripotent stem cells and extraembryonic stem cells. The methods and compositions described herein allow for the generation of synthetic embryos at various developmental stages leading up to early organogenesis and beyond. Also disclosed herein are embryo culture systems and methods of using the same.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 289,587, filed December 14, 2021, the contents of which are incorporated by reference in their entirety into this application for all purposes.

[0002] [Statement regarding federally funded R&D] This invention was made with Government support under Grant No. HD104575 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] [Reference to array list] This application has been submitted with a sequence listing in electronic format. The sequence listing is provided as a file entitled 30KJ-365851-WO_Sequence_Listing, created on December 13, 2022, and has a size of 9 kilobytes. The electronic format information of the sequence listing is incorporated by reference into this application in its entirety.

[0004] The present disclosure relates generally to the field of cell culture, and in particular to culturing embryos and stem cells. [Background technology]

[0005] 2. Description of Related Art During natural development, the zygote develops into the epiblast that forms the organism; the extraembryonic visceral endoderm (VE) that contributes to the yolk sac; and the extraembryonic ectoderm (ExE) that contributes to the placenta. Stem cells corresponding to these three lineages offer the possibility of completely regenerating the mammalian organism from multiple components rather than from a single totipotent zygote.

[0006] Embryonic stem cells (ES cells) derived from the epiblast show a remarkable ability to form embryo-like structures when aggregated, and when embedded in Matrigel, they can be induced to form trunk-like structures with somites, neural tubes, and gut. Although neurogenesis can be promoted in such "gastruloids" by suppressing the initial burst of Wnt activity, they do not accurately recapitulate the movements of gastrulation, nor do they fully represent the natural embryo anatomically. Other model embryoid bodies generated from ES cells aggregated with an ectopic morphogen signaling center can only generate the posterior midbrain, neural tube, cardiac tissue, and gut. Thus, these models do not recapitulate the entire development towards neural tube formation. Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need for stem cell-based embryo models that can capture natural stages of mammalian development in vitro, through gastrulation and neurulation, and beyond. [Means for solving the problem]

[0008] [overview] Disclosed herein are methods of generating synthetic embryos in vitro, which in some embodiments include: (a) co-culturing mammalian pluripotent stem cells and at least one extraembryonic stem cell in a first culture medium under first static conditions to allow the mammalian pluripotent stem cells and the extraembryonic stem cells to self-organize into a post-implantation embryo structure; (b) culturing the post-implantation embryo structure in a second culture medium under second static conditions to allow the post-implantation embryo structure to develop into a neurulating embryo structure; and (c) culturing the neurulating embryo structure under dynamic conditions in a culture chamber for at least one day to allow the neurulating embryo structure to develop into at least an early organogenic stage of a synthetic embryo.

[0009] The mammalian pluripotent stem cell may comprise a mammalian embryonic stem cell. In some embodiments, the at least one extraembryonic stem cell comprises a trophoblast stem cell, an induced extraembryonic endodermal stem cell, or both. In some embodiments, the induced extraembryonic endodermal stem cell, upon induction, can express a GATA transcription factor. The GATA transcription factor may be, for example, GATA4.

[0010] In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the first culture medium for up to 4 days.

[0011] In some embodiments, step (a) is from embryonic day E0 to E5.5. In some embodiments, the post-implantation embryo structure is a post-implantation pre-gastrulation embryo structure. In some embodiments, the post-implantation pre-gastrulation embryo structure resembles a natural embryo structure at E5.5. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in a substrate, e.g., the substrate comprises a dish, a U-plate, a flask, or a microwell plate. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in inverted pyramidal microwells. In some embodiments, 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.

[0012] In some embodiments, step (a) comprises culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in feeder cell (FC) medium, optionally passage the mammalian pluripotent stem cells and the extraembryonic stem cells in the feeder cell medium at least two times. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the FC medium for about 2 days. In some embodiments, step (a) comprises culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in in vitro culture (IVC) medium, optionally after culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the FC medium. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the IVC medium for about 2 days. In some embodiments, the FC medium and the IVC medium comprise a basal culture medium. The basal culture medium may comprise, for example, Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or a serum substitute thereof, a reducing agent, an antibiotic, L-glutamine or an analog thereof, or any combination thereof.

[0013] In some embodiments, the non-human serum or serum substitute is fetal bovine serum, bovine serum albumin, KnockOut TMIn some embodiments, the reducing agent comprises beta-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof. In some embodiments, the antibiotic comprises penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof. The FC medium may include, for example, sodium pyruvate, and / or one or more non-essential amino acids.

[0014] In some embodiments, the FC medium comprises DMEM, fetal bovine serum, sodium pyruvate, GlutaMax, MEM non-essential amino acids, 2-mercaptoethanol, penicillin and / or streptomycin, or any combination thereof. In some embodiments, the FC medium comprises DMEM, about 15% fetal bovine serum, about 1 mM sodium pyruvate, about 2 mM GlutaMax, about 1% MEM non-essential amino acids, about 0.1 mM 2-mercaptoethanol, about 1% penicillin and / or streptomycin, or any combination thereof. In some embodiments, the FC medium further comprises an anticoagulant, optionally heparin, fibroblast growth factor (FGF), optionally FGF2 and / or FGF4, or any combination thereof.

[0015] In some embodiments, the IVC medium comprises: 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. The insulin receptor agonist may be selected from IGF-I, IGF-II, analogs thereof, and any combination thereof. The estrogen receptor agonist may be, for example, β-estradiol, estrone, estriol, and estetrol, or analogs thereof.

[0016] The IVC medium may include transferrin, sodium selenide, ethanolamine, or analogs thereof, hi some embodiments, the IVC medium does not include sodium pyruvate.

[0017] In some embodiments, the IVC medium comprises DMEM / F12, fetal bovine serum, GlutaMax, ITS-X, β-estradiol, progesterone, N-acetyl-L-cysteine, penicillin and / or streptomycin, or any combination thereof. In some embodiments, the IVC medium comprises DMEM / F12, about 20% fetal bovine serum, about 2 mM GlutaMax, about 1× ITS-X, about 8 nM β-estradiol, about 200 ng / ml progesterone, about 25 μM N-acetyl-L-cysteine, about 1% penicillin and / or streptomycin, or any combination thereof.

[0018] In some embodiments, culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the first culture medium comprises increasing a serum concentration, optionally increasing the serum concentration from about 20% to about 30%, In some embodiments, step (a) comprises transferring the mammalian pluripotent stem cells and the extraembryonic stem cells from one substrate to another substrate.

[0019] In some embodiments, the post-implantation embryo structure is cultured in the second culture medium for up to 3 days. In some embodiments, step (b) is from embryonic day E5.5 to E8.0. In some embodiments, the neutralizing embryo structure resembles a natural embryo structure at E8.0. In some embodiments, the neutralizing embryo structure is cultured in a substrate, optionally comprising a dish, a U-plate, a flask, or a microwell plate. In some embodiments, the second culture medium is a post-implantation culture medium capable of supporting development of an embryo outside of the uterus. In some embodiments, the second culture medium comprises DMEM, non-human serum, human umbilical cord serum, L-glutamine or an analog thereof, an antibiotic, or any combination thereof. In some embodiments, the non-human serum comprises rat serum, and / or bovine serum. In some embodiments, the second culture medium comprises bicarbonate. In some embodiments, the second culture medium comprises HEPES. In some embodiments, the non-human serum is rat serum. In some embodiments, the second culture medium comprises DMEM, rat serum, human umbilical cord serum, GlutaMax, penicillin and / or streptomycin, HEPES, or any combination thereof. In some embodiments, the second culture medium comprises about 25% DMEM, about 50% rat serum, and about 25% human umbilical cord serum.

[0020] In some embodiments, step (b) comprises adding glucose to the second culture medium, e.g., at least 3 mg / ml glucose. In some embodiments, the glucose is added at least once, optionally on day 3 of culture. In some embodiments, step (b) comprises culturing the post-implantation embryonic structure in a medium containing about 1 mg / ml glucose for 2 days and culturing the post-implantation embryonic structure in a medium containing about 3 mg / ml glucose for 1 day.

[0021] In some embodiments, step (c) is from embryonic day E8.0 to at least E8.5. In some embodiments, the synthetic embryo resembles a natural embryo structure at E8.5. In some embodiments, the synthetic embryo resembles a natural embryo structure at E9.0. In some embodiments, the neutralizing embryo structure is cultured under dynamic conditions in the second culture medium. In some embodiments, the second culture medium in step (c) comprises at least 30% non-human serum.

[0022] In some embodiments, step (b) comprises supplementing said second culture medium with at least 3 mg / ml glucose, optionally at least 3.5 mg / ml glucose.

[0023] In some embodiments, the dynamic conditions include suspension agitation, optionally rotation. In some embodiments, step (c) is performed in a roller bottle. In some embodiments, each roller bottle includes two synthetic embryoid bodies in about 3 ml of medium. In some embodiments, the dynamic conditions include supplying a plurality of gases to the culture chamber. In some embodiments, the dynamic conditions include a gas pressure of about 0.5 to about 3 pounds per square inch (psi), optionally about 0.5 to 1 psi, optionally about 0.5 psi. In some embodiments, the dynamic conditions include supplying a constant concentration of oxygen to the culture chamber. In some embodiments, the dynamic conditions include supplying increasing concentrations of oxygen to the culture chamber. In some embodiments, the culture chamber has an atmosphere containing an increasing oxygen concentration from about 5% to about 25%, optionally from about 5% to about 13%, optionally from about 13% to about 18%, optionally from about 18% to about 21%.

[0024] In some embodiments, the synthetic embryo has an established brain region, a neural tube, a beating heart, and / or a gut tract, hi some embodiments, the synthetic embryo has developing somites and primordial germ cells.

[0025] In some embodiments, the methods do not include any in vivo steps.

[0026] In some embodiments, in culturing steps (a), (b), and (c), none of the mammalian pluripotent stem cells, the extraembryonic stem cells, the post-implantation embryonic structures, and the neutralizing embryo structures are present in an in vivo environment; and, optionally, wherein the in vivo environment comprises a tissue, an organ, an organism, or a combination thereof. In some embodiments, the synthetic embryo is a mouse embryo. In some embodiments, the synthetic embryo is a non-human mammalian embryo.

[0027] Any synthetic embryo obtained by the methods disclosed in this application is provided in this application.

[0028] Also provided herein is an in vitro culture (IVC) medium for generating a synthetic embryo in vitro according to the method of any one of claims 1-70, wherein the medium comprises: a basal culture medium comprising at least 20% non-human serum; insulin, an insulin analog, or an insulin receptor agonist; an estrogen, an estrogen analog, or an estrogen receptor agonist; and a progesterone, a progesterone analog, or a progesterone receptor agonist. The basal culture medium may comprise a reducing agent, an antibiotic, or a combination thereof. The non-human serum may comprise, for example, fetal bovine serum, bovine serum albumin, or both.

[0029] Disclosed herein are methods for investigating mechanisms involved in embryo development, comprising: any of the methods for generating a synthetic embryo in vitro; a method for identifying a compound useful for treating a disease, comprising contacting a synthetic embryo obtainable by any of the in vitro methods disclosed herein with the compound; a method for diagnosing or treating a disease or disorder in a subject, comprising: generating a synthetic embryo according to any of the in vitro methods disclosed herein; and implanting the synthetic embryo into the subject.

[0030] As disclosed herein, in some embodiments, the pluripotent stem cells and the at least one type of extraembryonic stem cell are obtained from the subject.

[0031] Also provided herein are methods for elucidating the role of a gene in embryonic development, comprising obtaining pluripotent stem cells and / or extraembryonic stem cells in which the gene has been modified or knocked out, and culturing the pluripotent stem cells and extraembryonic stem cells using any of the in vitro methods disclosed herein. [Brief description of the drawings]

[0032] [Figure 1]1A-1H show non-limiting exemplary embodiments and data relating to exemplary ETiX embryoid bodies described in the present application, which recapitulate natural mouse embryo developmental milestones up to embryonic day 8.5 (E8.5). FIG. 1A shows a schematic of ETiX embryoid body formation. ETiX embryoid bodies were formed by congregating ES cells, TS cells, and ES cells transiently expressing GATA4. They generated structures similar to natural E5.5 embryos at the post-implantation stage by day 4 (ETiX4). They then progressed to gastrulation (E6.5 / ETiX day 5 (ETiX5)) and neural tube formation (E8.0 / ETiX day 7 (ETiX7)) stages, and initiated organogenesis (E8.5 / ETiX day 8 (ETiX8)). Figure 1B-C show bright field images of natural mouse embryos (Figure 1B) and ETiX embryoid bodies (Figure 1C) at various time points, highlighting the morphological similarities (n = 1,197 ETiX4, 237 ETiX5, 170 ETiX6, 100 ETiX7, and 40 ETiX8, derived from 17 independent experiments). Scale bar, 100 µm. Figure ID shows uniform manifold approximation and projection (UMAP) analysis of scRNA-seq data at the indicated time points for E6.5, E7.5, and E8.5 natural embryos and ETiX embryoid bodies at days 5, 6, and 8 analyzed by inDrops sequencing (n = 29 ETiX5, 10 ETiX6, 7 ETiX8, 12 E6.5, 14 E7.5, and 9 E8.5). Figure IE shows annotated single-cell inDrops RNA-seq UMAPs showing cell types identified in natural embryos and ETiX embryoid bodies. AVE, anterior visceral endoderm; CLE, caudal lateral epiblast; NMP, neuro-mesodermal progenitors; PLPM, posterior lateral plate mesoderm.Figure IF shows the annotated and combined UMAPs of natural embryos cultured ex utero and harvested at the indicated time points (E7.5, E8, E8.5, E8.75 and E9.5) and of ETiX embryoid bodies (days 6 and 8), individually labeled and analyzed by tiny-sci-RNA-seq. n = 8 natural embryos from E7.5 to E9.5, n = 3 ETiX6, 2 poorly formed ETiX6, 5 ETiX8 and 4 poorly formed ETiX8. Figure IG shows the cell type composition of natural embryos at the indicated time points (E7.5, E8, E8.5, E8.75 and E9.5) and of ETiX embryoid bodies (days 6 and 8). FIG. 1H shows density plots highlighting the proportions of tissue types represented during development in natural and ETiX embryos. [Diagram 2]2A-2J show non-limiting exemplary embodiments and data relating to exemplary ETiX embryoid bodies described herein in the process of developing an anterior brain and patterned neural tube. FIG. 2A-2B show ventral views (main images) of an ETiX embryoid body at day 7 after static culture (FIG. 2A) and of a natural embryo at E8.0 (FIG. 2B), showing SOX1-positive neural folds and a Brachyury-positive neural tube extending anteriorly to posteriorly. Optical yz (bottom) and xz (right) cross sections show the notochord underlying the neural tube (n=11 day 7 ETiX embryoid bodies, n=3 E8.0 embryos from 4 experiments). Scale bar, 100 μm. Figure 2C shows dot plots depicting the average levels and percentages of cells expressing the indicated genes in selected tissues of natural embryos and ETiX embryoid bodies (derived from inDrops scRNA-seq data). Epi, epiblast. Figures 2D-E show lateral views of day 8 ETiX embryoid bodies (Fig. 2D) and E8.5 natural embryos (Fig. 2E) showing FOXG1 in the telencephalon and OTX2 in the forebrain and midbrain (from three experiments, n = 4 ETiX8; n = 2 E8.5 embryos). Scale bar, 100 μm. Figure 2F shows coronal views of neural tube cross sections showing dorsal-ventral patterning in day 8 ETiX embryoid bodies. Sections revealed pan-neuronal markers (SOX1 and SOX2), dorsal markers (PAX6 and PAX3), ventral markers (FOXA2, OLIG2, and NKX2-2), and neural crest markers (SOX10 and PAX3). Scale bar, 50 μm. n = 3 ETiX8 from three experiments. Figure 2G shows subclustered UMAPs of neural precursors highlighting neural subtypes by tiny-sci-RNA-seq. Figure 2H shows individual UMAPs showing the contribution of each time point to the global UMAP in Figure 2G. Figure 2I shows the percentage of cell types in Figure 2G in each individual day 8 ETiX embryoid body sequenced by tiny-sci-RNA-seq. MHB, midbrain-hindbrain boundary.Figure 2J shows ETiX8 embryos after culture in the absence (left) or presence (right) of 5-methyl-tetrahydrofolate (50 ng / ml) and stained for Sox1, Bry, and DNA (n=4 control ETiX, 3 treated ETiX from 2 independent experiments). Scale bar = 100 μm. [Diagram 3] 3A-3C show non-limiting exemplary embodiments and data related to Pax6 knockout in exemplary ETiX embryoid bodies described in this application, which summarize known mouse embryo phenotypes. FIG. 3A shows coronal sections of wild-type (WT) and Pax6-knockout (KO) ETiX embryoid bodies stained to reveal dorsal and ventral neural tube markers. Scale bar, 50 μm. FIG. 3B shows quantification of the images depicted in FIG. 3A, showing that there is no significant difference in the number of SOX1-positive cells in the neural tube, but there is an increase in the percentage of NKX2-2-positive cells after Pax6 knockout (from three experiments, three control day 8 ETiX and four day 8 Pax6-KO ETiX). Violin plots show medians and quartiles. Two-tailed Mann-Whitney U test, *P < 0.05. For SOX1-positive cells, P = 0.5382; for NKX2-2-positive cells, P = 0.0135. Figure 3C shows gene ontology (GO) analysis of enriched genes in two Pax6-knockout ETiX embryoid bodies at day 8 compared with five ETiX day 8 controls. NS, not significant (P > 0.05). [Figure 4]4A-L show non-limiting exemplary embodiments and data relating to ETiX embryoid bodies undergoing somitogenesis and cardiogenesis. 4A-B show lateral views of day 8 ETiX embryoid bodies (FIG. 4A) and natural E8.5 embryos (FIG. 4B) showing SOX2, Brachyury (BRY) and DNA (DAPI) and highlighting NMPs in the tailbud region (n=5 ETiX8, n=3 embryos from 4 experiments). Inset, schematic. 4C-D show dorsal views of day 7 ETiX embryoid bodies (FIG. 4C) and natural E8.0 embryos (FIG. 4D) after static culture showing SOX2, HOXB4 and DNA and highlighting somitogenesis flanking the neural tube. Right, magnified view of outlined area containing somites (n=9 day 7 ETiX embryoid bodies, n=5 E8.0 embryos from 4 experiments). Insets, schematic diagrams. Scale bars. 4A-D, 100 μm (main image), 50 μm (zoomed view 4A-B and upper zoomed view 4C), 20 μm (lower zoomed view 4C and zoomed view 4D). 4E shows quantification of somite pairs in natural E8.0 embryos and day 7 ETiX embryoid bodies. Violin plots show medians and quartiles. Two-tailed Mann-Whitney U test, P = 0.3020. 4F shows somite areas in E8.0 embryos and day 7 ETiX embryoid bodies. Violin plots show medians and quartiles. Two-tailed Mann-Whitney U test, P = 0.2717. For 4E and 4F, n = 9 day 7 ETiX embryoid bodies and n = 5 E8.0 embryos from 4 experiments. Figure 4G-H show day 8 ETiX embryoid bodies (Figure 4G) (lateral view) and natural E8.75 embryos (Figure 4H) (lateral view), showing OTX2, MYH2, and GATA4, highlighting the heart (n = 8 ETiX8, n = 2 natural embryos from 3 experiments). Outlined areas are enlarged on the right. Scale bars, 100 μm (main image), 20 μm (magnified view). Figure 4I shows a schematic of the mouse heart at E8.5, indicating the location of the section. Figure 4J shows a coronal section of a day 8 ETiX embryoid body, showing GATA4, NKX2-5, and MYH2. Scale bars, 100 μm.ETiX8, n = 3 from 3 independent experiments. Figure 4K shows dot plots indicating the levels and percentages of cells expressing the indicated genes in the indicated tissues from natural embryos (NE) and ETiX embryoid bodies by inDrops scRNA-seq. Figure 4L shows velocity plots of epiblast and mesoderm derived tissues over time in the inDrops sequencing dataset. Figure 4M shows a UMAP of the tiny-sci-RNA-seq dataset, showing cell types in the sub-clustered cardiac lineage. [Diagram 5]5A-5I show non-limiting exemplary embodiments and data relating to ETiX embryoid bodies in the process of developing gut pockets and primordial germ cells (PGCs). FIG. 5A-5B show sagittal sections of natural embryos at E8.5 (FIG. 5A) and ETiX embryoid bodies at day 8 (FIG. 5B) showing SOX2, SOX17, and GATA4. Scale bar, 100 μm. n=3 ETiX8, n=2 natural embryos from three experiments. fg, foregut; hg, hindgut. FIG. 5C shows dot plots showing the levels and percentages of cells expressing the indicated genes in selected tissues of natural embryos and ETiX embryoid bodies by inDrops scRNA-seq. FIG. 5D shows UMAPs of tiny-sci-RNA-seq dataset showing VE, gut, and early developmental cell types. Figure 5E shows ETiX embryoid bodies at day 6 (top) and natural E7.5 embryos (bottom), displaying STELLA, NANOG, and SOX2 and highlighting the presence of committed PGCs (from two experiments, n = 9 ETiX6, n = 4 embryos). Outlined areas are expanded on the right. Figure 5F shows quantification of developing PGCs in ETiX embryoid bodies (n = 9 ETiX6, 4 E7.5, 2 ETiX7, 4 E8.0, 4 ETiX8, and 3 E8.5). PGCs were scored for expression of STELLA, NANOG, and SOX2. Violin plots show medians and quartiles. Two-tailed Mann-Whitney U test, P = 0.3375 for all STELLA-positive cells in ETiX6 / E7.5, P = 0.3042 for triple-positive cells in ETiX6 / E7.5, P = 0.2277 for all STELLA-positive cells in ETiX8 / E8.5, and P = 0.2536 for triple-positive cells in ETiX8 / E8.5. Figure 5G-H show ETiX8 embryoid bodies (Figure 5G) and natural E8.75 embryos (Figure 5H), stained to reveal Sox17, Gata4, and Sox2, highlighting the formation of the gut in relation to the neural tube (from three independent experiments, n = 6 ETiX8, n = 3 embryos). Boxes indicate the magnified areas below each panel.Scale bar for Figures 5G-H = 100 μm. Scale bar for magnified boxes = 50 μm. Figure 5I shows an ETiX8 embryoid body sectioned coronally and stained to reveal Sox2, FoxA2, ​​and DNA, highlighting the formation of the gut pocket in relation to the neural tube. Scale bar = 100 μm. Scale bar for magnified boxes = 20 μm. [Figure 6] 6A-6J show non-limiting exemplary embodiments and data related to characterization of extraembryonic lineages in ETiX embryoid bodies. FIG. 6A shows a global UMAP of the tiny-sci dataset as shown in FIG. 1F. Selected cell clusters are highlighted. FIG. 6B shows gene expression of amniotic membrane marker Periostin (Postn) in natural embryos and ETiX embryoid bodies from the tiny-sci-RNA-seq dataset. FIG. 6C shows gene expression of allantoic membrane markers Tbx4 and Hoxa13 in natural embryos and ETiX embryoid bodies from the tiny-sci-RNA-seq dataset. FIG. 6D shows sub-clustered and annotated UMAP of extraembryonic endoderm from the tiny-sci-RNA-seq dataset. FIG. 6E shows a schematic of the dissection of chorioallantoic attachment of ETiX embryoid bodies. Figure 6F shows sagittal sections of chorioallantoic attachment and yolk sac of day 8 ETiX embryoid bodies, showing RUNX1 and DNA (left). Arrows highlight blood islands. Outlined areas are enlarged in middle and right panels. n = 3 ETiX8 from 3 experiments. Scale bars: 100 μm (left), 20 μm (middle and right). Figure 6G shows subclustered and annotated UMAPs of ExE and trophoblast cells from the tiny-sci-RNA-seq dataset. Figure 6H shows the contribution of individual time points to the subclustered UMAPs of ExE and trophoblast cells. Figures 6I-J show expression of selected prolactin genes in subclustered UMAPs of ExE and trophoblast cells of natural embryos (Figure 6I) and ETiX embryoid bodies (Figure 6J). [Figure 7]Figures 7A-7J show non-limiting exemplary embodiments and data relating to ETiX embryoid bodies that develop to comparable size and exhibit gene expression patterns similar to natural embryos with reproducible efficiency. Figure 7A shows quantification of the dimensions of ETiX embryoid bodies and natural embryos at comparable developmental time points (n=42 for ETiX4, 24 for ETiX5, 14 for ETiX6, 18 for ETiX7, 12 for ETiX8, 32 for E5.5, 18 for E6.5, 3 for E7.5, 8 for E8.0, 5 for E8.5, from 30 independent experiments). Figure 7B shows quantification of the efficiency of ETiX embryoid body formation from days 5 to 8 (n = 1197 for ETiX4, 237 for ETiX5, 170 for ETiX6, 100 for ETiX7, and 40 for ETiX8, from 17 independent experiments). Error bars represent SEM. Figure 7C shows brightfield images of ETiX embryoid bodies recovered at day 7 after static culture and before detailed analysis, highlighting the presence of the yolk sac. Scale bar, 100 μm (n = 100 for ETiX7, from 17 independent experiments). Figure 7D-E show isolated UMAPs of natural embryos (Figure 7D) and of ETiX embryoid bodies (Figure 7E) analyzed by inDrops scRNA-seq. Figure 7F shows stacked column graphs binning all sequenced cells in natural embryos and ETiX embryoid bodies according to germ layers and embryonic and extraembryonic origins at the indicated time points (inDrops scRNA-seq). Figure 7G shows stacked column graphs highlighting the proportion of tissue types that appeared during development in natural embryos and ETiX embryoid bodies (inDrops scRNA-seq). Figure 7H shows a Pearson correlation matrix showing the global similarity level between all identified tissues in natural embryos (rows) compared to ETiX embryoid bodies (columns) (inDrops scRNA-seq). Figure 7I shows a pairwise visualization of the proportion of cell types between natural embryos and ETiX embryoid bodies (inDrops scRNA-seq).FIG. 7J shows the Pearson correlation matrix indicating the global similarity level between all comparisons of tissue types in natural and ETiX embryos. [Figure 8] 8A-8C show non-limiting exemplary embodiments and data relating to examples of well-formed ETiX embryoid bodies, poorly formed ETiX embryoid bodies, and cultured natural embryos. FIG. 8A shows a typical day 8 ETiX embryoid body that was determined to have developed successfully. FIG. 8B shows a typical day 8 ETiX embryoid body that was poorly formed. FIG. 8C shows a natural embryo cultured ex utero from E6.5 to E8.5. All structures were stained with DAPI. Morphological features: Hf, headfold; H, heart; T, tail; All, allantois. (Some of these samples are also shown in the following panels: sample v of Figure 8A is also shown in Figure 4G; sample vi of Figure 8A is also shown in Figure 2D; sample viii of Figure 8A is also shown in Figure 13G; sample xi of Figure 8A is also shown in Figure 10A; sample xii of Figure 8C is also shown in Figure 11C; sample xiii of Figure 8C is also shown in Figure 4B; sample xvi of Figure 8C is also shown in Figure 4H; and sample xvii of Figure 8C is also shown in Figure 2E). [Figure 9]9A-9J show non-limiting exemplary embodiments and data related to the analysis of ETiX embryoid bodies and natural embryos by tiny sci-RNA-seq. FIG. 9A-9B show quality control of the first (FIG. 9A) and second (FIG. 9B) replicates for tiny sci-RNA-seq. Cells with abnormal percentages of reads mapping to exons and UMI counts per cell that were too high or too low were removed. FIG. 9C shows UMAPs of the first and second replicates for tiny sci-RNA-seq. The total number of cells in each dataset is shown. FIG. 9D shows batch variation of the first and second replicates for tiny sci-RNA-seq. FIG. 9E-9F show isolated UMAPs of natural embryos (FIG. 9E) and ETiX embryoid bodies (FIG. 9F) analyzed by tiny sci-RNA-seq. Figure 9G shows the contribution of each time point to the global UMAP of tiny sci-RNA-seq. Figure 9H shows the individual UMAPs of standard ETiX embryoid bodies analyzed at days 6 and 8 using tiny sci-RNA-seq. Figure 9I shows the PCA analysis of all natural embryos and standard ETiX embryoid body samples analyzed using tiny sci-RNA-seq. Figure 9J shows the correlation matrix after nonnegative least-squares (NNLS) regression analysis showing the global similarity level across all identified tissues in natural embryos (columns) compared to ETiX embryoid bodies (rows) in samples analyzed by tiny sci-RNA-seq. [Figure 10]Figures 10A-10E show non-limiting exemplary embodiments and data relating to the exemplary ETiX embryoid body tiny sci-RNA-seq dataset described in this application. The tiny sci-RNA-seq dataset was seamlessly integrated with a published single-cell sequencing dataset to highlight the differences between well-formed and poorly formed ETiX embryoid bodies. Figures 10A-10C show the integration of the tiny sci-RNA-seq dataset generated in this application with two published single-cell sequencing mouse datasets. In Figure 10A: 1 allantois, 2 anterior primitive streak, 3 blood precursor 1, 4 blood precursor 2, 5 cardiomyocyte, 6 caudal epiblast, 7 caudal mesoderm, 8 caudal neurectoderm, 9 definitive endoderm, 10 endothelium, 11 epiblast, 12 erythroid cell 1, 13 erythroid cell 2, 14 erythroid cell 3, 15 ExE ectoderm, 16 ExE endoderm, 17 ExE mesoderm, 18 forebrain / midbrain / hindbrain, 19 gut, 20 hemogenic endothelial precursors, 21 intermediate mesoderm, 22 mesenchyme, 23 mixed mesoderm, 24 nascent mesoderm, 25 neural crest, 26 NMP, 27 notochord, 28 paraxial mesoderm, 29 parietal endoderm, 30 PGC, 31 pharyngeal mesoderm, 32 primitive streak, 33 beak-like neuroectoderm, 34 semitic mesoderm, 35 spinal cord, 36 surface ectoderm, 37 visceral endoderm.In Figure 10B: 1 allantoic sac, 2 amniochorionic mesoderm A, 3 amniochorionic mesoderm 8, 4 anterior floor plate, 5 blood precursors, 6 definitive endoderm, 7 endothelium, 8 extraembryonic mesoderm, 9 extraembryonic visceral endoderm, 10 first heart field, 11 forebrain / midbrain, 12 fusion epithelium, 13 gut, 14 hematopoietic endothelial precursors, 15 hindbrain, 16 intermediate mesoderm, 17 neural crest, 18 neuromesoderm precursors, 19 notochord, 20 paraxial mesoderm A, 21 paraxial mesoderm B, 22 placodal area, 23 posterior floor plate, 24 Preepidermal keratinocytes, 25 primitive red blood cells, 26 PDCs, 27 second heart field, 28 somatic mesoderm, 29 spinal cord, 30 splanchnic mesoderm. In Figure 10C: 1 allantoic pouch, 2 amniotic mesoderm, 3 definitive endoderm, 4 early development, 5 endothelium, 6 extraembryonic ectoderm, 7 heart field, 8 hemogenic endothelial precursors, 9 lateral plate mesoderm, 10 megakaryocytes, 11 neural crest, 12 neuroectoderm, 13 neuromesoderm precursors, 14 paraxial mesoderm, 15 parietal endoderm, 16 primitive erythroid cells, 17 surface ectoderm, 18 visceral endoderm, 19 leukocytes. Figure 10D shows the cell type ratios for each individual well-formed (standard) and poorly-formed ETiX embryoid body (classified from abnormal morphology) sequenced at days 6 and 8 using tiny sci-RNA-seq (n = 3 for standard ETiX6, 2 for poorly-formed ETiX6, 5 for standard ETiX8, and 4 for poorly-formed ETiX8, from two independent experiments). Figure 10E shows the average cell type ratios for well-formed (standard) and poorly-formed ETiX embryoid bodies sequenced at day 8 using tiny sci-RNA-seq.The cell type proportions derived from each individual sample were also plotted. In each box plot, the center line indicates the median; box borders indicate the 25th and 75th percentiles, whiskers extend to the 5th and 95th percentiles; replicates are represented by dots. (n = 5 for standard ETiX8 and 4 for poorly formed ETiX8, derived from two independent experiments). In Figure 10E, 1, endothelium; 2, leukocytes; 3, megakaryocytes; 4, primitive erythroid cells; 5, heart field; 6, cardiac mesoderm; 7. Paraxial mesoderm; 8, neural mesoderm precursors; 9, neural crest; 10, neuroectoderm; 11, surface ectoderm; 12, gut; 13, parietal endoderm; 14, visceral endoderm; 15, amniotic mesoderm; 16, allantoic pouch; 17, embryonic ectoderm; 18, hemogenic endothelial precursors; 19, early development. [Figure 11]11A-11G show non-limiting exemplary embodiments and data related to exemplary ETiX embryoid bodies described in this application, which show neural folds and developing tail buds with comparable differentiation trajectories and timing. FIG. 11A shows a 7-day ETiX embryoid body recovered after static culture stained for SOX2, SOX1, and DNA, highlighting the formation of rostral neural folds (n=11 ETiX7, n=3 E8.0 natural embryos from 4 independent experiments). FIG. 11B shows dorsal views of day 7 ETiX embryoid bodies (left) and of natural E8.0 embryos (right) recovered after static culture, showing the formation of SOX1-positive neural folds, as well as BRY-positive notochord and tail buds (n = 11 ETiX7, n = 3 embryos from 4 independent experiments). Scale bar, 100 μm. FIG. 11C shows lateral views of day 8 ETiX embryoid bodies and of E8.5 natural embryos (FIG. 11C (continued)), showing expression of FOXG1 in the telencephalon and OTX2 restricted to the forebrain and midbrain (n = 4 ETiX8, n = 2 embryos from 3 independent experiments). Scale bar, 100 μm for FIG. 11A to FIG. 11C. FIG. 11D shows quantification of brain area in E8.5 natural embryos and in day 8 ETiX embryoid bodies. OTX2 was used to delineate the measurement areas. Each dot represents a sample (n = 6 E8.5 embryos and n = 17 ETiX8 from 7 independent experiments). Data are presented as violin plots with median and quartiles. Two-tailed unpaired t-test, ns = p > 0.05 (p = 0.5223). Figure 11E shows velocity plots of epiblast, neuroectoderm, and surface ectoderm at all time points analyzed in the inDrops sequencing dataset. Figure 11F shows latency analysis of epiblast, neuroectoderm, and surface ectoderm at all time points analyzed in the inDrops sequencing dataset.Figure 11G shows the latent time analysis of epiblast, neuroectoderm, and surface ectoderm at all time points analyzed in the inDrops sequencing dataset. [Figure 12] 12A-12J show non-limiting exemplary embodiments and data relating to the expression of markers selected to annotate neural tissues in natural embryos and ETiX embryoid bodies, and the regional expression of transcripts as revealed by sequential single molecule FISH. Expression of selected genetic markers within the annotated clusters is shown in FIG. 2G. FIGS. 12A-F provide plots showing the expression of genetic markers of cell populations representing the prosencephalon (FIG. 12A), the mesencephalon and the midbrain-hindbrain boundary (FIG. 12B), the hindbrain and spinal cord (FIG. 12C), the floor plate / roof plate (FIG. 12D), early neurons (FIG. 12E), and the neural crest (FIG. 12F) (tiny sci-RNA-seq). Figure 12G shows a schematic of sample sections for single-molecule fluorescence in situ hybridization (smFISH) and possible expression patterns for selected genes. Figures 12H-I show smFISH panels for n = 1 natural embryo (Figure 12H) and for n = 1 day 8 ETiX embryoid body (Figure 12I) cultured ex utero from E6.5 to E8.5. Scale bar = 200 μm. Figure 12J shows the percentage of cell types annotated in Figure 1F in each individual day 8 ETiX embryoid body and in Pax6 knockout ETiX embryoid body sequenced by tiny sci-RNA-seq. [Figure 13]13A-13J show non-limiting exemplary embodiments and data relating to the development of mesoderm in ETiX embryoid bodies into somites and cardiac tissue. FIG. 13A shows a lateral view of the ETiX embryoid body at day 8 shown in FIG. 3A, highlighting individual channels. The squared area is shown enlarged on the right. Scale bar, 100 μm. FIG. 13B shows the percentage of cells co-expressing BRY and SOX2 in natural embryos and in ETiX embryoid bodies (n=3 E8.5 embryos and n=4 ETiX8). Data are presented as violin plots with median and quartiles. Each dot represents a sample. Two-tailed Mann-Whitney U test, ns=p>0.05 (p=0.5182). FIG. 13C shows a dorsal view of the ETiX embryoid body at day 7 shown in FIG. 3C, highlighting individual channels. YZ and XZ views are also shown (n = 9 ETiX7, n = 5 E8.0 embryos from 4 experiments). Figures 13D-13E show dorsal (Figure 13D) and lateral (Figure 13E) views of day 7 ETiX embryoid bodies recovered after static culture, stained to reveal SOX2, HOXB4, and DNA, highlighting somitogenesis flanking the neural tube (n = 9 ETiX7 from 4 independent experiments). YZ and XZ views are also shown. Figure 13F shows an orthogonal view of the day 8 ETiX embryoid body shown in Figure 4G. Figure 13G shows a lateral view of day 8 ETiX embryoid bodies stained to reveal OTX2, MYH2, and DNA, highlighting cardiogenesis (n = 8 ETiX8 from 3 independent experiments). YZ and XZ views are also shown. Scale bars for Fig. 13A-D, 100 μm. Scale bars for enlarged areas, 50 μm. Fig. 13H shows day 8 ETiX embryoid bodies (top) and E8.5 natural embryos (bottom) sectioned coronally and stained to reveal GATA6 and MYH2 and highlight cardiac morphogenesis. Scale bars, 200 μm. Fig. 13I shows quantification of heart area in natural embryos and in ETiX embryoid bodies.The MYH2-positive area was used to measure the area of ​​cardiac or heart-like structures (n = 3 E8.5 embryos, n = 3 ETiX8). Data are presented as violin plots with medians and quartiles. Each dot represents a cross-section of the cardiac and heart-like regions. Two-tailed unpaired t-test ** = p ≤ 0.01 (exact p-value = 0.01). Figure 13J shows a dorsal view of a natural E8.0 embryo stained to reveal Sox2, HoxB4, and DNA to highlight somitogenesis flanking the neural tube (n = 2 embryos). Scale bar, 100 μm. [Figure 14] 14A-14F show non-limiting exemplary embodiments and data relating to developmental trajectories, timing of mesoderm differentiation, and expression of selected genes. FIG. 14A shows the latency of epiblast and mesoderm derived cells over time in the inDrops sequencing dataset. FIG. 14B shows quantification of the latency analysis of epiblast and all mesoderm derived products at all time points analyzed in the inDrops sequencing dataset. FIG. 14C shows an annotated UMAP of the tiny sci-RNA-seq dataset highlighting the paraxial mesoderm cluster. FIG. 14D shows expression of somitic markers Meox1, Meox2, and Pax3 in natural embryos and in ETiX embryoid bodies (tiny sci-RNA-seq dataset). Figure 14E shows the expression of cardiac markers Hand1, Hand2, atrial differentiation marker Nr2f2, ventricular differentiation marker Irx4, first heart field markers Tbx5, Hcn4, Nkx2-5, and second heart field marker Isl1 in natural embryos and ETiX embryoid bodies (tiny sci-RNA-seq dataset). Figure 14F shows UMAPs showing individual time points of natural embryos and ETiX embryoid bodies in the combined UMAP shown in Figure 4M. [Figure 15]15A-15F show non-limiting exemplary embodiments and data related to further characterization of the intestine of ETiX embryoid bodies, revealing similarities and differences when compared to natural embryos. FIG. 15A-15B show sagittal sections of E8.5 natural embryos (FIG. 15A) and of ETiX embryoid bodies at day 8 (FIG. 15B) stained to reveal GATA6. (n=3 ETiX8, n=2 embryos from 3 independent experiments). FIG. 15C-15D show sagittal sections of E8.5 natural embryos (FIG. 15C) and of ETiX embryoid bodies at day 8 (FIG. 15D) stained to reveal CDX2, NKX2.5, and FOXG1. (n=3 ETiX8, n=2 embryos from 3 independent experiments). Magnified areas on the right. Figures 15E-F show sagittal sections of E8.5 natural embryos (Figure 15E) and of day 8 ETiX embryoid bodies (Figure 15F) stained to reveal SOX2, OTX2, and FOXA2. Magnified regions of Figures 15E and 15F are shown on the right. Scale bars in Figures 15A-F, 100 μm. (n = 3 ETiX8, n = 2 embryos from three independent experiments). [Figure 16]16A-16L show non-limiting exemplary embodiments and data relating to developmental trajectories and timing of endodermal and extraembryonic contributions to gut formation. FIG. 16A shows velocity plots of epiblast, definitive endoderm, gut precursors, and primitive streak at all time points analyzed in the inDrops sequencing dataset. FIG. 16B shows latency analysis of epiblast, definitive endoderm, gut precursors, and primitive streak at all time points analyzed in the inDrops sequencing dataset. FIG. 16C shows quantification of latency analysis of epiblast, definitive endoderm, gut precursors, and primitive streak at all time points analyzed in the inDrops sequencing dataset. Figures 16D-16K show expression of selected marker genes for the contribution of embryonic (Figure 16D-16J) and extraembryonic (Figure 16K) endoderm to the gut (tiny sci-RNA-seq dataset). Figure 16L shows UMAPs showing the time series of individual natural embryos and ETiX embryoid bodies to combine the UMAPs of Figure 5D. [Figure 17]17A-17D show non-limiting exemplary embodiments and data relating to further examples of PGC formation in ETiX embryoid bodies. FIG. 17A-17C shows ETiX embryoid bodies at developmental day 7 after static culture (FIG. 17A and FIG. 17C) and natural embryos at developmental E8.0 (FIG. 17B) stained to reveal STELLA, NANOG, and SOX2 and highlight the presence of committed PGCs (n=2 ETiX7, n=4 embryos from two independent experiments). Boxes are enlarged at the bottom (FIG. 17A-17B) and on the right (FIG. 17C). Scale bars in FIG. 17A-17C, 100 μm for main panels, 50 μm for enlarged boxes. FIG. 17D shows ETiX-embryoid bodies at developmental day 8 stained to reveal STELLA, NANOG, and SOX2 and highlight the presence of committed PGCs. Scale bar in Figure 17D, 100 µm. (n = 4 ETiX8 from three experiments). [Figure 18]Figures 18A-O show non-limiting exemplary embodiments and data relating to characterization of yolk sac, endothelium, and extraembryonic ectoderm in ETiX embryoid bodies. Figures 18A-B show partially dissociated natural embryos cultured from E6.5 to E8.5 (Figure 18A) and day 8 ETiX embryoid bodies (Figure 18B), highlighting their development within the extraembryonic membranes. Legend: HF: headfold, H: heart, T: tail bud, All: allantoic. Scale bar, 100 μm. Figure 18C shows the sub-clustered UMAPs of extraembryonic endoderm in a tiny sci-RNA-seq dataset and the contribution of each individual time point to the sub-clustered UMAPs of extraembryonic endoderm. Figures 18D-18E show expression of selected parietal endoderm genes in sub-clustered UMAPs of extraembryonic endoderm in natural embryos (Figure 18D) and ETiX embryoid bodies (Figure 18E). Figures 18F-18G show expression of selected extraembryonic visceral endoderm genes in sub-clustered UMAPs of extraembryonic endoderm in natural embryos (Figure 18F) and ETiX embryoid bodies (Figure 18G). Figure 18H shows expression of selected endothelial markers Pecam1, Cd34, Icam1, Tek, Vegfa, and Cdh5 in natural embryos and ETiX embryoid bodies in the tiny sci-RNA-seq dataset. Figure 18I shows trophoblast precursor populations identified by Eomes, Cdx2, Sox2, Sox21, and Bmp4 in natural embryos in the tiny sci-RNA-seq dataset. Figure 18J shows expression of selected markers for the ectoplacental cone lineage. High levels of Hand1 (arrow on the left) indicate developmental progression of the ectoplacental cone (ECP) lineage toward spongiotrophoblast cells and trophoblast giant cells. Co-expression of Ascl2 and Chsy1 indicates committed ECP cells. Tpbpa identified mature spongiotrophoblast cells.Expression of Hand1 and prolactin genes (Figure 5I) indicated trophoblast giant cells. Figure 18K shows the expression of selected markers for chorionic precursors (right of the arrow in Hand1 UMAP in Figure 18J), chorion, and differentiated chorionic derivatives in natural embryos. Wnt7b indicates chorionic precursors, Tfrc indicates chorionic clusters, Epha4 identifies cells of syncytiotrophoblast layer I, and Gcm1 identifies cells of syncytiotrophoblast layer II. Figures 18L-18O show the expression of the marker genes presented in (Figures 18I-18K) in ETiX embryoid body UMAPs.

[0033] 19A-19B show non-limiting exemplary embodiments and data relating to the number of primordial germ cells (PGCs) and the development of extraembryonic tissues in ETiX embryos. FIG. 19A shows quantification of PGC formation at different stages of ETiX embryoid body development (n=9 for ETiX6, 2 for ETiX7, 4 for ETiX8). PGCs were scored for expression of Stella and co-expression of Nanog and Sox2. Data are presented as violin plots with median and quartiles. FIG. 19B shows sagittal sections of chorioallantoic attachment and yolk sac of ETiX8 embryos stained with keratin 18, Gata4, and DNA to visualize the chorion and yolk sac. Scale bar in FIG. 19B = 100 μm. Scale bar in enlarged square = 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description In the following detailed description, reference is made to the accompanying drawings, which form a part of this application. In the drawings, like symbols typically identify like elements unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 in this application. It will be readily understood that the aspects of the present disclosure, as generally described and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated and made a part of this disclosure.

[0035] All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced in this application are incorporated by reference in their entirety with respect to the relevant art.

[0036] definition Unless otherwise defined, technical and scientific terms used in this application 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:

[0037] The term "about" as used herein when referring to a measurable value, such as an amount of a compound, a dosage, a time, a temperature, and the like, is meant to include a variation of 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0038] As used herein, the term "differentiation" may refer to the process by which an unspecialized or less specialized cell acquires the characteristics of a specialized cell, such as a neuronal cell. A differentiated cell is one that has taken a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the process of differentiation refers to a cell that, under normal circumstances, will continue to differentiate into a particular cell type, or a subset of cell types, and has progressed down a differentiation pathway to the point where, under normal circumstances, it is unable 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 genetics of the cell; that is, what cells it came from and what cells it can give rise to. The lineage of a cell places the cell in the 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 a cell of a lineage of interest and may be used to assess the differentiation of an uncommitted cell into a lineage of interest.

[0039] As used herein, "marker," "lineage marker," or "lineage-specific marker" may refer to a nucleic acid molecule or a polypeptide molecule that is differentially expressed in a cell of interest. Differential expression may mean increased levels of a positive marker and decreased levels of a negative marker compared to undifferentiated cells. The detectable levels of the marker nucleic acid or marker polypeptide are sufficiently higher or lower in the cell of interest compared to other cells, such that the cell of interest can be identified and differentiated from other cells using various methods known in the art. In some embodiments, the marker may be enriched. As used herein, the term "enriched" has its ordinary meaning and may also refer to a statistically significant increase in the levels of a gene product (e.g., mRNA and / or protein) in one condition compared to another (e.g., one cell layer compared to another).

[0040] The term "concentration" as used in this application has its ordinary meaning and may also refer to (a) mass, molar, or volume concentration, mass, mole, or volume fraction, or (b) the ratio of the mass or volume of a component in a mixture or solution to the mass or volume of another component in the mixture or solution (e.g., ng / ml). In some embodiments, the concentration may refer to the ratio of active units per volume (e.g., U / ml).

[0041] The term "analog" as used herein refers to a compound that may be structurally related to the related molecule. The term "agonist" as used herein may refer to a compound that may not be structurally related to the related molecule. For example, an agonist may activate an associated receptor by altering the conformation of said receptor. Nevertheless, in both cases, the terms are used herein to refer to compounds or molecules that can mimic, reproduce, or otherwise generally substitute for a particular biological activity of the related molecule.

[0042] As used herein, the phrase "culture medium" refers to a liquid substance used to support stem cell and embryonic growth and development. The culture medium used in some embodiments of the invention may be a water-based medium containing a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, and / or proteins, such as cytokines, growth factors, and hormones, necessary for cell growth and embryonic development.

[0043] The present application discloses an in vitro method for generating synthetic embryos from stem cells. In some embodiments, the method includes culturing mammalian pluripotent stem cells and extraembryonic stem cells in a first culture medium under first static conditions to allow the mammalian pluripotent stem cells and the extraembryonic stem cells to self-organize into a post-implantation embryo structure, culturing the post-implantation embryo structure in a second culture medium under second static conditions to allow the post-implantation embryo structure to develop into a neurulation embryo structure, and culturing the neurulation embryo structure under dynamic conditions in a culture chamber for at least one day to allow the neurulation embryo structure to develop into at least an early organogenesis stage synthetic embryo. The present application also discloses synthetic embryo structures obtained by the methods disclosed herein.

[0044] Also disclosed herein is an in vitro culture (IVC) medium for generating a synthetic embryo in vitro according to the methods disclosed herein, the in vitro IVC medium comprising a basal culture medium containing at least 20% non-human serum, insulin, an insulin analog, or an insulin receptor agonist, estrogen, an estrogen analog, or an estrogen receptor agonist, and progesterone, a progesterone analog, or a progesterone receptor agonist.

[0045] Also disclosed herein are methods for investigating mechanisms involved in embryonic development according to the in vitro methods disclosed herein.

[0046] A method for generating synthetic embryos in vitro from stem cells Embryonic stem cells (ES cells) can undergo many aspects of mammalian embryonic development in vitro, but their developmental potential can be greatly expanded by interaction with extraembryonic stem cells, such as trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells, and / or inducible XEN cells (iXEN cells). Signals emanating from extraembryonic tissues play a key role in patterning the epiblast, driving establishment of the anterior-posterior axis.

[0047] Embryoid bodies have been previously assembled by assembling ES cells with TS cells, derived from extraembryonic ectoderm (ExE) precursors, and XEN cells, derived from extraembryonic visceral endoderm (VE) precursors. Replacement of XEN cells with ES cells transiently expressing the VE master regulator GATA4 (iXEN cells) has been shown to improve the efficiency and developmental potential of the resulting ETiX embryoid bodies. ETiX embryoid bodies specify the anterior organizer, the anterior visceral endoderm (AVE), which migrates to position the primitive streak and initiates gastrulation movements essential for subsequent development.

[0048] Methods and culture media are provided herein for assembling synthetic embryos in vitro from mammalian pluripotent stem cells and extraembryonic stem cells, such as ES cells, TS cells, and iXEN cells. In some embodiments, stem cell-derived synthetic embryos obtained using the methods and culture media disclosed herein progress development beyond gastrulation and neural tube formation and may correspond to natural embryos at or after 8.5 days post-fertilization. In some embodiments, stem cell-derived synthetic embryos establish all brain regions, neural tubes, beating hearts, and gut tubes. The neural tubes are sandwiched between developing somites, and primordial germ cells (PGCs) form in the tail region. This complete embryo model progresses development within an extraembryonic yolk sac that forms blood islands. In some embodiments, the self-organizing capacity of ES cells and extraembryonic stem cells to reconstruct mammalian development through gastrulation and beyond, to neural tube formation and early organogenesis, is demonstrated. The gastrulation and neurulation embryoid bodies generated using the methods and culture media disclosed herein can provide a powerful, physiologically relevant model of post-implantation embryo development. These complete embryoid bodies are also powerful in vitro models for analyzing the roles of various cell lineages and genes in development. Some of the methods and compositions disclosed herein are also disclosed in Amadei et al. "Embryo model completes gastrulation to neurulation and organogenesis." Nature 610, no. 7930 (2022): 143-153, the contents of which are incorporated herein by reference in their entirety.

[0049] Disclosed herein is an in vitro method for generating synthetic embryos from stem cells. In some embodiments, the method comprises culturing mammalian pluripotent stem cells and extraembryonic stem cells in a first culture medium under first static conditions, allowing the mammalian pluripotent stem cells and the extraembryonic stem cells to self-organize into a post-implantation embryo structure (e.g., a post-implantation pre-gastrulation embryo structure). The method further comprises culturing the post-implantation embryo structure in a second culture medium under second static conditions, allowing the post-implantation embryo structure to develop into a neurulation embryo structure. The method further comprises culturing the neurulation embryo structure under dynamic conditions in a culture chamber for at least one day, allowing the neurulation embryo structure to develop into at least an early organogenesis stage synthetic embryo. Synthetic embryo structures obtained by the disclosed methods are also disclosed herein.

[0050] In some embodiments, the methods disclosed herein do not include any in vivo steps. In some embodiments, none of the mammalian pluripotent stem cells, the extraembryonic stem cells, the post-implantation embryonic structures, and the neural tube embryonic structures are present in an in vivo environment during any of the culturing steps disclosed herein. The in vivo environment may include a tissue, an organ, an organism, or a combination thereof.

[0051] Stem cells and mammalian development Disclosed herein are methods and compositions for modeling, e.g., early mammalian embryonic development, by culturing in vitro a mammalian pluripotent stem cell and at least one extraembryonic stem cell (e.g., two extraembryonic stem cells). In some embodiments, the method includes: (a) culturing a mammalian pluripotent stem cell and at least one extraembryonic stem cell in a first culture medium under first static conditions to allow the mammalian pluripotent stem cell and the extraembryonic stem cell to self-organize into a postimplantation structure (e.g., an early postimplantation structure (e.g., E5.5)). In some embodiments, the mammalian pluripotent stem cell is an embryonic stem cell. In some embodiments, the mammalian pluripotent stem cells and at least one extraembryonic stem cell are capable of self-organizing into gastrulation embryonic structures under first resting conditions.

[0052] Although mammalian embryonic development has common features across all species, it is recognized that different mammalian species develop in different ways and at different rates. In general, however, the fertilized egg undergoes several division steps (going through two-cell, four-cell, and eight-cell stages) and then compacts to form a spherical mass of cells (called a morula, in which the 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 then develops into a blastocyst, which contains a fluid-filled vesicle surrounded by trophectoderm and has an inner cell mass at one end.

[0053] The term "embryo" as used herein refers to a mammalian organism from the single cell stage. Embryos as described herein are generated from in vitro culture steps from stem cells under appropriate conditions, and resemble (e.g., have similar morphology, length, weight, cell type composition, and expression of developmental marker genes) natural embryos of the corresponding stage generated in vivo.

[0054] Embryonic developmental stages are defined by the occurrence of specific structures and may be used to define equivalent stages in the development of other species. In some embodiments, embryonic developmental stages may be defined according to the "Carnegie stages", a standardized system for providing a unified time sequence of developmental events for vertebrate embryos. The earliest Carnegie stages are as follows in Table 1: [Table 1]

[0055] In some embodiments, the mammalian embryo generated in the present application is a mouse embryo. Theiler numbered stages of mouse development. The earliest stages apply to (C57BLxCBA)F1 mice and are described in the "emouse digital atlas" (www.emouseatlas.org) as Table 2. [Table 2-1] [Table 2-2]

[0056] The developmental stage of the synthetic embryos generated in this application is defined by their embryonic day of development. As used herein, the term "embryonic day (E)" in the context of a mammalian embryo (e.g., a mouse embryo) refers to an embryo that has the developmental characteristics of an in vivo (in the fallopian tube or in the uterus) mammalian embryo at a particular day after fertilization, where E0 is considered a fertilized egg.

[0057] In some embodiments, the methods and compositions described herein allow for culture up to or through a post-implantation stage corresponding to Theiler stages 7, 8, 9(a), 9(b), 10(a), 10(b), 10(c), 11(a), 11(b), 11(c), 11(d), 12(a), 12(b), 13, 14, 15, 16 and beyond, Carnegie stages (a), 5(b), 5(c), 6, 7, 8, 9 and beyond, and corresponding stages of other species. In some embodiments, the synthetic embryos generated herein may reach post-implantation stages of E4, E4.5, E5, E5.5, E6, E6.5, E7, E7.5, E8, E8.5, E9, E9.5 and beyond.

[0058] The methods and compositions described in this application can be applied to embryos of any suitable mammal, such as primates (e.g., humans, great apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, New World monkeys); rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (e.g., rabbits); cows; sheep; goats; horses; pigs; and other domestic, agricultural, laboratory, or household mammals.

[0059] The methods and compositions described herein can be applied to any of the non-human mammalian embryos described herein, including but not limited to those described herein. Thus, any of the culture media defined herein can support the development of a non-human mammalian embryo on a substrate from pre-implantation to post-implantation developmental stages.

[0060] The term "preimplantation stage" may be used herein to refer to a developmental stage earlier than a stage corresponding to Theiler stage 7, Carnegie stage 5(a), and corresponding stages in other species. As used herein, the term "postimplantation stage" may refer to a developmental stage later than a stage corresponding to Theiler stage 7, Carnegie stage 5(a), and corresponding stages in other species. The "postimplantation stage" may be determined by detecting up-regulation of one or more genes by the embryo. For example, such a stage may 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 Hand1.

[0061] Stem cells (e.g., mammalian pluripotent stem cells and extraembryonic stem cells) can be cultured using the media, kits, and methods described herein. As used herein, the term "stem cells" may refer to cells that can continue to retain some potential for differentiation even after cell division. Examples of stem cells include: embryonic (ES) stem cells with pluripotency obtained 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 the tissues of living organisms (e.g., liver stem cells, skin stem cells, and reproductive stem cells that underlie each tissue); pluripotent stem cells obtained from reproductive stem cells; pluripotent stem cells derived from nuclear reprogrammed somatic cells; totipotent stem cells and non-totipotent stem cells, etc. Partially committed stem cells (e.g., progenitor cells) may also be cultured using the media and according to the methods described herein.

[0062] The methods and compositions described herein can be applied to stem cells from any suitable mammal, including primates (e.g., humans, great apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, New World monkeys); rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (e.g., rabbits); cows; sheep; goats; horses; pigs; and other livestock, agricultural, laboratory, or domestic mammals. The methods and compositions described herein can be applied to stem cells from any non-human mammal described herein, including, but not limited to, the above. In some embodiments, the non-human mammal is a rodent.

[0063] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the potential to differentiate into all cells except the placenta. The pluripotent stem cell has 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, and urogenital system), or ectoderm (which forms epidermal tissue and the nervous system).

[0064] PSCs may be obtained from fertilized eggs, cloned embryos, germline stem cells, or stem cells in tissues. Also included are cells that have differentiation pluripotency similar to embryonic stem cells by artificially introducing multiple different genes into somatic cells (also called induced pluripotent stem cells or iPS cells). Induced pluripotent stem cells may be derived from any suitable source (e.g., hair follicles, skin cells, fibroblasts, etc.). Pluripotent stem cells may be prepared by methods known in the art. Any stem cell defined in this application may be derived from diseased or non-diseased tissue. Stem cells may be derived from any suitable mammal, including, but not limited to, primates (e.g., humans, great apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, New World monkeys); rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (e.g., rabbits); cows; sheep; goats; horses; pigs; and other domestic, agricultural, laboratory, or domestic mammals. The methods disclosed herein may be applied to stem cells of any non-human mammal described herein, including, but not limited to, the above.

[0065] In some embodiments, the PSC cells disclosed in the present application are mammalian embryonic stem cells (ESCs). The term "embryonic stem cell" (ES cell) as used in the present application refers to pluripotent stem cells derived from the inner cell mass of an early stage pre-implantation embryo, the blastocyst. Such cells may express genes involved in the naive pluripotency network (Oct4 / Nanog, Sox2, Klf4, etc.). Such cells may also have Oct4 proximal enhancer activity. They may contribute to all embryonic tissues in chimeras. The ES cells may be derived from mammalian embryos, obtained from iPS cells, or obtained from suitable cell lines. Non-limiting examples of the stem cells include embryonic stem cells of mammals and the like established by culturing early pre-implantation 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 introducing a plurality of 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 constituting an early embryo, embryonic stem cells established from primordial germ cells, cells isolated from a pluripotent cell population of early pre-implantation embryos (e.g., primitive ectoderm), and cells obtained by culturing these cells.

[0066] As will be appreciated by those skilled in the art, ES cells may be obtained from stem cell banks, such as the UK Stem Cell Bank, which provides human stem cell lines for research. The Jackson Laboratory in the United States (which provides the Jax mouse) also stores and derives mouse ES cells which are available commercially. Preferably, the ES cells are obtained, or are obtainable, by a method that does not involve the destruction of human animal embryos or of non-human animal embryos.

[0067] In some embodiments, the at least one extraembryonic stem cell comprises a trophoblast stem cell and an inducible extraembryonic endoderm stem cell. The extraembryonic stem cells in the methods described herein are genetically unmodified or genetically modified and capable of growth and self-renewal. In some embodiments, the cells may be derived from a mouse embryo. The cells are preferably self-renewing stem cells that represent the extraembryonic stem cell compartment of the early embryo.

[0068] The term "trophoblast stem cell" as used in this application refers to a stem cell derived from the trophoblast lineage of the embryo. The trophoblast stem cell is preferably not an extraembryonic cell derived from two cell types that are precursors of the human placenta: cytotrophoblast and syncytiotrophoblast. These cells may be derived from the late preimplantation stage E4.5 or early postimplantation stage (E5.5), but the resulting cell line represents the stem cell compartment present in the extraembryonic ectoderm of the mouse egg cylinder after implantation. Transcription factors such as Elf5, Eomes, and Tfap2c mark this lineage. TS cells may also be considered as cells that are precursors of the differentiated cells of the placenta. In the mouse, TS cells can be derived from either the polar trophectoderm of the blastocyst or from the products of the extraembryonic ectoderm (which arises from the polar trophectoderm after implantation).

[0069] The term "extra-embryonic endoderm stem cell" (XEN stem cell) as used herein refers to stem cells derived from the extra-embryonic endoderm of an embryo (e.g., a mouse embryo). The extra-embryonic endoderm is derived from hypoblast cells that normally migrate into the cavity of the blastocyst (beginning on day 8 of human embryonic development) and form the early and definitive yolk sac that lines the cavity. The extra-embryonic endoderm fills the remaining cavity of the blastocyst. In some embodiments, the XEN stem cells used herein include inducible XEN stem cells that are capable of expressing GATA transcription factors when induced (e.g., by doxycycline treatment). In some embodiments, the XEN cells may be derived from ESCs by overexpression of PrE-specific genes, GATA transcription factors (e.g., Gata4 / 6) or Sox17, or by treatment with growth factors. In some embodiments, the XEN stem cells used herein are inducible XEN stem cells that are capable of expressing GATA4 when induced.

[0070] In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in a first culture medium for 4 to 5 days (e.g., 4 or 5 days). In some embodiments, step (a) is at embryonic day E0-E5.5. The PSCs may be mammalian embryonic stem cells (ESCs). The mammalian ESCs may be mouse ESCs. The ESCs and / or extraembryonic stem cells may be genetically modified.

[0071] Synthetic embryos at various developmental stages Provided herein are methods, compositions, and culture media for modeling mammalian embryonic development by culturing stem cells, such as pluripotent stem cells (e.g., embryonic stem cells) and extraembryonic stem cells. The methods, compositions, and culture media disclosed herein allow for the generation of synthetic embryos through various developmental stages.

[0072] In some embodiments, the synthetic embryos produced using the methods and compositions described herein can reach a post-implantation (e.g., post-implantation pre-gastrulation stage). In some embodiments, the synthetic embryos produced herein can reach an early gastrulation stage. In some embodiments, the synthetic embryos produced herein can reach a late gastrulation stage. In some embodiments, the synthetic embryos produced herein can reach an early neurulation stage. In some embodiments, the synthetic embryos produced herein can reach a late neurulation stage. In some embodiments, the synthetic embryos produced herein can reach an early organogenesis stage or later.

[0073] Embryonic structures generated using the methods and culture media described herein may include post-implantation embryos, e.g., post-implantation pre-gastrulation embryonic structures. As used herein, the term "post-implantation pre-gastrulation" refers to an embryo after the implantation blastocyst stage and before the early gastrulation stage in the context of a mammalian embryo (e.g., a mouse embryo), characterized by an egg cylinder-shape before symmetry is broken. Post-implantation pre-gastrulation stage embryos may be defined as Theiler stages TS7 - TS8 (see Table 2). In some embodiments, post-implantation pre-gastrulation stage refers to E4.5-6.5, optionally E4.5-6, optionally E5-6.5, optionally E5-5.5. In some embodiments, post-implantation pre-gastrulation stage refers to E5.5.

[0074] In some embodiments, a post-implantation pre-gastrulation embryonic structure is generated by co-culturing mammalian pluripotent stem cells and at least one extra-embryonic stem cell in a culture medium (e.g., FC and / or IVC culture medium) under static conditions and allowing the mammalian pluripotent stem cells and the extra-embryonic stem cells to self-organize into a post-implantation pre-gastrulation embryonic structure. The co-culturing of the mammalian pluripotent stem cells and the at least one extra-embryonic stem cell may be for up to 4 or 5 days. The co-culturing of the mammalian pluripotent stem cells and the at least one extra-embryonic stem cell may be from embryonic day E0-E5.5.

[0075] The embryonic development stage of the synthetic embryonic structures generated using the methods and culture media disclosed in this application may be evaluated by comparing with in vivo natural embryos at the same developmental stage by multiple methods, including but not limited to morphology, length, weight, cell type composition, and expression of developmental marker genes (e.g., Oct4, Nanog, Sox2, Klf4, Cdx2, Gata4, Gata6, Brachyury, Otx2, Fgf5, and others described in the Examples and known in the art) using specific antibodies or primers, or transcriptional profiling, single-cell RNA sequencing, and other methods further described in the Examples section. In some embodiments, the post-implantation pre-gastrulation embryonic structures are similar to E5.5 natural embryonic structures. In some embodiments, the post-implantation pre-gastrulation embryonic structures and E5.5 natural embryonic structures have similar morphology, cell type composition, and gene expression characteristics. In some embodiments, the post-implantation, pre-gastrulation embryonic structure comprises a cavitated epithelial embryonic stem (ES) cell and trophoblast stem (TS) cell compartment, surrounded by a VE-like layer (see, e.g., FIG. 1A, E5.5 / ETiX4).

[0076] In some embodiments, the embryonic structures generated using the methods and culture media described herein include post-implantation embryos (e.g., early gastrulation embryonic structures). As used herein, the term "gastrulation" in the context of an embryo refers to an embryo after the expanded blastocyst stage and before the somitogenesis stage, characterized by the formation of a primitive streak and an epithelial to mesenchymal transition that forms the three germ layers. The process of gastrulation is generally considered to be a process in which a bilayered germinal disc changes to a trilayered germinal disc, as the endoderm emerges between the ectoderm and endoderm. As used herein, the term "early gastrulation" in the context of a mammalian embryo (e.g., a mouse embryo) refers to an embryo after the post-implantation pre-gastrulation stage and before the late gastrulation stage, characterized by an egg cylinder-shape with a primitive streak on the posterior side. Early gastrulation stage embryos may be defined as Theiler stages TS8 - TS10 (see Table 2). In some embodiments, the early gastrulation stage refers to E5-7.75, optionally E5-6.5, optionally E6.25-7.25, optionally 6.5-7.75, optionally E6.5-E7.5. In some embodiments, the early gastrulation stage refers to E6.5-E7.

[0077] In some embodiments, the early gastrulation embryo structure comprises a primitive amniotic cavity (resulting from the joining of cavities in the ES and TS cell compartments), a fully migrated AVE (as the boundary between the ES and TS cell compartments), and gastrulation, which may be manifested by an epithelial to mesenchymal transition and the formation of a cell layer between the ES cell and VE-like layers (see, e.g., FIG. 1A, E6.5 / ETiX5).

[0078] In some embodiments, culturing the embryonic structure from a post-implantation pre-gastrulation stage to an early gastrulation stage is accomplished by culturing the embryonic structure in culture medium (e.g., IVC medium, post-implantation culture medium, or both) for a suitable period of time. In some embodiments, culturing the embryonic structure from a post-implantation pre-gastrulation stage to an early gastrulation stage is performed for at least 1 day (e.g., 1 day, 2 days, or 3 days). In some embodiments, the culturing is from E5.5 to E6.6.

[0079] In some embodiments, the gastrulated embryo structures produced using the methods and culture media described herein resemble naturally gastrulated embryos, hi some embodiments, the synthetic gastrulated embryos and the naturally gastrulated embryos have similar morphology, cell type composition, and gene expression characteristics.

[0080] In some embodiments, the post-implantation pre-gastrulation embryo structure is continued to be cultured to its early gastrulation stage, allowing the post-implantation embryo structure to develop to a late gastrulation stage or complete gastrulation. As used herein, the term "late gastrulation stage" in the context of a mammalian embryo (e.g., a mouse embryo) refers to an embryo after the early gastrulation stage and before the early somite stage, characterized by an egg cylinder-shaped embryo with differentiated definitive endoderm, mesoderm, and ectoderm layers. Late gastrulation stage embryos may be defined as Theiler stages TS10 - TS11 (see Table 2). In some embodiments, the late gastrulation stage may correspond to E6.5-8, optionally E6.5-7.75, optionally E7.25-8, optionally E7-8.

[0081] In some embodiments, the post-implantation embryonic structure is continued in culture, allowing the post-implantation embryonic structure to develop through gastrulation and beyond until neural tube formation. Neural tube formation is generally considered to be the embryonic developmental process that begins when the notochord signals the overlying ectodermal germ layer to form a thick, flat neural plate, thereby inducing the formation of the central nervous system. The neural plate folds over itself to form the neural tube, which will later differentiate into the brain and spinal cord of the central nervous system. The neural tube embryos generated in the present application may be embryos resembling any of a series of morphological phases during the neural tube process. The neural tube embryos generated in the present application may be at an early neurulation stage, during a neurulation stage, or at a late neurulation stage.

[0082] In some embodiments, the neural tube forming embryonic structure is generated by culturing a post-implantation embryonic structure (e.g., a post-implantation pre-gastrulation embryonic structure) in a culture medium (e.g., a post-implantation culture medium) under conditions (e.g., static conditions) that allow the post-implantation embryonic structure to develop into a neural tube forming embryonic structure. In some embodiments, the culture of the post-implantation embryonic structure may be for up to 3 days. In some embodiments, the culture of the post-implantation embryonic structure may be from E5.5 to E8.0.

[0083] In some embodiments, the neuralized embryo structures produced using the methods and culture media described herein resemble naturally neuralized embryos, hi some embodiments, the synthetic neuralized embryos and naturally neuralized embryos have similar morphology, cell type composition, and gene expression characteristics.

[0084] In some embodiments, the neural tube formation embryo structure resembles a natural embryo structure at E7-9, optionally at E7.5-8.5, optionally at E7.5-8. In some embodiments, the neural tube formation embryo structure generated in the present application exhibits an anterior-posterior axis with branching neural folds that extend into the neural tube and terminate at a tail bud (a morphology similar to the early headfold stage of a natural embryo at E8.0). Posteriorly, the tail bud joins with allantoic tissue that connects to the developing chorion (see, e.g., FIG. 1C). In some embodiments, the embryoid body, allantoic, and chorion are contained within a fluid-filled sac that corresponds to the yolk sac. In some embodiments, the neural tube forming embryonic structures generated in the present application, and their corresponding natural embryonic counterparts, display a largely conserved distribution of cells among the different germ layers of the epiblast (ectoderm, mesoderm, and endoderm), as well as between embryonic and extraembryonic lineages.

[0085] In some embodiments, the neural tube-forming embryonic structures generated in the present application may express neuroectoderm and surface ectoderm markers (such as, but not limited to, Sox1, Sox2, Pax6, Pax3, Foxa2, Chordin, Shh, OTX2, FOXG1, PAX6, OLIG2, NKX2-2, SOX10, Brachyury, and others identifiable by one of skill in the art). Neuralized embryos contain a portion of SOX1-positive neural tube tissue that terminates in two neural folds, while the SOX1-negative, Brachyury-positive posterior portion displays a tail bud-like morphology. Neuralized embryos may have a Brachyury-positive notochord running underneath the neural tube.

[0086] In some embodiments, the neural tube embryonic structures generated herein include cells expressing markers of the hindbrain and spinal cord, the prosencephalon, the mesencephalon, and the midbrain-hindbrain boundary (e.g., Ets1, Sox10, Fezf1, Lhx2, Six3, En1, and Dmbx1, etc.).

[0087] In some embodiments, the neural tube embryonic structures may further develop into a somite stage embryo, or into a somitogenic embryo (e.g., from late gastrulation and / or neural tube stages to early somite stage). As used herein, the term "somitogenesis" in the context of a mammalian embryo (e.g., a mouse embryo) refers to an embryo after the late gastrulation stage and before the early organogenesis stage, characterized by the appearance of the first, 1 to 5 somites distinguishable by bright field microscopy. Early somitogenic embryos may be defined as Theiler stages TS12 - TS13 (see Table 2). In some embodiments, early somitogenesis refers to E7.5-9.25, optionally E7.5-8.75, optionally E8-9.25, optionally E8-9. In some embodiments, early somitogenic stage refers to E8.5.

[0088] In some embodiments, the synthetic embryonic structures described herein contain cells that express markers such as NKX2-5, GATA4, and / or MYH2 and / or have a gene expression signature that leads to somitogenesis. The neural tube forming embryonic structures may express cardiomyocyte markers such as troponin and myosin genes.

[0089] In some embodiments, the method may include culturing neural tube embryonic structures under dynamic conditions in a culture chamber for at least one day (e.g., 1, 2, 3, 4, or more days) to allow the neural tube embryonic structures to develop into a composite embryo at an organogenesis stage. In some embodiments, the method includes culturing somitogenic embryos (e.g., mouse embryos) under dynamic conditions under conditions that allow the embryos to develop into and beyond an organogenesis stage. In some embodiments, the organogenesis stage is an early organogenesis stage. The term "early organogenesis" in the context of mammalian embryos (e.g., mouse embryos) refers to embryos after the somitogenic stage and before the stage at which a heartbeat appears, characterized by the formation of a neural tube and migration of the mesoderm. In some embodiments, the early organogenesis stage refers to natural embryonic E8-9, optionally E8-8.5, optionally E8.5. Synthetic embryos at the early organogenesis stage may exhibit development of definitive endoderm that gives rise to the gut and associated organs. The presence of foregut and hindgut pockets may be observed in synthetic embryos at the early organogenesis stage. In some embodiments, development of the embryoid bodies described in this application may proceed further beyond the early organogenesis stage in culture.

[0090] In some embodiments, the synthetic embryo has developing somites and primordial germ cells. The synthetic embryos generated herein may have established brain regions, neural tubes, beating hearts, and / or gut tubes. The synthetic embryos generated herein may exhibit one or more of the following characteristics: a headfold with defined forebrain and midbrain regions, a beating heart-like structure, a trunk containing neural tubes and somites, a tail bud containing neuromesoderm precursors, a gut tube, and / or primordial germ cells.

[0091] Culturing embryonic cells in vitro from stem cells (e.g., mammalian pluripotent stem cells and at least one extraembryonic stem cell) may be performed until early organogenesis or any developmental stage in between is reached. In some embodiments, culturing the synthetic embryo in vitro is continued to allow the synthetic embryo at early organogenesis to further develop.

[0092] In some embodiments, the synthetic embryo produced using the methods and culture conditions described herein is a mammalian embryo. In some embodiments, the mammalian embryo is a non-human embryo (e.g., a mouse embryo or a rabbit embryo). In some embodiments, the mammalian embryo is a human embryo.

[0093] The culture conditions (e.g., medium, type, pressure, oxygen concentration, etc.) mentioned above for generating synthetic embryos at different developmental stages are described in the following sections and in a specific embodiment for mouse embryos in the Examples section.

[0094] The embryonic developmental stage of the synthetic embryos described in this application may be assessed by a number of methods (e.g., but not limited to, morphology, length, weight, expression of developmental marker genes using specific antibodies or primers, transcriptional profiling, etc.) in comparison to in vivo or natural embryo counterparts of the same developmental stage, as further described below and in the Examples section.

[0095] Morphological assessment of embryo development may be performed according to previously established morphological features (e.g., morphological features as described in the Carnegei 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), Theiler developmental stages (see, e.g., Table 2; www.emouseatlas.org)) or according to embryonic developmental dates.

[0096] In some embodiments, one or more developmental markers described in this application may be used to assess the developmental stage of the synthetic embryonic structure. A variety of methods exist in the art for detecting the presence, absence, or amount of marker gene products (e.g., mRNA and / or protein), as well as for detecting the localization or subcellular localization (e.g., nuclear and / or cytoplasmic) of marker gene products in the embryonic structure. Expression of the marker may be assessed by any of a wide variety of known methods for detecting 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.

[0097] 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. Expression of the marker can be monitored in a variety of ways, such as 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 can be a qualitative assessment of the level of gene expression, particularly in comparison to a control level. The type of level detected will be clear from the context.

[0098] In another embodiment, detecting or determining the expression level of the marker and its functionally similar homologs (e.g., fragments or genetic modifications (e.g., in its regulatory or promoter regions)) includes detecting or determining the RNA level of the marker of interest. In some embodiments, one or more cells from the synthetic embryonic structure are obtained and RNA is isolated from the cells. In some embodiments, RNA is obtained from a single cell. For example, cells may be isolated from a tissue sample by laser capture microdissection (LCM). This technique may be used to isolate cells from tissue sections (e.g., stained tissue sections), ensuring that the desired cells are isolated. It is also possible to obtain cells, for example, from a synthetic embryonic cell and culture the cells in vitro 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 isolated by methods known in the art (e.g., fluorescence activated cell sorting, microfluidics, etc.).

[0099] For example, when isolating RNA from synthetic embryonic structures at various developmental stages and / or from cells comprising said synthetic embryonic structures, it may be important to prevent any further changes in gene expression after removing the tissue or cells from the subject. Changes in expression levels are known to change rapidly after perturbation (e.g., heat shock or activation by lipopolysaccharide (LPS) or other agents). Furthermore, RNA within tissues and cells can degrade quickly. Thus, in a preferred embodiment, tissues or cells obtained from a subject are flash frozen as soon as possible.

[0100] RNA may be extracted from cells by various methods (e.g., lysis with guanidinium thiocyanate followed by CsCl centrifugation). Methods for obtaining RNA from single-cells are also known in the art. The RNA sample may then be enriched for a particular species. In some embodiments, poly(A)+ RNA is isolated from the RNA sample. Generally, such purification takes advantage of the poly-A tail on the mRNA. In particular, and as described above, poly-T oligonucleotides may be immobilized on a solid support to serve as affinity ligands for the mRNA. Kits for this purpose are commercially available (e.g., MessageMaker kit (Life Technologies, Grand Island, NY)). In some embodiments, the RNA population is enriched for a marker sequence. Enrichment may be performed, for example, by primer-specific cDNA synthesis or by multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription.

[0101] The RNA population, whether enriched for a particular species or sequence, may be further amplified. As defined in this application, 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 is used to amplify the mRNA, so that a signal becomes detectable or detection is enhanced. Such an amplification process is beneficial, especially when the biological, tissue, or tumor sample is of small size or volume.

[0102] A variety of amplification and detection methods can be used, such as reverse transcription of mRNA to cDNA followed by polymerase chain reaction (RT-PCR); or using a single enzyme for both steps (as described in U.S. Pat. No. 5,322,770), or reverse transcription of mRNA to cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) (as described in R.L. Marshall, et al., PCR Methods and Applications 4: 80-84 (1994)) are within the scope of the disclosed methods. Real-time PCR may also be used. Other known amplification methods that can be used in the present application include, but are not limited to, the so-called "NASBA" or "3SR" techniques [described in PNAS USA 87: 1874-1878 (1990) and also in Nature 350 (No. 6313): 91-92 (1991)]; Q-beta amplification [described in European Patent Application Publication (EPA) No. 4544610]; strand displacement amplification [described in GT Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315]; target mediated amplification [described in PCT Publication WO9322461]; PCR; ligase chain reaction (LCR) [see, e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., J. Am. Soc. Soc. Soc. Soc., 1999]; 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. Natl. Acad. Sci. USA 86, 1173 (1989)].Many techniques for determining absolute and relative levels of gene expression are known in the art, and commonly used techniques for use in the methods of the present disclosure include Northern analysis, RNase protection assays (RPA), microarrays, and PCR-based techniques (e.g., quantitative PCR and differential display PCR, etc.). For example, Northern blotting involves running a preparation of RNA in a denaturing agarose gel and transferring it to a suitable support, such as activated cellulose, nitrocellulose, glass, or nylon membrane. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed, and analyzed by autoradiography.

[0103] Visualization by in situ hybridization may be used, where a radioactively labeled antisense RNA probe is hybridized to a thin section of the sample, washed, cleaved with RNase, and exposed to a sensitive emulsion for autoradiography. The sample is stained with hematoxylin to reveal the histological composition of the sample, and the developed emulsion is visualized by dark field imaging with suitable light filters. Non-radioactive labels such as digoxigenin may also be used. In some embodiments, the probe is labeled with a fluorescent moiety.

[0104] Alternatively, mRNA expression may be detected on a DNA array, chip, or microarray. Labeled nucleic acids of a test sample obtained from a subject may be hybridized to a solid surface containing marker DNA. A positive hybridization signal is obtained in samples containing marker transcripts. Methods for preparing DNA arrays and their use are well known in the art (see, for example, U.S. Patent Nos. 66,186,796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. Patent Application No. 20030157485). Serial Analysis of Gene Expression (SAGE) may also be performed (see, for example, U.S. Patent Application No. 20030215858). In some embodiments, next generation sequencing (e.g., RNA-seq) may be used to analyze total mRNA expression from one (e.g., single-cell RNA-seq) or multiple cells. Nucleic acid target molecules labeled with barcodes (e.g., origin-specific barcodes) may be sequenced using the barcodes to generate a single read and / or contig that includes the sequence, or a portion of the sequence, of both the target molecule and the barcode. Exemplary next generation sequencing techniques include, for example, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing, among others. Methods for constructing sequencing libraries are known in the art.

[0105] The single-cell sequencing may be 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 to any particular theory, the single-cell RNA sequencing can efficiently and cost-effectively sequence thousands to tens of thousands of single-cells. In certain embodiments, the single-cell RNA sequencing includes pairing the single-cells in the droplet with oligonucleotides for reverse transcription, where the oligonucleotides are configured to provide a cell-of-origin specific barcode that uniquely identifies the transcripts from each cell, and a unique molecular identifier (UMI) that uniquely identifies each transcript. In certain embodiments, single-cell RNA sequencing involves pairing a single-cell within a droplet with a single microparticle bead coated with oligonucleotides for reverse transcription, where the oligonucleotides include a bead-specific barcode that uniquely identifies each bead and a unique molecular identifier (UMI) that uniquely identifies each primer. In some aspects of the present disclosure, unbiased classification of cells within a biological sample involves sequencing the transcriptomes of thousands of cells, preferably tens of thousands of cells (e.g., more than 1000 cells, or more than 10,000 cells).

[0106] The activity or level of a lineage marker protein can be detected and / or quantified by detecting or quantifying an expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means known to those of skill in the art. Any method known to those of skill in the art for detecting a polypeptide can be used, including, but 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.

[0107] Based on the present disclosure, non-limiting examples of techniques that may be used to detect marker proteins, depending on the practitioner's preference, are described below. One such technique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)), in which appropriately treated samples are run on an SDS-PAGE gel and then transferred to a solid support, such as a nitrocellulose filter. An anti-marker protein antibody (unlabeled) is then contacted with the support, followed by labeling with protein A or anti-immunoglobulin (suitably labeled, e.g., 125 The assay is then performed using secondary immunological reagents such as enzymes such as ELISA, horseradish peroxidase, alkaline phosphatase, fluorophores, etc. Chromatographic detection may also be used.

[0108] Immunohistochemistry may be used to detect expression of marker proteins. A suitable antibody is, for example, contacted with a thin layer of cells, washed, and then contacted with a second, labeled antibody. The label may be a fluorescent marker, an enzyme such as peroxidase, avidin, or a radiolabel. The assay is scored visually using a microscope. Anti-marker protein antibodies (e.g., intrabodies, etc.) may also be used for imaging purposes, for example to detect the presence of the marker protein in cells or, for example, 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 (e.g., fluorescein and rhodamine), and biotin. Antibodies that may 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 may be up to about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The term "specifically binds" refers to, for example, binding of an antibody to an epitope or antigen or antigenic determinant in such a manner that it may displace or compete with a second preparation of the same or similar epitope, antigen, or antigenic determinant. An antibody may preferentially bind to the marker protein relative to other proteins, such as related proteins.

[0109] Antibodies may be commercially available or prepared according to methods known in the art. Antibodies and derivatives thereof that may be used include polyclonal or monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, primatized (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, such as, but not limited to, Fv, Fab, Fab', and F(ab')2 fragments, may be used. Such fragments may be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage may produce Fab or F(ab')2 fragments, respectively. Other proteases with the required substrate specificity may also be used to produce 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 may be designed to include DNA sequences encoding the CH domain and hinge region of the heavy chain. In some embodiments, agents other than antibodies that specifically bind to marker proteins are used, such as peptides. Peptides that specifically bind to marker proteins can be identified by any means known in the art. For example, peptide phage display libraries can be used to screen for specific peptide binders of marker proteins.

[0110] Substrates for culturing synthetic embryos In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells, the post-implantation embryo, the gastrulation embryo, the neurulation embryo, and / or other synthetic embryos described herein are cultured in a substrate. In some embodiments, the method includes transferring the embryo from one substrate to another. The substrates used in the methods disclosed herein can be the same or different. For example, the mammalian pluripotent stem cells and the extraembryonic stem cells can be cultured in a first substrate to form a post-implantation pre-gastrulation embryo structure. The post-implantation pre-gastrulation embryo can be transferred to a second substrate to develop into a neurulation embryo. The neurulation embryo can be transferred to a third substrate to develop into a synthetic embryo of another developmental stage. The first substrate, second substrate, and third substrate can be of the same type or of different types. In some embodiments, the first substrate, the second substrate, and the third substrate are different types of substrates, for example, the first substrate and the second substrate can be microwell plates containing inverted pyramidal microwells, and the third substrate can be a rolling bottle culture unit, such as a rolling incubator.

[0111] Substrates used in the present application may include dishes, U-plates, flasks, or microwell plates. The microwell plates may include inverted pyramidal microwells. The size (e.g., depth and / or diameter) of each inverted pyramidal microwell may vary. Each inverted pyramidal microwell may be about 400 μm or about 800 μm in size. Each inverted pyramidal microwell may be about 400 μm or about 800 μm in diameter. In some embodiments, each inverted pyramidal microwell may be about 100, 200, 300, 400, 500, 600, 700, 800, 900 μm, 1 mm in size and / or diameter, or a value or range between any two of these values. Each microwell (e.g., receptacle) may have a depth of about 250 μm to about 400 μm (e.g., about 300 μm to about 350 μm). Additionally or alternatively, the plurality of vessels may have an average depth of about 250 μm to about 400 μm (e.g., about 300 μm to about 350 μm). In particular, when the vessels are wells, they may be arranged on the substrate in a row, i.e., in a grid pattern with regular spacing in substantially orthogonal directions. Whatever the topography of the substrate, the substrate may bear one or more embryos. When the substrate comprises one or more vessels, each vessel may independently contain one or more embryos (e.g., 2, 3, 4, or 5 or more embryos). In some embodiments, each embryo structure is located in a different respective well. In alternative embodiments, each vessel may contain multiple embryos (e.g., 2, 3, 4, or 5 or more embryos).

[0112] The method disclosed in the present application can be applied to any suitable size of culture volume. For example, the culture volume per embryo can be about 50 μl to about 10 ml, optionally about 100 μl to about 5 ml, optionally about 250 μl to about 5 ml, optionally about 1 ml to about 5 ml. The culture volume 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.

[0113] Conditions for culturing synthetic embryos The stem cells and / or embryos may be cultured under static conditions or dynamic conditions. In some embodiments, the stem cells and / or embryos described herein are cultured under static conditions. As used herein, the term "static conditions" refers to cell culture conditions in which the culture is performed without agitation. For example, the mammalian pluripotent stem cells and the extraembryonic stem cells may be cultured under a first static condition in a static incubator for a period of time (e.g., 1, 2, 3, 4 days or more) to form a post-implantation embryo (e.g., a post-implantation pre-gastrulation embryo). The post-implantation embryo may be cultured under a second static condition in a static incubator for a period of time (e.g., 1, 2, 3 days or more) to develop into a neural tube forming embryo. The first static condition and the second static condition may be the same or different. For example, different culture media may be used for the two (or more) static conditions. The stem cells and embryos may be cultured under static conditions for 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments, the stem cells and / or embryos are cultured under static conditions for up to 7 days. In some embodiments, the stem cells and / or embryos are cultured under static conditions from E0 to E8.0.

[0114] In some embodiments, the method includes culturing in static conditions followed by dynamic conditions. Thus, the methods disclosed herein include culturing embryos (e.g., embryos undergoing neural tube formation) under dynamic conditions in a culture chamber. As used herein, the term "dynamic conditions" refers to cell culture conditions involving agitation (e.g., suspension agitation) (e.g., but not limited to, rotating, rolling, shaking, inverting, etc.) of the culture. Non-limiting examples of dynamic cultures or dynamic culture conditions include roller cultures (culture on a rolling device), shaker cultures (culture on a shaker, e.g., an orbital shaker), or other dynamic cultures identifiable to one of skill in the art.

[0115] In some embodiments, the dynamic conditions include providing a plurality of gases to the culture chamber. The plurality of gases may include O2, N2, CO2, H2, H2O, or combinations thereof. In some embodiments, a mixture of gases is provided into a rotating culture chamber containing one or more embryos. In some embodiments, the dynamic conditions include a gas pressure in the culture chamber of about 0.5 to about 3 pounds per square inch (psi). In some embodiments, the gas pressure in the culture chamber is less than atmospheric pressure at sea level. For example, the gas pressure in the culture chamber is about 0.5 to less than 1 psi. In some embodiments, the gas mixture is provided to the culture chamber at about 0.5 psi.

[0116] In some embodiments, the dynamic conditions include supplying a constant concentration of oxygen to the culture chamber. In some embodiments, the oxygen is supplied to the culture chamber at an increasing concentration. The oxygen concentration may be increased throughout the culturing step, starting at 5%, to 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or more, at any suitable time interval (e.g., daily intervals). In some embodiments, the oxygen concentration is increased gradually from about 5% to about 13%, to about 18%, to about 21% at daily intervals. In some embodiments, the culture chamber may have an atmosphere containing an increasing oxygen concentration, from about 5% to about 25% oxygen level, optionally from about 5% to about 13%, optionally from 13% to about 18%, optionally from about 18% to about 21%. In some embodiments, the increase is by 1.5-2.5 fold, or by 1.5-2 fold per increase step. In some embodiments, the oxygen level in the culture chamber is less than 30%. In some embodiments, the dynamic conditions used in culturing neural tube forming embryos do not include a step of decreasing the oxygen concentration throughout the culturing step. In some embodiments, the increase in oxygen concentration is every 0.5-2 days, every 0.5-1.5 days, every 1-2 days, or every 1-1.5 days of the culture. In some embodiments, the increase is every 20-28 hours of the culture. In some embodiments, the increase is at daily intervals.

[0117] The neural tube-forming embryo may be cultured under dynamic conditions as described herein for at least one day (e.g., one, two, three, four or more days). In some embodiments, the neural tube-forming embryo is cultured under dynamic conditions from E8.0 to at least E8.5 (e.g., E8.5, E9, or later). In some embodiments, the neural tube-forming embryo may be cultured under dynamic conditions in a culture chamber for at least one day to allow development to proceed into a synthetic embryo at least at an early organogenesis stage.

[0118] Culture medium for culturing synthetic embryos In some embodiments, the method includes co-culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in feeder cell (FC) medium, and optionally passaging the mammalian pluripotent stem cells and the extraembryonic stem cells at least twice (e.g., 2, 3, 4 or more times) in the feeder cell medium. The mammalian pluripotent stem cells and the extraembryonic stem cells may be cultured in the FC medium for 1, 2, 3, or 4 days. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the FC medium for about 2 days.

[0119] In some embodiments, the method includes co-culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in in vitro culture (IVC) medium, optionally after culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the FC medium. The mammalian pluripotent stem cells and the extraembryonic stem cells may be cultured in the IVC medium for 1, 2, 3, or 4 days. In some embodiments, the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the IVC medium for about 2 days, optionally after culturing in the FC medium for about 2 days.

[0120] In some embodiments, the method includes partially replacing an amount of FC medium (e.g., at least half of the medium) with fresh FC medium or with IVC medium. The replacement may occur every 20-28 hours (e.g., every 24 hours) of the culturing step. In some embodiments, the method includes partially replacing an amount of IVC medium (e.g., at least half of the medium) with fresh IVC medium.

[0121] In some embodiments, the method includes culturing the post-implantation embryonic structure in a second culture medium under second static conditions, allowing the post-implantation embryonic structure to develop into a neural tube-forming embryonic structure. The second culture medium is a post-implantation culture medium capable of supporting embryo development outside of the uterus. The post-implantation embryonic structure may be cultured in the post-implantation culture medium for up to 3 days or more (e.g., 1, 2, 3 days or more). The volume of the culture medium may be increased every 20-28 hours (e.g., every 24 hours) by providing the embryo with an amount of fresh culture medium (e.g., 200-500 μl per embryo). In some embodiments, the method includes culturing the neural tube-forming embryonic structure under dynamic conditions in the same post-implantation culture medium. In some embodiments, the post-implantation culture medium comprises human umbilical cord serum. In some embodiments, the post-implantation culture medium comprises bicarbonate or HEPES.

[0122] The culture medium disclosed in the present application may include a basal culture medium. The basal medium may include water, salts, amino acids, a carbon source, vitamins, lipids, and a buffer. Suitable carbon sources may be assessed by one skilled in the art from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. The basal culture medium may include any of the following: Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or serum substitutes thereof, antibiotics, L-glutamine or analogs thereof (e.g., GlutaMAX® ... TM ), or any combination thereof.

[0123] Non-human serum or serum substitutes include fetal bovine serum, bovine serum albumin, rat serum, KnockOut TMThe antibiotics may include penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof. In some embodiments, the culture medium (e.g., FC medium and IVC medium) includes a reducing agent. The reducing agent may include beta-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof.

[0124] The concentration or amount of one or more components in the solution or medium may be varied, for example, non-human serum or serum substitutes, antibiotics, reducing agents, and / or L-glutamine (e.g., GlutaMAX TMThe amount of may vary and, in some embodiments, may be subject to adjustments as needed by one of ordinary skill in the art. In some embodiments, the amount of non-human serum or serum substitute thereof can be 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 value or range between any two of these values) volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. In some embodiments, the amount of antibiotic can be 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 / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. For example, the amount of the 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 any value or range between any two of these values). TM) may vary. For example, in some embodiments, the concentration of L-glutamine in the culture medium may be about 0.1 mM to about 40 mM, about 0.2 mM to about 20 mM, about 0.5 mM to about 10 mM, about 1 mM to about 5 mM, or about 1.5 mM to about 2.5 mM (e.g., about 2 mM). When percentages are used for agents, ingredients, and compounds, they may be % w / w, % w / v, or % v / v, of the total formulation, unless otherwise indicated.

[0125] Each component of the culture medium described herein is present in an amount such that the culture medium is suitable to support stem cell self-organization into a post-implantation embryonic structure and / or further development of the post-implantation embryonic structure. In some embodiments, the post-implantation embryonic structure is an embryonic structure undergoing gastrulation. In some embodiments, the post-implantation embryonic structure is at a pre-gastrulation stage.

[0126] In embodiments of the in vitro culture medium provided herein, the culture medium may be free, substantially free, or essentially free of one or more of: epidermal growth factor (EGF) receptor agonists or analogs thereof (e.g., EGF or EGF substitutes); fibroblast growth factor family (FGF) receptor agonists or analogs thereof (e.g., FGF or FGF substitutes); leukemia inhibitory factor (LIF) receptor agonists or analogs thereof (e.g., LIF or LIF substitutes); bone morphogenetic protein (BMP) receptor agonists or analogs thereof (e.g., BMP or BMP substitutes); WNT receptor agonists or analogs thereof (e.g., WNT or WNT substitutes). Additionally, the culture medium may be free, substantially free, or essentially free of TGFβ receptor agonists or analogs thereof. Unless otherwise specified, the culture medium may be free, substantially free, or essentially free of members of the nodal, activin, stem cell factor, or hedgehog families of proteins.

[0127] The stem cells described herein, e.g., mammalian pluripotent stem cells and at least one extraembryonic stem cell, may be cultured separately prior to co-culture as described herein, e.g., in a suitable culture medium as described in U.S. Publication No. 2022 / 0308041, the contents of which are incorporated by reference in their entirety into this application.

[0128] The culture media described herein may contain other components or analogs thereof. As used herein, the term "analog" may refer to a biologically active analog of any component of the culture medium. Such analogs may be natural or synthetic.

[0129] The specific biologically active ligands and compounds, e.g., insulin, progesterone, etc., used in the media defined in this application are used for illustrative purposes. However, one of skill in the art will readily recognize that analogs of such ligands and compounds can be used as substitutes as well, so long as they retain the relevant biological activity. One of skill in the art can routinely identify other biologically active compounds suitable for use as substitutes. For example, these can be naturally occurring compounds or compounds that can be made by synthetic or semi-synthetic methods.

[0130] FC medium In some embodiments, the FC medium may further comprise an effective amount of sodium pyruvate, e.g., at a concentration of about 0.05 mM to about 20 mM (e.g., about 0.05, 0.1, 0.2, 0.3, 0.4, 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 mM, or a value or range between any two of these values). In some embodiments, the FC medium comprises about 1 mM sodium pyruvate.

[0131] The FC medium may also include an effective amount of an 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 non-essential amino acid may be, for example, about 0.1% to about 2% (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) volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. In some embodiments, the FC medium includes about 1% of the non-essential amino acid. Non-essential amino acids may be included in the culture medium, for example glycine (from about 1 mg / ml to about 25 mg / ml, or from about 5 mg / ml to about 10 mg / ml, for example about 7.5 mg / ml), L-alanine (from about 1 mg / ml to about 25 mg / ml, or from about 5 mg / ml to about 10 mg / ml, for example about 9 mg / ml), L-asparagine (from about 5 mg / ml to about 30 mg / ml, or from about 10 mg / ml to about 15 mg / ml, for example about 13.2 mg / ml), L-aspartic acid (from about 5 mg / ml to about 30 mg / ml, or from about 10 mg / ml to about 15 mg / ml, for example about 13 mg / ml), L-glutamic acid (from about 5 mg / ml to about 50 mg / ml, or from about 10 mg / ml to about 20 mg / ml, for example about 15 mg / ml), L-proline (from about 5 mg / ml to about 50 mg / ml, or from about 10 mg / ml to about 20 mg / ml, for example about ... and / or L-serine (from about 5 mg / ml to about 30 mg / ml, or from about 10 mg / ml to about 15 mg / ml, e.g., about 11 mg / ml).In some embodiments, the culture medium may contain L-glycine at a concentration of about 7.5 mg / ml, L-alanine at a concentration of about 9 mg / ml, L-asparagine at a concentration of about 13 mg / ml, L-aspartic acid at a concentration of about 13 mg / ml, L-glutamic acid at a concentration of about 14.5 mg / ml, L-proline at a concentration of about 11.5 mg / ml, and L-serine at a concentration of about 10.5 mg / ml.

[0132] The non-human serum in the FC medium may vary. In some embodiments, the FC medium may comprise non-human serum (e.g., fetal bovine serum) in 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 any value or range between any two of these values) volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. In some embodiments, the FC medium comprises about 15% to about 20% non-human serum (e.g., fetal bovine serum). In some embodiments, the FC medium comprises about 15% fetal bovine serum. In some embodiments, the FC medium comprises about 20% fetal bovine serum.

[0133] The FC medium may include an effective amount of L-glutamine or an analog thereof. L-glutamine may be included 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 (e.g., about 2 mM). In some embodiments, L-glutamine is included in the FC medium at a concentration of about 2 mM.

[0134] The FC medium may include an effective amount of a reducing agent. In some embodiments, the concentration of the reducing agent in the FC medium may 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 FC medium at a concentration of about 0.1 mM. In some embodiments, the FC medium includes β-mercaptoethanol (BME) at a concentration of about 0.1 mM.

[0135] In some embodiments, the FC medium comprises an effective amount of 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 (AS1892802), 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-Benzodioxine-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), Rock inhibitors, 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; Rock antibodies available from Santa Cruz Biotechnology, selected from the group consisting of: Rock-1 (B 1), Rock-1 (C-19), Rock-1 (H-11), Rock-1 (G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2 (D-11), Rock-2 (N-19), Rock-2 (H-85), Rock-2 (30-J); ROCK CRISPR / Cas9 knockout plasmids selected from 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 Plasmids and / or shRNA Lentiviral Particle gene silencers 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.

[0136] The FC medium may contain an effective amount of a ROCK inhibitor, for example, from about 0.1 nM to about 100 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 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 In some embodiments, the FC medium comprises a ROCK inhibitor at a concentration of about 7.5 nM.In some embodiments, the FC medium comprises from about 1 nM to about 100 nM Y-27632 (e.g., about 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 nM, or any value or range between any two of these values). In some embodiments, the FC medium comprises about 7.5 nM Y-27632. In some embodiments, the FC medium does not contain a ROCK inhibitor.

[0137] In some embodiments, the FC medium comprises DMEM, fetal bovine serum, sodium pyruvate, GlutaMax TM, MEM non-essential amino acids, 2-mercaptoethanol, penicillin and / or streptomycin, or any combination thereof. In some embodiments, the FC medium comprises DMEM, about 15% fetal bovine serum, about 1 mM sodium pyruvate, about 2 mM GlutaMax, about 1% MEM non-essential amino acids, about 0.1 mM 2-mercaptoethanol, about 1% penicillin and / or streptomycin, or any combination thereof. In some embodiments, the FC medium comprises Dulbecco's modified essential medium (Gibco 41966052), about 15% fetal bovine serum (Cambridge Stem Cell Institute), about 1 mM sodium pyruvate (Gibco 11360039), about 2 mM GlutaMAX (Gibco 35050038), about 1% MEM non-essential amino acids (Gibco 11140035), about 0.1 mM 2-mercaptoethanol (Gibco 31350010), and about 1% penicillin / streptomycin (Gibco 15140122). The FC medium may further comprise one or more anticoagulants, such as heparin, and fibroblast growth factors (FGFs) (e.g., FGF2 and / or FGF4), or any combination thereof.

[0138] IVC medium The IVC medium includes the basal medium described in this application. The basal medium may include water, salts, amino acids, a carbon source, vitamins, lipids, and a buffer. Suitable carbon sources may be evaluated by one of skill in the art from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. Basal media may be purchased, for example, under the trade names Advanced DMEM / Fl2 (Gibco, 12634-010) and CMRL-1066 (Invitrogen or Sigma). The basal medium may include Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Roswell Park Memorial Institute (RPMI) medium 1640, Neurobasal (登録商標) Neurobasal (登録商標) A, Connaught Medical Research Laboratory 1066 (CMRL-1066), or any combination thereof.

[0139] The IVC medium may 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.

[0140] The amount of insulin, estrogen, progesterone, or their analogs or receptor agonists present in the IVC medium may vary. For example, in some embodiments, the IVC medium has a concentration of 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, In some embodiments, the composition may comprise one or more hormones (e.g., progesterone) at concentrations (e.g., 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), and / or one or more growth factors (e.g., insulin or insulin-like growth factor).In some embodiments, the IVC medium comprises 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, 0.5mM, 1 The therapeutic agent may include a hormone (eg, estrogen) in the amount of 0.1 mM, or a value or range between any two of these values, and / or insulin, or an insulin-like growth factor.

[0141] In some embodiments, the insulin receptor agonist is selected from the group including IGF-I, IGF-II, analogs thereof, or any combination thereof. The estrogen receptor agonist may be selected from the group including β-estradiol, estrone, estriol, and estetrol, or any analogs thereof. The IVC medium may include transferrin, sodium selenate, ethanolamine, or any analogs thereof. The IVE medium may include insulin-transferrin-selenium-ethanolamine (ITS-X). In some embodiments, the IVC medium further includes an agonist of the activin type 1 receptor or of the activin type 2 receptor. The IVC medium may include a reducing agent. In some embodiments, the reducing agent may include N-acetyl-L-cysteine, dithiothreitol (DTT), β-mercaptoethanol (BME), or any combination thereof.

[0142] The IVC medium may include non-human serum, the concentration of which may vary in different embodiments. In some embodiments, the IVC medium may include non-human serum (e.g., fetal bovine serum) at 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 any value or range between any two of these values) volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. In some embodiments, the IVC medium comprises about 20% to about 30% non-human serum (e.g., fetal bovine serum). In some embodiments, the IVC medium comprises about 20% fetal bovine serum. In some embodiments, the IVC medium comprises about 30% fetal bovine serum. In some embodiments, culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the IVC medium comprises increasing a serum concentration, optionally increasing the serum concentration from about 20% to about 30%.

[0143] The IVC 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 (e.g., about 2 mM). In some embodiments, L-glutamine is contained in the culture medium at a concentration of about 2 mM.

[0144] The IVC medium may include an effective amount of a reducing agent. In some embodiments, the concentration of the reducing agent in the IVC medium may 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 any value or range between any two of these values). In some embodiments, the reducing agent is included in the IVC medium at a concentration of about 25 μM. In some embodiments, the IVC medium includes N-acetyl-L-cysteine ​​at a concentration of about 25 μM.

[0145] Penicillin may be included in the culture medium at a concentration of about 1 unit / ml to about 500 units / ml, about 2 units / ml to about 250 units / ml, about 5 units / ml to about 100 units / ml, about 10 units / ml to about 50 units / ml, or about 20 units / ml 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 μg / ml to about 500 μg / ml, about 2 μg / ml to about 250 μg / ml, about 5 μg / ml to about 100 μg / ml, about 10 μg / ml to about 50 μg / ml, 25 or about 20 μg / ml to about 30 μg / ml, for example about 25 μg / ml. The culture medium may contain penicillin at a concentration of about 25 units / ml, and / or streptomycin at a concentration of about 25 μg / ml.

[0146] The culture medium, as described herein, may comprise a basal medium (e.g., Advanced DMEM / Fl2) supplemented with an insulin receptor agonist (e.g., insulin (e.g., about 2 mg / ml to about 25 mg / ml)), transferrin (e.g., about 1 mg / ml to about 10 mg / ml), selenium (e.g., sodium selenate (e.g., about 0.001 mg / ml to about 0.01 mg / ml)), ethanolamine (e.g., about 0.5 mg / ml to about 10 mg / ml), an estrogen receptor agonist (e.g., estradiol (e.g., about 5 nM to about 10 nM)), a progesterone receptor agonist (e.g., progesterone (e.g., about 50 ng / ml to about 500 ng / ml)), and a reducing agent (e.g., N-acetyl-L-cysteine ​​(e.g., about 17.5 μM to about 40 μM)). In some embodiments, the IVC medium is free or substantially free of sodium pyruvate.

[0147] In some embodiments, the IVC medium comprises DMEM / F12, fetal bovine serum, GlutaMAX, ITS-X, β-estradiol, progesterone, N-acetyl-L-cysteine, penicillin and / or streptomycin, or any combination thereof. In some embodiments, the IVC medium comprises DMEM / F12, about 20% fetal bovine serum, about 2 mM GlutaMAX, about IX ITS-X, about 8 nM β-estradiol, about 200 ng / ml progesterone, about 25 μM N-acetyl-L-cysteine, about 1% penicillin and / or streptomycin, or any combination thereof.

[0148] Post-implantation medium In some embodiments, the post-implantation embryo is cultured in a post-implantation medium capable of supporting embryo development outside the uterus. The method described herein includes culturing the post-implantation embryo structure in a post-implantation medium under static conditions, allowing the post-implantation embryo structure to develop into a neural tube-forming embryo structure. The method also includes culturing the neural tube-forming embryo in a post-implantation medium under dynamic conditions in a culture chamber for at least one day, allowing the neural tube-forming embryo structure to develop into a synthetic embryo at least at an early organogenesis stage.

[0149] In some embodiments, the post-implantation embryo comprises a basal medium as described herein, such as Dulbecco's Modified Eagle Media (DMEM) or DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum (e.g., rat and / or bovine serum), human umbilical cord serum, L-glutamine or an analog thereof (e.g., GlutaMAX TM ), antibiotics, or any combination thereof.

[0150] The DMEM or DMEM / F12 may be present in the medium at 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 / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w) of the medium. In some embodiments, the DMEM or DMEM / F12 is present in the medium at about 25%. In some embodiments, the post-implantation medium comprises DMEM at about 25%.

[0151] The post-implantation medium may comprise an effective amount of non-human serum from about 5% to about 60% (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%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or any value or range between any two of these values), volume / volume (% v / v), weight / volume (% w / v) or weight / weight (% w / w). In some embodiments, the post-implantation medium comprises about 50% non-human serum. In some embodiments, the post-implantation medium comprises about 50% rat serum.

[0152] The amount of human umbilical cord serum in the post-implantation medium may vary. In some embodiments, the post-implantation medium comprises an effective amount of human umbilical cord serum, 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 any value or range between these values) of the medium, volume / volume (% v / v), weight / volume (% w / v), or weight / weight (% w / w). In some embodiments, the post-implantation medium comprises about 25% human umbilical cord serum.

[0153] In some embodiments, the post-implantation medium comprises about 25% DMEM, about 50% rat serum, and about 25% human umbilical cord serum.

[0154] In some embodiments, the post-implantation medium further comprises an effective amount of bicarbonate, which may be present in the post-implantation medium at a concentration of from about 0.1 mM to about 30 mM, optionally from 1 mM to about 20 mM.

[0155] In some embodiments, the post-implantation medium comprises an effective amount of HEPES, which may be present in the post-implantation medium at a concentration of from about 0.1 mM to about 30 mM, optionally from 1 mM to about 20 mM.

[0156] The post-implantation medium may comprise an effective amount of L-glutamine or an analog thereof (e.g., GlutaMAX TM L-glutamine may be included 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 (e.g., about 2 mM).

[0157] The antibiotic may be included in the post-implantation medium at a concentration of about 1 unit / ml to about 500 units / ml. For example, penicillin may be included in the culture medium at a concentration of about 1 unit / ml to about 500 units / ml, about 2 units / ml to about 250 units / ml, about 5 units / ml to about 100 units / ml, about 10 units / ml to about 50 units / ml, or about 20 units / ml 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 μg / ml to about 500 μg / ml, about 2 μg / ml to about 250 μg / ml, about 5 μg / ml to about 100 μg / ml, about 10 μg / ml to about 50 μg / ml, 25 or about 20 μg / ml to about 30 μg / ml (e.g., about 25 μg / ml). The post-implantation medium may include penicillin at a concentration of about 100 units / ml, and / or streptomycin at a concentration of about 100 μg / ml.

[0158] The post-implantation medium may include, for example, DMEM, rat serum, human umbilical cord serum, GlutaMAX, penicillin and / or streptomycin, HEPES, or any combination thereof. In some embodiments, the post-implantation medium includes about 25% DMEM, about 50% rat serum, and about 25% human umbilical cord serum. The post-implantation medium is further supplemented with 1x Glutamax (GIBCO, 35050061), 100 units / ml penicillin and 100 μg / ml streptomycin, and 11 mM HEPES (GIBCO 15630056).

[0159] In some embodiments, culturing the post-implantation embryo includes adding glucose to the post-implantation medium. The glucose may be added to the post-implantation medium or to the basal culture medium at an increasing concentration throughout the culturing step. The glucose concentration may be increased from at least about 3 mg / ml to about 4-5 mg / ml. In some embodiments, the glucose is added to the post-implantation medium from about 3 mg / ml to about 3.5 mg / ml. The glucose may be added at least once (e.g., 1 time, 2 times, 3 times, or more times) at a concentration of at least 3 mg / ml (e.g., 3, 3.5, 4, 4.5, 5, or more). The post-implantation medium may be supplemented with at least 3 mg / ml glucose on about day 3 of culture in the post-implantation medium. In some embodiments, at the time the embryo reaches the neural tube stage and thereafter, the medium contains at least about 3 mg / ml glucose, and the next day contains at least about 3.5 mg / ml glucose.

[0160] In some embodiments, the glucose is added to the post-implantation medium at an increasing concentration throughout the culturing step. In some embodiments, the glucose is added to the post-implantation medium at a constant concentration and then increased in concentration. In some embodiments, the glucose is added to the post-implantation medium at a constant concentration throughout static conditions and then increased in concentration throughout dynamic conditions. In some embodiments, the glucose may be added to the post-implantation medium at a constant concentration under static conditions and then increased in concentration at the same or different static concentration. For example, culturing the post-implantation embryo may include culturing the post-implantation embryo structure in a medium containing about 1 mg / ml glucose for about 2 days under static conditions and culturing the post-implantation embryo structure in a medium containing about 3 mg / ml glucose for about 1 day.

[0161] In some embodiments, the glucose is added at increasing concentrations when the embryo is transferred from static to dynamic conditions. For example, the method may include culturing the post-implantation embryo structure in a medium containing about 3 mg / ml glucose under static conditions for about 1 day (e.g., when the embryo reaches the neurulation stage), followed by culturing in a medium containing about 3.5 mg / ml glucose under dynamic conditions for at least 1 day. In some embodiments, the glucose is added in the medium at increasing concentrations throughout the dynamic conditions.

[0162] Also disclosed herein are embryo culture systems (eg, embryo culture devices) suitable for generating synthetic embryos in vitro using any of the methods disclosed herein.

[0163] Purpose Also provided herein are synthetic embryos obtainable by the in vitro methods described herein for use in methods of diagnosing, preventing, or treating disease in a patient in need thereof. For example, embryonic cells obtainable from the present invention may be used in stem cell therapy (e.g., cancer treatment, tissue replacement, reconstructive surgery, tissue repair, wound healing, bone marrow transplantation, treatment for stroke, alopecia, blindness, deafness, diabetes, heart disease, bowel disease, arthritis, skeletal injuries, tooth replacement, neurological disease, and any other condition in which replacement cells or tissues may be beneficial). The cells may also be used to screen therapeutic compounds for efficacy and safety, as will be appreciated by those skilled in the art.

[0164] In some embodiments, the synthetic embryo structure may be used for transplantation into a patient in need thereof for use in a method of diagnosing, preventing, or treating a disease in the patient as described herein. In certain embodiments, the pluripotent stem cells used to obtain the embryo were originally obtained from the patient, which is believed to reduce the likelihood of rejection by the patient's immune system. Thus, pluripotent stem cells (e.g., embryonic stem cells and extraembryonic stem cells obtained from a patient) may be cultured using the methods described herein to provide material for transplantation back into the patient to prevent or treat a condition. For example, the embryo may be used to grow a replacement organ or tissue for the patient to restore organ or tissue function after loss of function due to degeneration, aging, and / or disease in the patient.

[0165] Also disclosed herein is a method for providing a transgenic non-human animal, comprising gestating an embryo obtained from cells cultured using the in vitro methods described herein. Such transgenic non-human animals may be useful in drug screening or in disease research. For example, model animals may be produced to study certain conditions. Using the methods provided herein, it is believed that transgenic and chimeric embryos can develop more efficiently (which currently rely on, for example, the labor-intensive process of harvesting blastocysts and manually replacing the inner cell mass).

[0166] Disclosed herein is a method for studying the effect of a test agent on embryo development. In some embodiments, the method comprises: a) generating a synthetic embryo using a method described herein; b) contacting the synthetic embryo with a test agent; and c) determining the effect of the test agent on the synthetic embryo. In some embodiments, the determining step comprises comparing the phenotype or genotype of the synthetic embryo in the presence of the test agent with the phenotype or genotype of the synthetic embryo in the absence of the test agent. The method may comprise contacting the mammalian pluripotent stem cell and at least one extraembryonic stem cell with the test agent during or after step (a) and before step (b), during or after step (b) and before step (c), or during or after step (c).

[0167] The method may include determining the subsequent effect on the formation of a synthetic embryo at various developmental stages. The determining step may be performed using any method known in the art. For example, the method may include recording one or more images of the embryonic structure.

[0168] Disclosed herein are methods for investigating mechanisms involved in embryo development. In some embodiments, the methods include any of the in vitro methods for generating synthetic embryo structures at various developmental stages described herein. Investigating mechanisms involved in embryo development may include any method known in the art. For example, the investigating step may include investigating the effect of a test agent on embryo development, as described above. In some embodiments, investigating mechanisms involved in embryo development may include determining the effect of genetic perturbations on the embryo structures.

[0169] The method may include recording a plurality of images of the synthetic embryonic structure, the plurality of images being recorded over a predetermined period of time, thereby 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.

[0170] Typically, fluorescent markers (e.g., fluorescent dyes or fluorescent marker proteins, etc.) are used in imaging embryo development. Such markers may be added to the culture system. For example, fluorescent dyes may be added to visualize specific molecules or cellular structures. For example, DAPI may be used to stain DNA or MitoTracker (Invitrogen) may be used to stain mitochondria. Additionally or alternatively, the embryonic structure may endogenously produce such fluorescent markers (e.g., the embryonic structure may include one or more cells that express a fluorescent marker protein). Such cells may be genetically modified to be endowed with the ability to express such marker proteins. Thus, fluorescent imaging devices may be particularly suitable for the described methods. Thus, imaging devices may include fluorescent microscopes (e.g., confocal microscopes) (including, but not limited to, wide field, scanning and spinning disc confocal, and light sheet microscopes).

[0171] Confocal microscopes allow for the generation of 2D or 3D images by imaging a single point of a sample at any given time, but by scanning different points in the sample in a regular raster to provide image data that can be assembled into a 2D or 3D image. For example, by scanning a sample in a single plane, a 2D image of a slice through the sample can be generated. By combining multiple or "stacks" of such 2D images, a 3D image can be generated. Spinning disk confocal microscopes offer further advantages over confocal laser scanning microscopes. Furthermore, light sheet microscopes can also be used to better image embryo development.

[0172] Also disclosed herein is a method for elucidating the role of genes in embryonic development, comprising obtaining pluripotent stem cells and / or extraembryonic stem cells in which the genes have been modified or knocked out, and culturing the pluripotent stem cells and extraembryonic stem cells using the in vitro methods described herein, which can thus aid in the development of treatments for conditions related to embryonic development, such as infertility treatments.

[0173] Also disclosed herein is a method of imaging a developing embryo, comprising culturing mammalian pluripotent stem cells, and extraembryonic stem cells, or synthetic mammalian 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 or a three-dimensional image. Multiple images of the same embryo may be recorded. The imaging device may include a microscope device and a suitable recording device. The imaging device may further 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. EXAMPLES

[0174] 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 disclosure in any way.

[0175] Example 1 Materials and Methods In this example, we provide the general experimental materials and methods used in Examples 2-7 described below:

[0176] Cell lines and culture conditions All cell lines used in this application are mouse cell lines and include: CAG-GFP / tetO-mCherry mouse ES cells (constitutive GFP expression in the membrane; transient mCherry expression upon Dox treatment). The parental CAG-GFP / tetO-mCherry ES cell line was derived from an existing mouse line that constitutively expresses CAG-GFP and transiently expresses mCherry upon Dox induction. This line was generated by crossing CAG-GFP reporter mice with tetO-mCherry histone mice. In this application, an independent Dox-inducible Gata4-expression cassette was introduced into the CAG-GFP / tetO-mCherry ES cell line by piggyBac-based translocation. Thus, mCherry and Gata4 are controlled by two independent Dox-responsive promoters. CAG-GFP / tetO-mCherry / tetO-Gata4 ES cells were generated in-house. Cerl-GFP ES cells (GFP expression under the control of the Cerl promoter) were derived from a published Cerl-GFP mouse line. Cerl-GFP / tetO-Gata4 ES cells were generated in-house. Wild-type CD1 TS cells were generated in-house. Wild-type CD1 ES cells were donated. CD1 / tetO-Gata4 ES cells were generated in-house. Sox2-Venus / Brachyury-mCherry / Oct4-ECFP ES cells, Blimp1-GFP ES cells, and BVSC ES cells were donated.

[0177] For the experiments described in this application, ETiX embryoid bodies were successfully generated using the following ES cell lines: wild-type CD1 ES cells; Sox2-Venus / Brachyury-mCherry / Oct4-ECFP ES cells; CAG-GFP / tetO-mCherry ES cells; Blimp1-GFP ES cells; and BVSC ES cells.

[0178] In addition to the above lineages, five more lines were also examined that could progress to days 5 and 6 but could not progress beyond that. These lines were: Lfng reporter (LuVeLu) ES cells; Msgn1-Venus ES cells; Hes7-Achilles ES cells; Sox1-GFP ES cells; mTmG ES cells (generated in-house). The unsuccessful results generated with these five lines are not described in this application.

[0179] Most of the constructs presented here were generated using wild-type CD1 ES cells, wild-type CD1 TS cells, and CD1 / tetO-Gata4 ES cells. The gender of the cell lines was unknown as the cells were not genotyped to determine their gender. All cell lines were routinely tested every two weeks to ensure they were free of mycoplasma contamination. Mouse ES and TS cells were cultured as detailed in “Inducible Stem-Cell-Derived Embryos Capture Mouse Morphogenetic Events In Vitro”, Amadei et al., 2021, Developmental Cell 56, 366-382 ( / / doi.org / 10.1016 / j.devcel.2020.12.004) (Amadei 2021), the contents of which are incorporated by reference in their entirety into this application. The CD1 tetO-Gata4 Dox-inducible cell line was established as described elsewhere. The cell line was not authenticated.

[0180] Culture of mouse ES and TS cells Mouse ES and TS cells were cultured as detailed in Amadei 2021. Briefly, mouse ES cells were cultured on gelatinized plates at 37°C, 5% CO2, 21% O2 in N2B27 (containing 50% Neurobasal-A (Gibco 10888022), 50% DMEM / F-12 (Gibco 21331020), 0.5% N-2 (in-house), 1% B-27 (Gibco 10889038), 2 mM GlutaMAX (Gibco 35050038), 0.1 mM 2-mercaptoethanol (Gibco 31350010) and 1% penicillin / streptomycin (Gibco 15140122)). N2B27 was supplemented with 3 μM CHIR99021 (Cambridge Stem Cell Institute), 1 μM PD0325901 (Cambridge Stem Cell Institute), and 10 ng / ml leukemia inhibitory factor (Cambridge Stem Cell Institute). Mouse TS cells were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs, Insight Biotechnology, ASF-1201) in RPMI 1640 (Sigma) containing 20% ​​fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM 2-ME, 1 mM sodium pyruvate, and 1% penicillin-streptomycin (TS cell medium) supplemented with 25 ng / ml FGF4 (R&D Systems 7486-F4-025) and 1 μg / ml heparin (Sigma-Aldrich H3149-25KU) at 37°C and 5% CO2.MEFs were cultured in feeder cell (FC) medium containing Dulbecco's modified essential medium (Gibco 41966052), 15% FBS (Cambridge Stem Cell Institute), 1 mM sodium pyruvate (Gibco 11360039), 2 mM GlutaMAX (Gibco 35050038), 1% MEM non-essential amino acids (Gibco 11140035), 0.1 mM 2-mercaptoethanol (Gibco 31350010), and 1% penicillin / streptomycin (Gibco 15140122). ES and TS cells were passaged when they reached 70% confluency as follows: cells were washed once with 1x PBS (Life Technologies 10010056) and trypsinized (trypsin-EDTA 0.05% Life Technologies 25300054) for 3 min at 37°C. The reaction was stopped by adding 2 ml of FC or TS cell medium, respectively. Cells were disaggregated by gentle pipetting 4-5 times and centrifuged at 200 x g for 4 min. TS cells were then resuspended in TS cell / F4H culture medium and plated on MEF-coated plates at a dilution of 1:20 or 1:10. ES cells were washed once with 1 ml of 1×PBS, centrifuged again, resuspended in N2B27 2iLIF, and plated onto gelatin-coated plates at a dilution of 1:10 or 1:20.

[0181] Knockout of Pax6 using CRISPR-Cas9 Pax6 was targeted within a 104 bp region immediately preceding the homeobox sequence in exon 6. Guide RNA (gRNA) oligonucleotides were designed using an online CRISPR design tool. The ones with the lowest off-target potential were selected based on the software's predictions. gRNAs were annealed with their respective reverse oligonucleotides, cloned into PX459, and transformed into DH5α cells as previously described. Minipreps were Sanger sequenced using the sequencing primer 5′-TGCATATACGATACAAGGCTGTTAG-3′ (SEQ ID NO: 9). Wild-type ES cells (CD1 background) were transfected using Lipofectamine 3000 according to the manufacturer's instructions. Briefly, cells were seeded at a density of 25,000 cells per well in 24-well plates the day before transfection. The next day, the cells were transfected with the gRNA pair in PX459 (500 ng per plasmid). A PIP Fucci construct without the antibiotic resistance cassette in a separate well was used as a transfection control. A negative control (no DNA) was also run in parallel. After 2 days of selection with 1 μg / ml antibiotic, the cells were washed with fresh medium and removed from the antibiotic. Individual clones were isolated and cultured in 96-well plates until colonies were visible. Single colonies grew in 18 of the 46 wells. Each of these was passaged and split into three new wells (each in a different 96-well plate). Two of these plates were trypsinized and frozen using FC medium + 10% DMSO + 25% FBS. Colonies in the remaining plates were grown until confluent.Genomic DNA was extracted from each single clone and genotyped using Platinum Taq DNA polymerase (Invitrogen, 13001012) and the following primers: FW: 5′-AAGAGACCTTGCGAGAGCAC-3′ (SEQ ID NO: 7); RV: 5′-GAACTTTCCCACCAGGAGCA-3′ (SEQ ID NO: 8). A standard 25 μl reaction was prepared with 12.5 μl Platinum Taq PCR master mix, 2 μl template DNA, 0.5 μl of 10 μM stock of each primer, and 9.5 μl H2O. PCR cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 40 s. The PCR products were then examined using gel electrophoresis. Promising clones that migrated smaller than the wild type (<715 bp) were subjected to Sanger sequencing. Immunofluorescence was performed to confirm the deletion, following the neural differentiation protocol.

[0182] Formation of ETiX embryoid bodies Formation of ETiX embryoid bodies was performed as previously described in Amadei 2021. Briefly, to prepare AggreWell plates (STEMCELL Technologies 34415), 500 μl of anti-adhesion rinse solution (STEMCELL Technologies 07010) was added to each well. The plates were then centrifuged at 2,000 xg for 5 minutes and incubated at room temperature for 20 minutes. The rinse solution was then aspirated from the wells. 1 ml of PBS was added to wash each well. After aspirating the PBS, 500 μl of FC medium was added to each well.

[0183] To generate ETiX embryos, doxycycline (1 μg / ml) (Sigma-Aldrich D9891-5G) was added to CAG-GFP tetO-Gata4 ES cells for 6 hours. TSCs were trypsinized and added to gelatinized plates for 20 minutes at 37°C to remove MEFs. CAG-GFP WT ES cells and CAG-GFP tetO-Gata4 ES cells were then trypsinized. ES cells were washed once with 1×PBS and trypsinized with 0.05% trypsin-EDTA (ThermoFisher Scientific) for 3 minutes at 37°C. The reaction was stopped by adding 2 ml of FC medium. Cells were gently disaggregated by pipetting 4-5 times and centrifuged at 200×g for 4 minutes. The cell pellet was washed once with 1 x PBS, centrifuged again, and resuspended in 1-2 ml of FC medium. A cell suspension containing 19,200 TS cells, 6,000 CAG-GFP WT ES cells, and 6,000 CAG-GFP tetO-Gata4 ES cells was mixed and pelleted by centrifugation. The cell pellet was resuspended in 1 ml of FC medium containing 7.5 nM ROCK inhibitor (Y27632, STEMCELL Technologies 72304). After the cell mixture was dropped into the AggreWell, the plate was centrifuged at 100 xg for 3 minutes, and the cells were seeded into the AggreWell (day 0).

[0184] The next day (day 1), two medium changes were performed by removing 1 ml of medium from each well and adding 1 ml of fresh FC medium without ROCK inhibitor. On day 2, one medium change was performed, replacing 1 ml of medium with 1 ml of fresh FC medium. On day 3, 1 ml of medium was removed from each well and after equilibration in the incubator for 20 min, 1.5 ml of In Vitro Culture Medium 1 (IVC1) (containing 20% ​​v / v FBS) was added. IVC1 was made with advanced DMEM / F12 (Gibco, 21331-020) supplemented with 20% (v / v) FBS, 2 mM GlutaMAX, 1% v / v penicillin-streptomycin, 1 x ITS-X (Thermo Fisher Scientific, 51500-056), 8 nM β-estradiol, 200 ng / ml progesterone, and 25 μM N-acetyl-L-cysteine. On day 4, ETiX embryoid bodies in the AggreWell were transferred to Cellstar 6-well multiwell plates for suspension culture (Greiner Bio-One 657185) with 5 ml of IVC1 (containing 30% v / v FBS) per well.

[0185] Ex utero culture of mouse embryos was as described previously. DRH medium consisted of 25% DMEM, 50% rat serum, and 25% human umbilical cord serum, and allowed development to proceed up to the somite stage in static culture and beyond the somite stage after transfer to a Precision roller bottle culture apparatus (BTC Engineering). Glutamine and antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin) were added to glutamine-free bicarbonate-buffered DMEM (Gibco 11054). Low glucose DMEM (1 mg / ml) was supplemented with 3 mg / ml glucose, as glucose depletion has been implicated as the primary cause of abnormal formation and growth retardation. Embryos were routinely cultured in phenol red-free medium, as phenol red is fluorescent. The medium contained the same ratios of DMEM (Gibco 11880), rat serum, and human umbilical cord serum as DRH, but was buffered with HEPES rather than bicarbonate (and was renamed ex utero culture medium (EUCM)). EUCM contained 25% DMEM (GIBCO 11880) plus 50% rat serum and 25% human umbilical cord serum, supplemented with 1×Glutamax (GIBCO, 35050061), 100 units / ml penicillin, and 100 μg / ml streptomycin, and 11 mM HEPES (GIBCO 15630056). Rat whole embryo culture serum was from Charles River. Human umbilical cord serum was provided by the Cambridge Blood and Stem Cell Biobank, which is supported by the Cambridge NIHR Biomedical Research Centre, the Wellcome Trust-MRC Stem Cell Institute, and the Cambridge Experimental Cancer Medicine Centre, UK. Human and rat serum was heat-inactivated (from frozen) at 56°C for 35 min and sterilized by filtration.

[0186] Typically, on day 5, IVC1 was replaced with DRH or EUCM containing 1 x Glutamax (GIBCO, 35050061), 100 units / ml penicillin, and 100 μg / ml streptomycin, and 11 mM HEPES (GIBCO 15630056). Each ETiX embryoid body was transferred to a single well of a 24-well, non-adherent dish (Greiner 662102) containing 250 μl of DRH or EUCM. On day 6, an additional 250 μl of DRH or EUCM was added to each ETiX embryoid body. On day 7, the samples were transferred to a roller bottle culture chamber apparatus, and the low glucose medium was supplemented with 3.0 mg / ml D-glucose (Sigma G8644). Each roller bottle contained 2 ml of medium and three ETiX embryoid bodies. The medium was further supplemented with 3.5 mg / ml D-glucose on day 8. In each roller bottle, two ETiX embryoid bodies were cultured in 3 ml of medium.

[0187] A device for regulating gas pressure and gas mixture on a roller bottle has been described previously. The device was modified to generate pressure rather than gas mixture. Thus, embryoid bodies were allowed to proceed through development to day 8 using only the pressure-generating component of the device. A device was constructed in this application to deliver a defined gas mixture at 0.5 psi rather than the 6.5 psi previously described. Embryoid bodies and natural embryos were successfully cultured for sequential smFISH using this device. Providing a gas mixture at this lower pressure was effective in promoting development when 21% oxygen was continuously delivered during culture or when the oxygen concentration was gradually increased from 5%, to 13%, to 18%, to 21%, at daily intervals. DRH medium required the use of 5% CO2.

[0188] Mouse model and embryo collection Mice used in this study (6-week-old CD-1 males obtained from Charles River and transgenic females bred in-house) were housed in the animal room in accordance with national and international guidelines. All experiments performed were regulated by the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and reviewed by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). The experiments were also approved by the Home Office. Mice were kept in an animal facility with a 12:12 photoperiod and were provided with food and water ad libitum.

[0189] Natural mating of 6-week-old transgenic females with CD-1 males was performed. Mouse embryos were collected by dissection from the deciduae in M2 medium (Sigma M7167) at embryonic days E5.5, E6.5, and E7.5. Embryos at E6.5 were cultured in EUCM in static conditions until E8.5 in the same previously reported manner as ETiX embryoid bodies. At E8.5, embryos were transferred to roller bottles in DRH or EUCM supplemented with 3.0 mg / ml D-glucose. Each bottle contained 2 ml of DRH or EUCM medium and 3 embryos. Both male and female embryos were used. Embryos were randomly assigned. The investigator was not blinded to the assignment of embryos.

[0190] Plasmids and transfection Gata4 cDNA was PCR amplified from pSAM2-mCherry-Gata4 using Gata4 / AttB primers (see Table 3). The primers were designed as outlined in the Gateway cloning manual. As the plasmid already contained an AttB site, it was not necessary to incorporate any part of the Gata4 open reading frame during primer design. pSAM2-mCherry-Gata4 was donated (Addgene plasmid # 72690; / / n2t.net / addgene:72690; RRID:Addgene_72690). It was then cloned into PB-tetO-hygromycin by Gateway technology (Thermo Fisher Scientific) according to the manufacturer's instructions. Clones were verified by sequencing. Transformation was performed using 5α-competent E. coli (New England Biolabs C2987I). To generate ES cells with doxycycline-inducible Gata4, PB-tetO-hygro-Gata4, pBAse, and rtTA-Zeocin (0.25 μg each / reaction) were transfected into 12,000 CAG-GFP ES cells using Lipofectamine 3000 transfection reagent (Invitrogen L3000001), followed by antibiotic selection with hygromycin (1:250; Gibco 10687010) and zeocin (1:1000; InvivoGen ant-zn-1) for 7 days. The PB-tetO-hygro, pBAse, and rtTA-Zeocin were obtained as gifts from the Stem Cell Institute (Cambridge, UK). [Table 3]

[0191] Immunofluorescence ETiX embryoid bodies and natural embryos were processed for immunofluorescence as previously reported in Amadei 2021. Briefly, ETiX embryos and natural mouse embryos were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times with PBST (PBS containing 0.1% Tween 20). They were then permeabilized with permeabilization buffer (PBST containing 0.1 M glycine and 0.3% Triton X-100). For embryos up to E7.5 and ETiX embryos up to day 6, permeabilization was performed for 30 min at room temperature, followed by three washes with PBST for 5 min each. Natural embryos from E7.5 onwards and ETiX embryoid bodies from day 6 onwards were permeabilized for 35 min. Samples were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (PBS containing 10% FBS and 0.1% Tween 20). After washing three times with PBST, they were incubated with secondary antibodies and DAPI overnight at 4 degrees or for 2 hours at room temperature and washed another three times with PBST before imaging.

[0192] Cryosectioning and slide immunofluorescence Post-fixation native embryo and ETiX embryoid body samples were cryoprotected in 30% sucrose / PBS (w / v) overnight at 4°C. Samples were then transferred to cryomolds filled with optimal cutting temperature (OCT) compound (Agar Scientific) and frozen on a metal block cooled with dry ice. The samples were cut at 12 μm thickness on a cryostat, collected onto lysine-coated slides, and stored at -80°C until ready for immunofluorescence. To remove the OCT compound, slides were washed in PBS for 5-10 min and then briefly air-dried for 5-10 min. Samples were permeabilized with permeabilization buffer (PBST containing 0.1 M glycine and 0.3% Triton X-100) for 10 min at room temperature and then blocked with blocking buffer (PBS containing 10% FBS and 0.1% Tween 20) for 1 h at room temperature. After permeabilization and blocking, samples were processed as described above. Slides were mounted with Vectashield, sealed with nail polish, and allowed to air dry in the dark overnight before imaging.

[0193] inDrops scRNA-seq Sample Preparation and Isolation After recovery, natural embryos and ETiX embryoid bodies were disaggregated for single-cell sequencing as previously described in Amadei 2021. Briefly, natural embryos and ETiX embryoid bodies were cut into small pieces, transferred to Falcon tubes, centrifuged, washed with PBS, incubated in Tryple Express (Gibco™ 12604013) for 15 min, and disaggregated into single cells by vigorous pipetting 20 times every 5 min. If clumps remained, the incubation was extended for an additional 5 min and the samples were pipetted further. Samples were filtered through a 40 μm filter to remove large clumps, centrifuged at 200 x g for 5 min, and then resuspended in PBST (PBS with 0.02% Tween 20). To determine the percentage of live and dead cells, the suspensions were quantified with Trypan Blue (Sigma T8154) at a 1:1 ratio and then processed for encapsulation (see below). Samples analyzed: n = 29 for ETiX5, n = 10 for ETiX6, n = 7 for ETiX8, n = 12 for E6.5, n = 14 for E7.5, and n = 9 for E8.5.

[0194] inDrops scRNA-seq Library Preparation and Sequencing Libraries were prepared following the inDrops v3 workflow using the v3 barcoding scheme. Briefly, polyacrylamide beads were generated and barcoded to obtain a diversity of 147,456 barcodes. Single-cell suspensions were diluted to a concentration of 100,000 cells per ml and co-encapsulated with barcoded beads and reverse transcriptase and lysis mixture. Fractions of approximately 1,000 cells were collected in 1.5 ml Eppendorf tubes pre-filled with 200 μl mineral oil, subjected to UV photolysis, and incubated at 50°C for 2 h and 70°C for 20 min. The droplets were then de-emulsified and further amplified using double-strand synthesis and in vitro transcription. The libraries were then fragmented and reverse transcribed. Final libraries were amplified using a unique 8-bp index and limited-cycle PCR and quantified using the Qubit High Sensitivity Kit (Invitrogen) and Bioanalyzer High Sensitivity DNA Kit (Agilent). Libraries were pooled in equimolar ratios and purified using a 1.5x volumetric ratio of AmpureXP beads. The libraries were sequenced with a Nextseq 75 cycle 400M read high output kit using 5% PhiX spike-in as an internal control. The distribution of read cycles was as follows: read 1, 61 cycles; index 1, 8 cycles; index 2, 8 cycles; read 2, 14 cycles.

[0195] scRNA-seq bioinformatics analysis BCL files were converted to Fastq files using Illumina's bcl2fastq software. Sequenced libraries were quality-checked using the FastQC tool v0.11.9 (www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and de-multiplexed using the Pheniqs tool in biosails v2.1.0. The fastq files were further filtered and mapped to the mouse GRCm38.99 reference genome with GRCm38.99 gtf annotations, and duplicates were removed using the zUMIs pipeline v2.9.7. The count matrix containing exon and intron counts was then used as input for downstream analysis using Seurat version 3. Cells were filtered based on the number of genes detected (between 700 and 4,000), unique molecular identifiers (UMIs) detected (<7,500), the percentage of UMI counts mapping to mitochondrial genes (between 1% and 15%), and the doublet score (<0.3) calculated using Scrublet v0.1, resulting in a total of 26,748 cells. Natural and synthetic embryo datasets were merged in Seurat. Shared embeddings were corrected for batch effects (both system and harvested time points) using Harmony v4.3.12. Louvain clustering was performed on the shared embeddings. Cell types were annotated using markers calculated using the FindAllMarkers function in Seurat. Pearson correlation coefficients between cell types, single-cell velocity profiles, and latencies for each system were calculated using Scanpy v1.0 and scVelo v0.2.4 tools.Plots were generated using Scanpy (written in Python for the dot plots and velocity), and Seurat (written in R for the UMAP plots), and ggplot2 for the remainder of the plots (written in R for the bar graphs and ratio scatter plots).

[0196] Generating single-cell sequencing data using tiny-sci-RNA-seq A simplified version of sci-RNA-seq3, further optimized for 'tiny' samples, was performed. Briefly, 100 μl of hypotonic PBS-based lysis buffer containing DEPC as an RNase inhibitor was added to each tube. The resulting nuclei were then fixed with four volumes of a mixture of methanol and dithiobis (succinimidyl propionate) (DSP). After careful rehydration and washing with sucrose / PBS / triton / MgCl2 buffer (SPBS™), the nuclei were distributed into two 96-well plates for reverse transcription, with eight wells per embryo. After reverse transcription, the nuclei were pooled, washed with SPBS™, and redistributed into a new plate for ligation of the second index primer using T4 DNA ligase. Nuclei were then pooled again, washed, and redistributed onto three final plates for second strand synthesis, extraction, tagmentation, and PCR amplification to add a third index and a plate index. Products were pooled per PCR plate, size-selected, and sequenced in two Illumina NextSeq runs (NextSeq-1 and NextSeq-2). Samples analyzed: n = 8 natural embryos from E7.5 to E9.5, n = 3 for ETiX6, n = 2 for failed ETiX6, 5 for ETiX8, 4 failed ETiX8, and 2 Pax6 knockout ETiX8.

[0197] Processing sequence reads from tiny-sci-RNA-seq data For each NextSeq run of newly generated tiny-sci-RNA-seq data, read alignment and generation of gene count matrices were performed using a slightly modified pipeline developed for sci-RNA-seq3: base calls were converted to fastq format using Illumina bcl2fastq v2.20 and demultiplexed using the maximum likelihood demultiplexing package deML with default settings based on PCR i5 and i7 barcodes. Downstream sequencing processing and generation of single-cell digital expression matrices were similar to sci-RNA-seq, except that reverse transcription (RT) indexes were combined with hairpin adapter indexes. Mapped reads were then split into constituent cell indexes by demultiplexing reads using both RT indexes and ligation indexes (Levenshtein edit distance (ED) < 2, including insertions and deletions). Briefly, demultiplexed reads were filtered based on RT index and ligation index (ED < 2, including insertions and deletions) and adaptor-clipped using trim_galore v0.6.5 with default settings. Trimmed reads were mapped to the mouse reference genome (mm10) of mouse embryo nuclei using STAR v2.6.1d with default settings and gene annotation (Gencode VM12 for mouse). Uniquely mapped reads were extracted. Duplicates were removed using UMI sequences (ED < 2, including insertions and deletions), RT index, hairpin ligation adaptor index, and read 2 end-coordinate (i.e., reads with UMI sequences less than 2 edit distances. RT index, ligation adaptor index, and tagmentation sites were considered as duplicates).Finally, the mapped reads were split into constituent cell indexes by further demultiplexing the reads using RT index and ligation hairpins (ED < 2, including insertions and deletions). To generate digital expression matrices, the number of strand-specific UMIs for each cell that map to the exonic and intronic regions of each gene was calculated using the Python v2.7.13 HTseq package. For multiple mapped reads, the read was assigned to the nearest gene unless another intersecting gene was within 100 bp of the end of the nearest gene (in which case the read was discarded). Most analyses included both expected strand intronic and exonic UMIs in the single-cell expression matrix for each gene. After generating single-cell gene count matrices, we filtered out doublets and potentially low-quality cells (by examining the number of UMIs and the percentage of reads mapping to exonic regions per cell), leaving 285,640 cells (n = 130,611 cells for NextSeq-1, n = 155,029 cells for NextSeq-2; Figure 9A-D). The following popular and freely available data analysis software was used in this project: scrublet version 0.1, Scanpy version 1.6.0, Monocle versions 2, 3, and 3-alpha, Seurat version 3, and ggplot2 version 3.3.5.

[0198] Subclustering Conventional scRNA-seq data processing was performed using Seurat v3: (1) normalizing UMI counts by total counts per cell and performing log transformation; (2) selecting the 2,500 most variable genes and scaling their expression to zero mean and unit variance; (3) applying principal component analysis and using the top 30 principal components to create a k-nearest neighbor graph followed by Louvain clustering (resolution = 1); (4) performing UMAP visualization in 2D space (dims (dimensions used as input features) = 1:30, min_dist = 0.3). Adjacent clusters were manually merged if there were a limited number of differentially expressed genes between them. For sub-clustering, we took a subset of cells of interest (e.g., cardiac mesoderm) and followed the approach above to identify more detailed cell populations.

[0199] Identification of cell types correlated between ETiX embryoid bodies and natural embryos using NNLS regression Transcriptional profiles of cells within each cell type were first aggregated for either ETiX embryoid bodies or natural embryos in the newly generated tiny-sci-RNA-seq data. NNLS regression was applied to the phenotypes of all cell types (M b ) based on gene expression of the target cell type (T a ) predicted gene expression in:T a = β 0a + β 1a M b (Here, β 0a is the intercept of the regression, β 1a is the β-coefficient of the regression), the 3,000 most highly expressed genes in the target cell type, and the 3,000 most highly specific genes. Next, the roles of Datasets A and B were swapped. Thus, all cell types in Dataset A (M a ) gene expression in the target cell type (T b ) predicted gene expression in:Tb = β 0b + β 1b M a Finally, for each cell type a in dataset A and each cell type b in dataset B, we combined the two correlation coefficients: β = 2(β ab + 0.01)(β ba + 0.01), resulting in a statistic whose high values ​​reflect both reciprocal and specific predictability.

[0200] Selection criteria for ETiX embryoid bodies All ETiX embryoid bodies were retrieved from the AggreWell for analysis on day 4 of development and analyzed under a stereomicroscope. ETiX embryoid bodies with cylindrical morphology and two clearly defined cell compartments (ES cell compartment and TS cell compartment) surrounded by an outer cell layer, a VE-like layer, were selected. The ES cell compartment was expected to be epithelial with a lumen. The TS cell compartment was more variable in appearance. Therefore, a broader range of appearances was selected for the TS cell compartment, although an epithelial-looking TS cell compartment similar to the ExE of natural embryos would also be desirable. The majority of ETiX embryoid bodies were generated using a wild-type, unlabeled stem cell line, so the selection was based on morphology alone. ETiX embryoid bodies with the correct body plan of the ES cell compartment and of the TS cell compartment surrounded by a VE-like layer were then transferred to equilibration medium and culture continued. However, during the selection on day 5, the following additional criteria were incorporated: (1) the lumens of the ES cell compartment and of the TS cell compartment were expected to integrate; (2) ideally, the onset of gastrulation could be observed on one side of the ETiX embryoid body; (3) the AVE was expected to migrate to the ES cell-TS cell boundary and be on the opposite side of the forming streak; (4) ETiX embryoid bodies in which the AVE was anchored to the tip of the structure or not at the boundary were excluded; and (5) when using triple reporter cells, the one with higher Sox2-Venus expression was ensured. On day 4, 10%-15% of the structures formed in the pyramidal microwells were harvested. From day 4 to day 5, 20% of the structures harvested on day 4 were cultured.

[0201] Image acquisition, processing, and analysis Images were acquired using Leica SP5 and SP8 confocal microscopes (Leica Microsystems) equipped with 40x oil and 25x water immersion objectives, respectively. A 405-nm diode laser (DAPI), a 488-nm argon laser (Alexa Fluor 488), a 543-nm HeNe laser (Alexa Fluor 568), and a 633-nm HeNe laser (Alexa Fluor 647) were used to excite fluorophores. Images were taken with a z-step of 1.2-5 μm. The images were processed and analyzed using FIJI with NDSAFIR 3.0, Smart Denoise (Gurdon Institute). Area measurements used for quantification were also collected using Fiji. Figures were assembled using Adobe Illustrator v26.0.1.

[0202] The images provided are representative of multiple experiments analyzed. As with natural embryos, the described features of ETiX embryoid bodies can be observed under a conventional stereomicroscope. For example, beating heart-like structures were clearly visible in 87% of the resulting structures. Headfolds and somites were also easily visible and recognizable. Brightfield microscopy of such morphology predicted reproducibility of staining. Furthermore, analysis of individual ETiX embryoid bodies by single-cell sequencing showed remarkable reproducibility between these structures. Quantification suggested that the organs and regions analyzed were comparable in size to those in natural embryos, although a larger size variation was observed between ETiX embryoid bodies. Finally, it is likely that some of the variation observed in ETiX embryoid bodies was caused by the ex utero culture itself, since it also caused variation in the development of natural embryos.

[0203] Sequential smFISH - Primary probe design Gene-specific primary probe sets were designed as previously described, with some modifications. Briefly, a 35-nt binding site probe set was generated for each gene using exonic sequences from the consensus region of all spliced ​​isoforms. For genes that did not yield sufficient targets (>40), intronic and 5' untranslated region sequences were also used. The University of California Santa Cruz (UCSC) masked genome and annotation database was used to obtain gene sequences and extract 35-bp sequences with 45-75% GC content and no regions with repeated 5-nt bases of the same kind. Each probe sequence was then run against a BLAST database constructed from intron and mRNA sequences reversed with GENCODE. Any probe that showed a BLAST hit with at least a 15-nt match to any sequence other than the target gene was considered an off-target hit and was removed from the probe set. All probe sets for each gene were then trimmed down to a maximum of 40 probes by removing all probes furthest from the 55% GC content of the target.

[0204] Sequential smFISH - Readout probe design The readout probes were previously designed. Briefly, a set of 20-nt probe sequences was randomly generated by combination of A, T, G, and C nucleotides. Sequences with 45-60% GC were selected and run against the BLAST database to eliminate any sequences that matched any closely homologous sequence longer than 14 nt to the mouse transcriptome. The reverse complements of these readout sequences were included in the primary probes with AA or TAAT linkers as follows: [readout]-AA-[readout]-AA-[readout]-TAAT-[probe binding sequence]-TAAT-[readout]-AA-[readout]-AA-[readout]. Thus, each probe was 141 nt long.

[0205] Sequential smFISH - Construction of primary and readout probes Primary probes were ordered from IDT as oligonucleotide pools with a concentration of 50 pmol per probe and 5'-phosphate modifications. Readout probes were ordered from IDT as 250 nmol DNA oligonucleotides purified by HPLC and with 5'-fluorophore modifications (5' Alexa Fluor 647N, 5' Alexa Fluor 488N, or 5' Alexa Fluor 546N).

[0206] Sequential smFISH - Coverslip functionalization Coverslips were functionalized by treatment with 1 M HCl for 1 h at room temperature, rinsing once with water, treatment with 1 M NaOH for 1 h at room temperature, and then immersion in 1% bind-silane (GE-Healthcare, 17-1330-01) prepared in 10% (v / v) acidic ethanol solution at pH 3.5 for 30 min. The coverslips were then rinsed thoroughly three times with 100% ethanol, placed on a glass slide, and heat cured in a >90°C oven for 30 min. The coverslips were allowed to cool. The area of ​​the coverslip intended for the placement of the final tissue section was covered with 100 μg / μl poly-D-lysine (Gibco, A3890401) for >1 h. The coverslips were then washed thoroughly three times with water and dried in a tissue culture hood with UV-sterilization. For long term storage, the coverslips were stored dry at 4°C and used within 2 weeks.

[0207] Sequential smFISH - Sequential smFISH experiments on ETiX embryoid bodies ETiX embryoid bodies and natural embryos were fixed in 4% paraformaldehyde overnight at 4°C. They were then washed twice in PBST (PBS with 0.1% Tween 20) at 4°C, washed in a series of graded methanol / PBST for 10 min each at 4°C, and dehydrated in graded methanol. Samples were stored overnight at -20°C, then rehydrated in a series of graded methanol / PBST washes and washed twice in PBST for 10 min at 4°C. Embryonic samples were then immersed in 30% sucrose / PBS (w / v) overnight at 4°C or until the samples sunk to the bottom of the tube. Samples were transferred to cryomolds, carefully placed in OCT compound solution (Agar Scientific), and frozen in dry ice-ethanol. Samples were stored at -80°C until sectioning.

[0208] Tissue blocks were cut at 20-μm thickness using a micron cryostat and placed on top of the functionalized coverslips. After drying for >15 min, the coverslips were placed at -80°C for long-term storage. After storage at -20°C for at least 1 day, the tissue sections were permeabilized in 70% ethanol at 4°C for >1 h, then dried and cleared in 1 ml of 8% SDS (Invitrogen, AM9822) in 1x PBS for 30 min at room temperature. After rinsing twice with PBS and once with nuclease-free water, a custom-made flow cell (fluid volume approx. 30 μl) made of a glass slide (25 x 75 mm) with a thickness of 1 mm and a hole of 1 mm diameter, and a PET film (total thickness 0.25 mm) coated on both sides with acrylic adhesive (Grace Bio-Labs, RD481902) was attached to the cover glass. Using the hole of 1 mm diameter, the tissue sample was incubated overnight at 37°C in 30% hybridization buffer (Molecular Instruments) containing 3.3 nM of each probe. To prevent evaporation during incubation, the hole of the flow cell was covered using a sticker (Grace Bio-Labs, GBL629200). The samples were then washed 4 times for 1 h with 30% wash buffer (Molecular Instruments) and then rinsed 5 times with 4× saline-sodium citrate buffer (SSC) (Thermo Fisher, 15557036).

[0209] Sequential smFISH - Sequential smFISH in control embryos After every step of the ETiX embryoid body smFISH experiment, an additional clearing step was performed to improve the signal-to-noise ratio in the resulting images. In addition to the gene marker probes, poly-30-T LNA oligonucleotides with 5'-acrydite modifications (IDT) were hybridized at 2 μM. After washing with 30% washing buffer, a 100 μm thick sticker with a 3 mm diameter circle cut out was applied to the sample. A gel solution containing 4% acrylamide and 0.2% bis-acrylamide (Bio-Rad, 1610154) with 0.25% VA-044 (Fujifilm, LB-VA044-50GS) was prepared on ice and treated with nitrogen for >5 min. 20 microliters of the solution was dropped onto the sample (contained within the circle cut out in the sticker) and a 22 x 22 mm coverslip was placed over the sample. The samples were then placed in a humidified sealed chamber and flushed with nitrogen for >10 min to remove all oxygen. The samples were incubated overnight at 4°C to allow the hydrogel to permeate the samples, and then placed at 37°C for 3.5 hours to allow the hydrogel to set. The small glass coverslip and sticker were then removed. The samples were treated with 1:100 proteinase K (NEB, P8107S), 50 mM pH 8.0 Tris-HCl (Thermo Fisher, 15568025), 1 mM EDTA (Thermo Fisher, 15575020), 0.5% Triton X-100 (Sigma, 93443), 500 mM NaCl (Sigma, S5150), and 1% SDS (Invitrogen, AM9822) for 2.5 hours at 37 degrees in a humidified chamber. The samples were washed with 2×SSC for 15 min and treated with Label-X (0.1 μg / μl Acryloyl-X SE (Thermo Fisher A20770) and 0.1 μg / μl Label-IT reagent (Mirus, MIR3900)) as previously described for 45 min at 37°C.The samples were washed with 2×SSC and then re-embedded in hydrogel as described above for further stabilization for long-term imaging.

[0210] Sequential smFISH - Microscope setup All imaging experiments were performed using the imaging platform and fluid delivery system as previously described. The microscope (Ti Eclipse) was equipped with a confocal scanner unit (Yokogawa CSU-W1), sCMOS camera (Andor Zyla 4.2), 60x oil immersion objective (Nikon Plan / Apo, NA 1.4, WD 0.13), motorized stage (ASI MS-2000), and a 7-wavelength Nikon LUNF XL laser launcher. The following filters were used: 435 / 26 bp for 405 nm (Chroma), 525 / 36 bp for 488 nm (Chroma), 588-700 bp for 561 nm (Chroma 59007 dual band pass), and 705 / 72 bp for 647 nm (Chroma). A custom-made autosampler was used to move designated readout probes in hybridization buffer (10 nM per readout probe in 2 x SSC, 10% ethylene carbonate (Sigma, E26258), 10% dextran sulfate (Sigma, D4911), and 0.1 μg / mL DAPI (Thermo Fisher, D1306)) from a 2.0 ml 96-well plate through a multichannel fluidic valve (IDEX Health and Science, EZ1213-820-4) to the custom-made flow cell using a syringe pump (Hamilton Company, 63133-01).Other buffers, such as 2 x SSC, 10% wash buffer (2 x SSC, 10% formamide (Thermo Fisher, AM9342), 0.1% Triton X-100 in nuclease-free water), 55% wash buffer (2 x SSC, 55% formamide, 0.1% Triton X-100 in nuclease-free water), and anti-bleaching buffer (3 mM Trolox (Sigma, 238813), 1% w / v D-glucose (Sigma, G7528), 1:100 diluted catalase (Sigma, C3155), 1.0 mg / ml glucose oxidase (Sigma, G2133), and 50 mM pH 8.0 Tris-HCl) were also pumped into the custom-made flow cell through a multichannel fluidic valve using a syringe pump. Integration of the imaging and automated fluid delivery system was controlled by μManager and custom-written scripts in Python using a Jupyter notebook.

[0211] Sequential smFISH - Imaging Sequential hybridization and imaging routines were performed as previously described, with some modifications. The samples with the custom-made flow cell were first connected to an automated fluidics system on a motorized stage on the microscope. Regions of interest (8 x 8 tile scans with 25% overlap for ETiX embryoid bodies and 8 x 9 tile scans with 20% overlap for control embryos) were identified and used for sequential rounds of hybridization and imaging as follows: Hybridization buffer containing readout and DAPI was poured over the samples and incubated for 60 min, followed by washing with 10% wash buffer for 1 min and three washes with 2 x SSC, after which anti-bleaching buffer was applied for image acquisition. Z-stacks with sections of 0.65 μm optical thickness were acquired for each tile, using exposure times of 500-2,000 ms for all lasers used. After image acquisition, the readout probes were washed from the samples three times with 55% wash buffer (with a 5 min incubation period between each wash) and once with 2 x SSC. This process was repeated for all rounds of genetic markers. For the final round of smFISH imaging, the readout probes were stripped from the samples and incubated in 0.1 μg / ml DAPI in 2 x SSC for 30 min before imaging in all channels.

[0212] The hybridization and stripping routines for each readout probe took approximately 1.5 h. The imaging time per tile took approximately 8 min each. As a result, the entire tile scan took approximately 8.5 h. Thus, it took approximately 5 days to complete the 11 rounds of hybridization and imaging routines for each experiment.

[0213] Sequential smFISH - Image analysis. To increase the signal-to-noise ratio in the ETiX embryoid body images, the last round of imaging (DAPI only, no readout probe) was used to subtract the sample background signal. This was not done for the control embryo FISH images because the background in that experiment was minimal due to the addition of a hydrogel clearing step. Therefore, for the ETiX embryoid body images, each tile of the background round was registered to the corresponding tile of each smFISH round using the phase_cross_correlation function (without normalization) in the skimage package written in Python, using the DAPI channel as a reference to generate a shift vector. The shift vector was then applied to the remaining channels of the background round (488 nm, 561 nm, and 647 nm), and the registered sample background was subtracted from the corresponding channel for each tile of each smFISH ETiX embryoid body round.

[0214] For both ETiX embryoid body and control embryo FISH images, background signal was further reduced for each tile using the ImageJ rolling ball background subtraction algorithm with a radius of 50 pixels. Each DAPI tile was then max-projected, and the tiles for each round were then stitched together using the ImageJ Grid / Collection stitching algorithm with a regression threshold of 0.01, no overlap calculation, and other standard settings. To stitch the other channels as well as the DAPI channel, the remaining channels were then max-projected. The tiles for each round were stitched together using settings taken from the tile configuration file generated from stitching the DAPI tiles.

[0215] An Ilastik classifier was then trained and used to classify the signals of each channel (405 nm, 488 nm, 561 nm, and 647 nm) of each FISH round into foreground and background. A DAPI foreground mask was extended using a disk structuring element with a radius of 10 pixels and used to mask out any spurious foreground signals from the 488 nm, 561 nm, and 647 nm channels that were far outside the nucleus-positive imaging area. The foregrounds of the 488 nm, 561 nm, and 647 nm channels were distance transformed and used to find local minima, which were then used to perform watershed to obtain the final label image (in which each label represents one unique detected transcript). Next, to visualize gene expression across different imaging rounds, the stitched DAPI images were registered across different rounds to obtain a shift vector for each round. This shift vector was then applied to the label positions corresponding to each round. For visualisation in the figures, the centre of each label was plotted as a disk with a 7-pixel radius.

[0216] Gene ontology analysis Gene Ontology (GO) of the extraembryonic endoderm was performed using the online platform DAVID. Differentially expressed genes in pairwise comparisons were selected by choosing genes with adjusted p-values ​​<0.05 and enriched in one or the other sample of the pairwise comparison. The list was then uploaded in the DAVID user interface and analyzed using the Gene Functional Annotation Clustering tool and Gene Functional Annotation Table. The first 20 clusters with the highest enrichment scores (-log P-values) were graphed. GO analysis was performed to explore the functional roles of differentially expressed mRNAs between natural embryos and ETiX embryoid bodies, and between wild-type and Pax6 knockout ETiX embryoid bodies, in terms of “biological process”, using the GO Enrichment Analysis online tool (GO Ontology Database). Differentially expressed genes for pairwise comparisons were selected as above. GO terms were entered into Excel for graph generation.

[0217] statistical analysis Data were tested for normality using the Shapiro-Wilk test. Normally distributed data were analyzed with unpaired t-tests as indicated in the figure legends. Non-normally distributed data were analyzed with the Mann-Whitney U test. All tests were performed with Prism GraphPad software v9.2. The sample numbers for the statistical tests are indicated in the Brief Description of Figures section above. All tests were performed as two-tailed tests. For each test, different samples were used, except for Figure 4F (where multiple somites per sample were measured to determine area) and Figure 13I (where multiple sections of the heart sample were measured for area measurement). All replicates were biological replicates, not technical replicates. Sample sizes were not pre-determined. Sample allocation was random. Investigators were not blinded to the type of sample they were working with.

[0218] Example 2 Development of embryoid bodies through neural tube formation This example reports that ETiX8 embryoid bodies cultured ex utero can develop through neural tube formation with high efficiency. Morphological, gene expression, and cell type analyses of ETiX8 embryoid bodies were similar to those of E8.5 spontaneous embryos, which are known to develop through neural tube formation.

[0219] To investigate anterior brain development in ETiX embryoid bodies, ES, TS, and iXEN cells were seeded and allowed to self-organize. On day 4, ETiX embryoid bodies with correct post-implantation morphology were transferred to suspension culture (Figure 1A). Typically, 2-4 wells of AggreWell plates were set up to obtain 100-150 embryo-like structures on day 4, in which a VE-like layer surrounded hollowed epithelial ES and TS cell compartments. These ordered structures constituted 10-15% of all structures recovered from any given well. This variability reflects random collisions between the three types of stem cells in the microwells and variability in the expression of different cadherins among these cell types. On day 5, ETiX embryoid bodies that possessed a primitive amniotic cavity (resulting from the merging of the ES and TS cell compartments), had a fully migrated AVE (at the border of the ES and TS cell compartments), and were undergoing gastrulation (as evidenced by an epithelial-to-mesenchymal transition and the formation of a cell layer between the ES cell and VE-like layers) were further cultured ex utero under conditions capable of supporting embryonic development beyond embryonic day (E) 7.5, including supplementing the medium with glucose on day 7 and transferring the gastrulating embryoid bodies to roller culture bottles for an additional day (from day 7 to day 8) (FIG. 1A and Methods).

[0220] Gastrulating ETiX embryoid bodies were very similar to natural gastrulating embryos (Fig. 1B-C), although ETiX embryoid bodies showed greater size variation (Fig. 7A). The efficiency of ETiX embryoid body development from day 4 to day 5 was 21% on average. For constructs selected for further culture at day 5, the transition efficiency from day 5 to day 6, day 6 to day 7, and day 7 to day 8 exceeded 70% at each transition (Fig. 7B). Notably, at day 7, neurulation-prone ETiX embryoid bodies cultured under static conditions showed an anterior-posterior axis that branched neural folds extending into the neural tube and terminating in the tail bud, a morphology similar to the early headfold stage of natural embryos at E8.0. Posterior to this, the tail bud joined with the allantoic tissue, which connected to the developing chorion (Fig. 1C). The embryoid bodies, allantois, and chorion were contained within a fluid-filled sac equivalent to the yolk sac (Figure 7C). These conditions therefore allowed ETiX embryoid bodies to develop through gastrulation and beyond, up to neural tube formation.

[0221] To monitor development by examining gene expression changes at single-cell resolution, ETiX embryoid bodies were isolated on days 5, 6, and 8, and natural embryos were dissected on E6.5, E7.5, and E8.5 (n = 29 for ETiX5, 10 for ETiX6, 7 for ETiX8, 12 for E6.5, 14 for E7.5, and 9 for E8.5). ETiX embryoid bodies and natural embryos were dissected into single-cells and sequenced by single-cell RNA sequencing (scRNA-seq) using the inDrops method (see Example 1). UMAP analysis revealed similar cell contributions to developmental lineages in natural embryos and ETiX embryoid bodies (Figure 1D). To determine cell types, the cell populations were subclustered using Seurat and then annotated based on published datasets (Figure 1E). Based on gene expression patterns, 26 cell types were identified, all of which were clearly represented in both the natural embryos and ETiX embryoid bodies data sets. Clustering of the natural embryos and ETiX embryoid bodies individually showed similar local cluster topographies in UMAP (Figure 7D-E). Only one cluster in the natural embryos was not represented in the ETiX embryoid bodies. This missing cluster corresponded to the junctional zone of the placental cluster in the natural embryos. During development, this cell population gave rise to trophoblast giant cells and spongiotrophoblasts. Several other cell types (notably PGCs and neural crest cells) could not be detected by scRNA-seq in either the natural embryos or the ETiX embryoid bodies, but were observed by immunofluorescence.

[0222] Natural embryos and ETiX embryoid bodies showed highly conserved distribution of cells among the various germ layers of the epiblast (ectoderm, mesoderm, and endoderm) and among the embryonic and extraembryonic lineages (epiclastoderm, ExE, extraembryonic mesoderm, and extraembryonic endoderm) (Figure 7F). As expected, in natural embryos, the cell type complexity increased over time, corresponding to the formation of differentiated tissues and organs. For example, cardiomyocytes and neuroectoderm began to appear from E7.5. The increase in cell type complexity and spatiotemporal maturation of all identified populations were similar between natural embryos and ETiX embryoid bodies, indicating that the embryoid bodies during neural tube formation followed a similar developmental timeline (Figure 7G and Figure 1H). For example, both systems showed the development of the three germ layers and their derivatives (neurectoderm, surface ectoderm, and gut precursors), the initiation of organogenesis (cardiomyocytes), and the formation of extraembryonic tissues such as the amnion and allantoic membrane (Figure 1E and Figure 1H). Pearson correlation matrices showed high similarity of gene expression between cell-type clusters of natural embryos and those of ETiX embryoid bodies (Figure 7H and Figure 7J). Comparing cell-type ratios at different time points between natural embryos and ETiX embryoid bodies, they were broadly similar within individual clusters, although some variation was observed (Figure 7I). Thus, neuralizing embryoid bodies recapitulated the generation of multiple tissues of neuralizing embryos, as evidenced by both their morphology and their cell type-specific patterns of gene expression.

[0223] To further evaluate the reproducibility of embryoid body formation during neural tube formation, we performed additional rounds of single-cell sequencing, in which individual ETiX embryoid bodies with apparently correct morphology, as well as individual natural embryos cultured in vitro from E6.5, taken at different times during development, were individually barcoded and analyzed by tiny-sci-RNA-seq (hereafter referred to as 'tiny-sci'; general methods in Example 1 above) (a combinatorial indexing-based method for single-nucleus RNA-sequencing profiling from small amounts of starting material). Furthermore, to understand why some ETiX embryoid bodies did not develop properly, we also included individual examples of morphologically abnormal embryoid body development at days 6 and 8 (examples of 'badly formed' embryoid bodies, well-formed ETiX embryoid bodies, and natural embryos cultured in vitro (Figure 8A-C)).

[0224] After data processing and quality control, this new dataset contained profiles of 285,640 cells with no discernible batch effects between sequencing rounds (Fig. 9A-D). Annotation of this dataset yielded 19 clusters present in both natural embryos and ETiX embryoid bodies (Fig. 1F). Notably, in contrast to the previous dataset, the presence of neural crest cell populations was clearly detected, whereas PGCs remained undetected. In this dataset, clustering of natural embryos and ETiX embryoid bodies individually also showed similar local cluster topographies in UMAP (Fig. 9E-F). Individual ETiX embryoid bodies had very similar cell-type composition per sample (Fig. 1G). From the UMAPs at different time points, it was clear that the ETiX embryoid bodies at day 6 and day 8 were most similar to natural embryos at E7.5 and E8.5 or E8.75, respectively (Figure 9G), and this finding was also observed in the individual replicates (Figure 9H). The similarity between natural embryos and ETiX embryoid bodies was also confirmed by principal component analysis, where samples were aligned by developmental age along PC1, and ETiX embryos at day 6 were most similar to E7.5 natural embryos, and ETiX embryos at day 8 were most similar to E8.5-E8.75 natural embryos (Figure 9I). Notably, E9.5 natural embryos were intermingled with these samples, suggesting that the natural embryos did not substantially progress in development outside the uterus beyond E8.75. Day 6 ETiX embryoid bodies and E7.5 spontaneous embryos separated along PC2, whereas day 8 ETiX embryoid bodies and E8.5-9.5 spontaneous embryos did not (Figure 9I). To assess how similar each particular cluster was overall to all other clusters within the dataset, the dataset was analyzed with a non-negative least-squares (NNLS) regression matrix (Figure 9J) (Example 1 General Methods).This revealed that every cluster in the natural embryo dataset showed the highest similarity to its ETiX embryoid body counterpart (e.g., the natural heart field cluster was most similar to the ETiX embryoid body heart field cluster; Figure 9J). Furthermore, this tiny-sci dataset integrated very well with the public dataset, confirming that the same populations reported previously were captured upon sampling of natural embryos and ETiX embryoid bodies (Figures 10A-C). The differences between good ETiX embryoid bodies and poorly formed structures were not readily apparent, as even poorly formed embryoid bodies with morphological abnormalities appeared to have a very similar overall cellular composition to well formed embryoid bodies and natural embryos (Figure 10D), possibly reflecting some limitations of the RNA-seq analysis and / or failure of morphogenetic events despite continued appropriate gene expression. However, the poorly formed structures tended to contain smaller proportions of paraxial mesoderm, neuroectoderm, and surface ectoderm at the expense of a larger proportion of ExE cells (Figure 10E).

[0225] Next, GO analysis was performed to identify global transcriptional differences between natural embryos and ETiX embryoid bodies. Natural embryos showed enrichment of terms related to uterine development, implantation, and re-regulation of the endothelial compartment, whereas ETiX embryoid bodies showed terms related to embryo morphogenesis (Tables 2-5). GO analysis did not reveal any obvious stress signatures or metabolic differences between natural embryos and ETiX embryoid bodies.

[0226] Table 4 lists the differentially expressed genes that were enriched in natural embryos cultured ex utero from E6.5 to E7.5 by GO analysis and analyzed by tiny-sci. [Table 4]

[0227] Table 5 lists the differentially expressed genes that were enriched in ETiX-embryoid bodies analyzed with tiny-sci at day 6 of development by GO analysis. [Table 5]

[0228] Table 6 lists the differentially expressed genes that were enriched in natural embryos cultured ex utero from E6.5 to E8.5 by GO analysis and analyzed by tiny-sci. [Table 6]

[0229] Table 7 lists the differentially expressed genes that were enriched in ETiX-embryoid bodies analyzed by tiny-sci at day 8 of development by GO analysis. [Table 7]

[0230] Example 3 Embryoid body development in the forebrain and midbrain regions This example reports on the investigation of neurogenesis in the synthetic embryoid bodies described in this application.

[0231] During natural development, the anterior epiblast retains its epithelial characteristics, upregulates the neuroectodermal marker Sox1 from E8.0 onwards, and initiates the formation of the nervous system. The neuroectodermal lineage gives rise to the forebrain, midbrain, hindbrain and spinal cord.

[0232] To investigate neurogenesis in embryoid bodies, the expression of well-established neuroectoderm markers was analyzed by immunofluorescence. SOX1 and SOX2 were expressed in the neuroepithelial cell population along the entire anterior-posterior axis of the day 7 neural tube forming embryoid bodies in a pattern similar to that of natural E8.0 embryos (Fig. 2A-B and Fig. 11A-B). SOX1-positive neural tube tissue occupied two-thirds of the length of the day 7 neural tube forming embryoid bodies and terminated in two neural folds (Fig. 11A). Meanwhile, the SOX1-negative, Brachyury-positive posterior showed a tailbud-like morphology similar to that of natural E8.0 embryos (Fig. 2A-B). A Brachyury-positive notochord ran underneath the neural tube and was easily recognizable in both the neural tube forming natural embryos and the ETiX embryoid bodies (Fig. 2A-B).

[0233] We next examined scRNA-seq data for the expression of key markers of the neuroectoderm, the surface ectoderm that gives rise to most epithelial tissues, the notochord, which is important in patterning the neuroectoderm and neural tube, and the epiblast, the precursor of all these tissues. We observed similar expression of Foxa2, Chordin, and Shh, which mark the notochord, in ETiX embryoid bodies and natural embryos (Figure 2C). Furthermore, natural embryos and ETiX embryoid bodies expressed similar levels of Sox1, Sox2, Pax6, and Pax3 in the neuroectoderm and showed a similar surface ectoderm signature for keratin gene expression. Thus, the tissue-specific gene expression patterns of embryoid bodies during neural tube formation strongly resembled those of natural embryos.

[0234] The transcription factor OTX2, which contributes to midbrain and forebrain patterning, showed localized expression in the most anterior third of the headfold in ETiX embryoid bodies at day 8 of neural tube formation (Fig. 2D-E). This region corresponded to the forebrain and midbrain in E8.5 natural mouse embryos. EMBs at day 8 of neural tube formation also expressed the transcription factor FOXG1, which plays an important role in brain development, in the same region as natural E8.5 embryos (Fig. 2D-E and Fig. 11C). The brain regions demarcated by OTX2 expression were similar in natural embryos and ETiX embryoid bodies (Fig. 11D). The neural tube in EMBs at day 8 of neural tube formation was closed and showed clear neural precursor regions within the neural tube, demarcated by the expression of markers PAX6, OLIG2, and NKX2-2 (Fig. 2F). FOXA2 was expressed in cells lining the ventral midline of the neural tube, marking a population of floor plate cells (Figure 2F). PAX3, on the other hand, was expressed in the dorsal neural tube, somatic mesoderm, and neural crest cells (Figure 2F). SOX10 expression confirmed the identity of neural crest cells migrating from the neural tube, likely governing their detachment and migration during natural brain development (Figure 2F). We also determined whether neuroectoderm and surface ectoderm formed in a similar manner in embryoid bodies during neural tube formation compared to natural embryos. To this end, we calculated transcriptional trajectories using RNA velocity (RNA velocity integrates the ratio of spliced ​​to unspliced ​​RNA over time to infer how the starting population evolved and differentiated). Comparison of RNA velocities between natural embryos and embryoid bodies undergoing neural tube formation showed similar differentiation trajectories from the epiblast to neuroectoderm and surface ectoderm, suggesting that specification of these two tissues follows similar developmental transcriptional programs in these two systems (Figure S11E).To determine whether these tissues were formed at comparable times, latency analysis was performed to assign random combinations of pseudotimes to measure when specific tissues or subpopulations appeared. Latency analysis indicated that the neuroectoderm appears to be specified later in neural tube-forming embryoid bodies than in natural embryos. This was also observed for the surface ectoderm, although the difference was significantly smaller (Figure 11F-G). Taken together, these findings suggested that the neuroectoderm and surface ectoderm were specified with similar transcriptional trajectories, but with somewhat different timing, in neural tube-forming ETiX embryoid bodies and natural embryos.

[0235] The tiny-sci dataset was further used to explore the different neural cell types present in ETiX embryoid bodies during neural tube formation. Using well-established markers, these subclusters were assigned to specific neural identities (Figure 12A-E). Subclustering and annotation of all neuroectoderm-derived cell types showed the presence of cells expressing markers of the hindbrain and spinal cord, prosencephalon, mesencephalon, and midbrain-hindbrain boundary. Cells expressing genes indicative of tectal plate and floor plate identity were also observed (Figure 2G-I). However, without spatial organization data, it cannot be concluded that there was dorsal-ventral patterning in the brain. The presence of neural populations similar to those observed in E8.5 embryos was observed in ETiX embryoid bodies during neural tube formation (Figure 2G). These neural types were almost completely absent at E7.5, consistent with neural induction occurring primarily in an explosive manner at E8.0. Thus, no neurons were observed in ETiX embryoid bodies at day 6, very similar to E7.5 embryos, whereas in ETiX embryoid bodies at day 8, all of these neural subtypes were present, in good agreement with natural embryos at E8.5 and E8.75 (Figure 2H). Finally, examination of individual ETiX embryoid bodies at day 8 showed that the presence of these neural subtypes was largely recapitulated in each structure (Figure 2I). However, populations of early neurons appeared in three of the five samples examined, suggesting that specimens with neurons may have been at a somewhat more advanced stage with respect to the initiation of neurogenesis. Neural crest cells expressed the known marker Ets1 in addition to Sox10 (Figure 12F). To confirm that the formation of these neural subtypes was accompanied by regionalized patterns of gene expression, sequential smFISH was performed on sectioned native embryos and ETiX embryoid bodies (Figures S12G-I).In both, expression of Fezf1, Lhx2, and Six3 in the forebrain, and regional expression of En1 and Dmbx1 in the midbrain was observed, consistent with published results.

[0236] To test whether the neural tube of embryoid bodies during neural tube formation responds to developmental insults in the same way as the neural tube of natural embryos, we generated embryoid bodies from a transgenic ES cell line that does not express PAX6. PAX6 is a transcription factor required for neural tube patterning, as well as brain and eye development. Pax6-knockout ETiX embryoid bodies had similar cell-type ratios as control structures (Figure 12J). Consistent with the development of natural Pax6-knockout embryos, Pax6-knockout embryoid bodies showed no change in the total number of SOX1-positive cells in the neuroectoderm, but an increased proportion of NKX2-2-positive cells, suggesting an expansion of the ventral domain of the neural tube (Figures 3A-B). To determine other developmental consequences of Pax6 deletion, we examined global changes at the transcriptional level in the absence of Pax6. Consistent with previous results, there was an enrichment of transcripts associated with neurogenesis, and axon development and formation (FIG. 3C).

[0237] Nervous system-related defects are the second most frequent developmental abnormality in humans, including brain defects such as anencephaly, and spinal defects such as spina bifida. In one particular batch of serum, 75-80% of developing ETiX embryos showed abnormal twisting and kinking of the neural tube (Figure 2J). Similar neural tube defects in both mice and humans are largely rescued by supplementing the maternal diet with folic acid. To test whether folic acid treatment rescued neural tube defects in developing ETiX embryos, the ETiX embryos were cultured in the presence of 5-methyl-THF (50 ng / ml), a metabolically active form of folic acid. 5-methyl-THF substantially rescued neural tube defects in ETiX embryos (Figure 2J), indicating that ETiX embryos can serve as relevant disease models.

[0238] Taken together, the results in this example demonstrate that embryoid bodies during neural tube formation undergo headfold morphogenesis in a manner similar to that observed in natural embryos and may serve as relevant disease models.

[0239] Example 4 Somitogenesis and heart development in embryoid bodies This example reports that neural tube-forming embryoid bodies can undergo somitogenesis to the stage where a beating heart develops. Morphology similar to that of natural embryos and expression of markers associated with somite and heart development were observed.

[0240] During natural embryogenesis, neuro-mesodermal progenitors (NMPs) contribute to the derivation of the neural tube and paraxial mesoderm. To determine whether neural tube-forming ETiX embryoids form NMPs, we performed immunofluorescence to detect expression of the NMP markers SOX2 and Brachyury in a domain spanning the posterior region of the tail bud in day 8 neural tube-forming ETiX embryoids (Fig. 4A). In contrast, more anterior regions of day 8 ETiX embryoids express either SOX2 but not Brachyury - marking the neural lineage - or Brachyury but not SOX2 - marking the mesoderm lineage (Fig. 4A and Fig. S13A). This pattern of marker expression was similar to that of E8.5 natural embryos (Figure 4B) and is consistent with the reported differentiation trajectory of these cells in the embryos. Co-localization of Brachyury and SOX2 in tailbud cells was similar between ETiX embryoid bodies and natural embryos (Figure 13B). These findings were consistent with the reported differentiation trajectory of these cells in natural embryos.

[0241] The paraxial mesoderm then gave rise to somites, paired cell masses formed along the anterior-posterior axis of the embryo, which were required for the segmentation of skeletal muscle, blood vessels, and skin. Somites pairs expressing the homeobox protein HOXB4 were observed on either side of the SOX1- and SOX2-positive neural tube mass in both day 7 ETiX embryoid bodies and natural E8.0 embryos (Fig. 4C-D and Fig. 13C-E). Quantification of the number of somite pairs and the area of ​​somites in ETiX embryoid bodies and natural embryos showed no significant differences (Fig. 4E-F). These expression patterns indicate key features of somitogenesis, thus recapitulating the corresponding natural process.

[0242] A unique set of cells fated to form the heart also emerged from the primitive streak during gastrulation. In natural embryos, this developmental event occurred around E8.0. Heartbeat was established when cardiac mesoderm differentiated into cardiomyocytes. The formation of a beating structure was observed at the bottom of the brain region in day 8 neural tube forming embryoid bodies. The beating region of this neural tube forming embryoid body expressed myosin heavy chain II (MYH2) and the transcription factor GATA4, which are required for cardiac development (Figures 4G-H and 13F-G), with a spatiotemporal profile similar to that of natural embryos. Immunostaining of the indicated sections in day 8 neural tube forming embryoid bodies showed triple-positive compartments for NKX2-5, GATA4, and MYH2 (Figures 4I-J). The MHY2-positive region also expressed the transcription factor GATA6 (Figure S13H). Comparison with the hearts of age-matched E8.5 natural embryos showed that the adjacent cavities observed in the MYH2-positive region of ETiX embryoid bodies were very similar to those of natural embryos. However, no obvious cardiac looping was observed. Furthermore, the area of ​​the cardiac domain was reduced in ETiX embryoid bodies compared to natural embryos (Figure S13I).

[0243] The scRNA-seq data from mesoderm and its derivatives confirmed and extended the findings from immunofluorescence (Figure 4K). In neuralizing embryoid bodies, gene expression signatures leading to somitogenesis were clearly visible, but the transcript levels of presomitic identification genes (Tbx6, Hes7, and Msng1), Notch pathway genes (Notch1 and Lfng), and a somite marker (Meox1) were lower than in natural embryos. Both natural embryos and neuralizing embryoid bodies expressed Gata4, as well as other key regulators of heart development (e.g., Gata6, Meis1, Tbx5, and Hand1, etc.) (Figure 4K). Similarly, neural tube-forming embryoid bodies expressed cardiomyocyte markers such as troponin genes (Ttn and Tnnt2) and myosin genes (Myh7, Myh6, Myl3, and Myl7), suggesting that the development of mesoderm in neural tube-forming embryoid bodies was remarkably similar to that in natural embryos. To further confirm this, RNA kinetic analysis was performed on the epiblast and all its mesoderm derivatives in both neural tube-forming embryoid bodies and natural embryos. The differentiation trajectories between the two were highly similar (Figure 4L). Similarly, latency analysis showed that these mesoderm derivatives appeared in neural tube-forming embryoid bodies in a temporal order that was broadly consistent with natural embryos (Figure 14A). Of the tissues examined, only the caudal lateral epiblast and NMP appeared to appear slightly later in the embryoid bodies during neural tube formation, whereas notochord formation appeared to occur slightly earlier than in natural embryos (Figure 14B).Paraxial mesoderm-derived cell clusters in the tiny-sci dataset were examined.Cultured natural embryos expressed somite markers Meox1, Meox2, and Pax3, whereas ETiX embryoid bodies expressed Pax3 but instead showed relatively weak expression of Meox1 or no expression of Meox2 (Figure 14C-D), which may suggest a difference in somite formation between natural embryos and ETiX embryoid bodies.

[0244] Using the tiny-sci dataset, cardiac tissue was further analyzed to determine whether additional cell populations could be identified. Subclustering (FIG. 4M) of the cardiac lineage, which expresses Hand1 and Hand2 (FIG. 14E), allowed the identification of a first cardiac field, characterized by robust expression of canonical markers Tbx5, Nkx2-5, and Hcn4 (FIG. 14E), and a second cardiac field, characterized by regional expression of Isl1 (FIG. 14E). The atrial marker Nr2f2 and the ventricular differentiation marker Irx4 were detected in both cultured natural embryos and ETiX embryoid bodies (FIG. 14E). Consistent with latency analysis of pooled data provided by inDrops sequencing, the tiny-sci dataset also confirmed that cardiac cell types appeared in neural tube-forming embryoid bodies in a conserved temporal manner that reflects the developmental order of natural embryos. Indeed, no substantial cardiac lineage was observed in either day 6 ETiX embryoid bodies or E7.5 natural embryos. Instead, the cardiac lineage of natural embryos appeared mainly from E8.5 onwards. In this respect, the development of day 8 ETiX embryoid bodies captured the cellular contributions of E8.5, E8.75 and E9.5 natural embryos (Figure 14F).

[0245] The results in this example demonstrate that the ETiX embryoid bodies described in this application can proceed through neurulation with the formation of a neural tube, the initiation of somitogenesis, and the generation of mesodermal structures (e.g., heart-like structures, etc.).

[0246] Example 5 Initiation of intestinal development in synthetic embryoid bodies This example reports that the synthetic embryoid bodies described in this application are capable of giving rise to definitive endoderm that gives rise to the gut and related organs.

[0247] After observing the extensive development and morphogenesis of the ectoderm and mesoderm, we also determined the extent to which neural tube-forming embryoid bodies exhibited the development of definitive endoderm, which gives rise to the gut and associated organs. Sox17 is required for gut endoderm development. Whole mount immunofluorescence of ETiX day 8 embryoid bodies revealed a Sox17-positive region beneath the heart, suggesting a primitive gut population (Fig. 5G-H). In contrast, the heart was Sox17-negative and Gata4-positive, quite similar to natural embryos (Fig. 5G-H). Sectioning of ETiX day 7 embryoid bodies revealed an open gut pocket beneath the neural tube that expressed the transcription factors Sox2 and FoxA2, ​​both essential for gut development (Fig. 5I). Sagittal sections of E8.5 natural embryos and day 8 ETiX embryoid bodies revealed the presence of foregut and hindgut pockets (Fig. 5A-B). In addition to being expressed in the brain and neural tube, the transcription factor SOX2 was also expressed in the foregut of natural embryos, a pattern of expression conserved in neural tube-forming embryoid bodies (Fig. 5A-B). Similarly, SOX17 was expressed in the hindgut of both natural and neural tube-forming embryoid bodies (Fig. 5A-B), as well as in scattered cell populations suggestive of endothelial precursors. However, within the time frame of our observations, the intestine of ETiX embryoid bodies was not as fully developed as that of natural embryos. GATA4 was expressed prominently in the hearts of both natural embryos and ETiX embryoid bodies, whereas it was expressed in the hindgut of natural embryos but not in the hindgut of ETiX embryoid bodies (Fig. 5A-B). In contrast, GATA6 was expressed in both the hearts and hindgut of natural embryos and ETiX embryoid bodies (Fig. 15A-B). Further characterization showed that the foreguts of both ETiX embryoid bodies and natural embryos expressed FOXG1 and OTX2 (Fig. 15C-F), and the hindguts of both expressed CDX2 (Fig. 15C-F).However, in contrast to natural embryos, no expression of the transcription factor FOXA2 was observed in the intestine of ETiX embryoid bodies (Figure 15F). Expression of NKX2-5 was not detected in natural embryos or in ETiX embryoid bodies, even though its mRNA was shown to be expressed in the intestine (Figure 15C-D).

[0248] To further characterize the gut and associated endoderm tissues, the scRNA-seq dataset was further analyzed. InDrops scRNA-seq data from natural embryos and ETiX embryoid bodies identified definitive endoderm (Cer1 + Sox17 + Gata6 + ) and VE intestinal precursors (Rhox5 + Cldn6 + Apoa1 +), revealing a gene expression signature corresponding to the epiblast-to-gut differentiation trajectory (Fig. 5C). However, RNA kinetics analysis indicated possible differences in the epiblast-to-gut differentiation trajectory between natural embryos and ETiX embryoid bodies (Fig. 16A). In contrast, latency analysis indicated that the timing of emergence of these lineages was very similar in embryos and embryoid bodies (Fig. 16B-C). Taken together, these data indicate that gut formation in ETiX embryoid bodies likely proceeds with a similar timing to gut formation in natural embryos, but with some differences in the developmental trajectory. Analysis of gut and endoderm clusters in the tiny-sci dataset indicated that the gut cluster has both embryonic and extraembryonic contributions (Fig. 5D). In the embryonic part of the gut cluster, higher expression of genes related to definitive endoderm origin was observed (Fig. 5D and Fig. 16D-J), while the putative VE-derived part of the gut cluster (Fig. 5D) expressed higher levels of Ttr, which is expressed by enterocytes of extraembryonic origin (Fig. 16K). No cell populations corresponding to liver, pancreas, small intestine or large intestine precursors were observed in either ETiX embryoid bodies or natural embryos at this stage, suggesting that neither had progressed beyond an uncommitted endoderm state under the applied culture conditions, consistent with the initiation of organ-specific identity in the gut at E8.75. The VE of ETiX embryoid bodies at day 6 was very similar to that of natural embryos at E7.5. However, cells in day 8 ETiX embryoid bodies appeared to be largely absent from the VE of E8.5 to E9.5 embryos ( Fig. 16L ).

[0249] Despite observations showing the establishment of endodermal precursors of the gut, the further development of the endodermal precursors of the gut and its associated organs has not been studied. To achieve this, it may be necessary to optimize culture conditions. Given the appropriate culture conditions, the development of ETiX embryoid bodies is not expected to proceed further in culture.

[0250] Example 6 Development of PGCs in synthetic embryoid bodies This example reports that PGCs can appear in the proximal-posterior region of the epiblast in E6.5 mouse embryos, approximately contemporaneous with the onset of Brachyury expression.

[0251] Committed PGCs are characterized by the expression of STELLA, which was detected at the ES cell-TS cell border in ETiX embryoid bodies at day 6, similar to natural embryos at E7.5 (Figure 5E). PGCs were also detectable at later time points in the development of ETiX embryoid bodies (Figures 17A-17D). During the developmental process, PGCs reactivated the pluripotency markers SOX2 and NANOG, as observed to occur in ETiX embryoid bodies (Figures 5E and 17A-17D). PGCs were found in close proximity to the allantois in ETiX embryoid bodies at days 7 and 8, similar to natural embryos at E8.0 (Figures 17A-17D). Quantification of the number of PGCs in natural embryos at E7.5 and E8.5, and in ETiX embryoid bodies at days 6 and 8, showed that STELLA + or STELLA + NANOG + SOX2 + It was shown that there was no significant difference between the total numbers of cells (Figure 5F). Quantification of the number of PGCs also showed that there were 30-100 PGCs in day 8 ETiX embryos, which is similar to the reported number of PGCs in natural E8.5 embryos (Figure 19A). Thus, the inability to detect PGCs in the single-cell dataset likely reflects their very low numbers.

[0252] Example 7 Development of yolk sac and blood islands in synthetic embryoid bodies This example reports the establishment of yolk sac and blood islands around the ETiX embryoid bodies described in this application. The development of yolk sac and blood islands was also confirmed by the expression of amniotic and amniotic mesoderm markers.

[0253] ETiX embryoid bodies developed inside a membrane similar to the amnion and yolk sac, which provide nutrition to the embryo until the fetal-maternal circulation is established. Both cultured embryos and ETiX embryoid bodies had to be dissociated from their yolk sac for immunostaining (Fig. 18A-B). In both sequencing datasets, the amnion and amniotic mesoderm constituted clusters of cells expressing the amniotic marker Postn (Fig. 6A-B). Furthermore, in both cultured natural embryos and ETiX embryoid bodies, clusters of cells representative of allantoic tissue, marked by expression of Tbx4 and Hoxa13, were detected (Fig. 6C).

[0254] During development, the yolk sac originated from parietal endoderm and VE-derived cells, respectively. Both of these populations were present in the dataset disclosed in this application (Figure 6A). To further understand the diversity of cell types present in these two extraembryonic endoderm lineages, subclustering analysis was performed (Figure 6D and Figure 18C). The extraembryonic endoderm of ETiX embryoid bodies closely matched native embryos (left and rightmost UMAP), but also contained cells that were largely absent in native embryos (ETiX-only extraembryonic endoderm) (Figure 6D and Figure 18C). These cells began to appear in embryoid bodies at day 6, with the majority appearing by day 8 (Figure 18C). In the subclustered UMAPs, the parietal endoderm cluster expressed high levels of collagen (Col4a1 and Col4a2) and laminin (Lama1 and Lamb1) genes, as previously reported and as seen in earlier stage ETiX embryoid bodies (Fig. 18D-E). Consistently, this cluster also expressed the parietal yolk sac marker Pga5 (Fig. 18D-E). The earliest clusters in the dataset, 'Early VE' and 'Early VE 2', expressed genes enriched in E7.5 embryonic VE (e.g., Spink1) as well as genes associated with extraembryonic VE (ExVE) (Afp, Trf, Ttr, and Car4) (Fig. 18F-G). Taken together, this suggested that the ExVE genes were predominantly expressed in the left side of the subclustered UMAP, with the prevailing ExVE signature predominating (Figure S18C). GO analysis of the 'early VE' and 'differentiated yolk sac' clusters highlighted terms consistent with the role of the visceral yolk sac in nutrient transport, lysosomal function, and lipid and cholesterol metabolism (Tables 6 and 7). The 'immature yolk sac' clusters were predominantly present in ETiX embryoid bodies and lacked a clear expression pattern, suggesting that they may be an unusual population.The 'Early VE 2' cluster showed expression of the pro-haematopoietic factor Runx1, which is present in both natural embryos and ETiX embryoid bodies and is known to be restricted to the VE at the embryo-ExE boundary. Taken together, subclustering of extraembryonic endoderm cells suggests that ETiX embryoid bodies are capable of giving rise to parietal yolk sac cells and acquiring a mature visceral yolk sac identity. However, subclustering of extraembryonic endoderm cells also identified cells that were too immature to undergo the correct developmental program.

[0255] Table 8 lists genes that were enriched in the "early VE" cell population within the extraembryonic endoderm subcluster of ETiX-embryoid bodies by GO analysis. [Table 8]

[0256] Table 9 lists genes that were enriched in the "differentiated yolk sac" cell population of ETiX embryoid bodies by GO analysis. [Table 9]

[0257] The extraembryonic portion of the developing yolk sac in ETiX embryoid bodies was attached to structures resembling the chorion and allantois (Figure 6E). The yolk sac supported primitive hematopoiesis in the embryo. Notably, RUNX1-positive blood islands were observed within the mesoderm of the yolk sac and at the base of the allantois in day 8 neural tube forming embryoid bodies (Figure 6F). Consistent with the formation of blood islands, endothelium-related genes were expressed in the tiny-sci dataset (Figure 18H). Furthermore, the allantois in day 8 ETiX embryoid bodies was connected to chorion-like tissue (which expresses Gata4 and keratin 18) and distinct from the yolk sac in natural embryos (which expresses only Gata4) (Figure 19B).

[0258] Finally, we characterized the trophoblast compartment in natural embryos to determine whether ETiX embryoid bodies are also capable of generating the cell populations necessary to form a functional placenta. During development, the proximal portion of the ExE differentiates into ECPs (the precursors of trophoblast giant cells and spongiotrophoblasts). The portion of the ExE that retains stem cell properties differentiates into the chorion and ultimately forms the syncytiotrophoblast layer of the labyrinth, whose surface mediates gas and nutrient exchange between the fetus and the mother. Subclustered UMAPs of ExE (Figure 6G) were annotated using known markers (Figures S18I-O) and showed the presence of trophoblast precursors, committed and uncommitted ECPs, trophoblast giant cells, spongiotrophoblast cells, chorionic precursors, chorion, and first and second layer syncytiotrophoblasts.

[0259] In ETiX embryoid bodies, the continued presence of trophoblast precursors dividing into ECP and chorion lineages was observed, as in natural embryos. However, the chorion lineage in ETiX embryoid bodies did not completely cluster with that of natural embryos (Fig. 6G-H, see Fig. 18I-O for specific markers). Furthermore, the ECP lineage in ETiX embryoid bodies did not fully develop due to altered or absent expression of the ECP gene (ETiX-only ECP). Furthermore, the absence of prolactin gene expression indicated the absence of trophoblast giant cells and spongiotrophoblast cells (Fig. 6I-J). This analysis showed that while the development of the chorion lineage was extensive, the extraembryonic lineage derived from the ECP was largely absent in ETiX embryoid bodies.

[0260] The results in this example show that the embryonic lineage of ETiX embryoid bodies closely captures natural development, with variability observed in extraembryonic embryonic lineages that may reflect lack of contact with the maternal environment (natural embryos were collected when the ECP had already begun to develop).

[0261] usage In at least some of the above described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment (unless such substitution is technically impossible). Those skilled in the art will appreciate that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter, as defined by the appended claims.

[0262] With respect to the use of substantially any plural and / or singular terminology in this application, one of ordinary skill in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth in this application. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. In this application, references to "or" are intended to encompass "and / or" unless specifically stated otherwise.

[0263] Those skilled in the art will understand that, in general, the terms used in this application, and in particular in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, for example, but not limited to," the term "having" as "having at least," the term "includes" as "includes but is not limited to," etc.). Those skilled in the art will further understand that, where a specific number is intended in the introduced claim recitation, such intent will be expressly recited in the claim, and in the absence of such recitation, no such intent exists. For example, to aid in understanding, the appended claims below may use the preamble phrases "at least one" and "one or more" to recite the claims. However, the use of such phrases should not be interpreted as meaning that reciting a claim with the indefinite article "a" or "an" limits any particular claim containing such a claim recitation to embodiments containing only one such recitation, even if the same claim includes the preface 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 recite a claim. In addition, those skilled in the art will recognize that when a particular number is explicitly recited in an introduced claim recitation, such recitation should be interpreted to mean at least the recitation number (e.g., a minimum recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, when language similar to "at least one of A, B, and C, etc." is used, such configurations are generally intended in the sense that one of ordinary skill in the art would understand the language (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When language similar to "at least one of A, B, or C" is used, such configurations are generally intended in the sense that one of ordinary skill in the art would understand the language (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that nearly any disjunctive word and / or phrase presenting two or more alternative terms, whether in this specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms.

[0264] In addition, when features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also by such description described in terms of any individual members or subgroups of members of that Markush group.

[0265] As one of ordinary skill in the art would understand, for any and all purposes, including in terms of providing a written description, all ranges disclosed in this application also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be at least equally divided into halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed in this application can be readily divided into a lower third, middle third, upper third, etc. As one of ordinary skill in the art would also understand, all language such as "up to," "at least," "greater than," "less than," etc., refers to a range that is inclusive of the recited numbers and that can then be divided into sub-ranges as described above. Finally, as one of ordinary skill in the art would understand, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to groups having 1, 2, 3, 4, or 5 items, and so on.

[0266] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for producing a synthetic embryo in vitro, said method comprising: (a) co-culturing mammalian pluripotent stem cells and at least one extraembryonic stem cell in a first culture medium under first static conditions to allow the mammalian pluripotent stem cells and the extraembryonic stem cells to self-organize into post-implantation embryonic structures; (b) culturing the post-implantation embryonic structure in a second culture medium under second static conditions to allow the post-implantation embryonic structure to develop into a neural tube-forming embryonic structure; and (c) culturing the neural tube forming embryonic structures under dynamic conditions in a culture chamber for at least one day to allow the neural tube forming embryonic structures to develop into synthetic embryos at least at an early organogenesis stage.

2. 10. The method of claim 1, wherein the mammalian pluripotent stem cells comprise mammalian embryonic stem cells.

3. 10. The method of claim 1, wherein the at least one extraembryonic stem cell comprises a trophoblast stem cell, an inducible extraembryonic endodermal stem cell, or both.

4. The method of claim 3, wherein the induced extraembryonic endoderm stem cells are capable of expressing a GATA transcription factor upon induction.

5. The method of claim 4, wherein the GATA transcription factor is GATA4.

6. 6. The method of any one of claims 1 to 5, wherein the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the first culture medium for up to 4 days.

7. 6. The method of any one of claims 1 to 5, wherein step (a) is from embryonic day E0 to E5.

5.

8. 6. The method of any one of claims 1 to 5, wherein the post-implantation embryonic structure is a post-implantation pre-gastrulation embryonic structure.

9. 9. The method of claim 8, wherein the post-implantation pre-gastrulation embryonic structure resembles a natural embryonic structure at E5.

5.

10. 6. The method of any one of claims 1 to 5, wherein the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in a substrate, optionally wherein the substrate comprises a dish, a U-plate, a flask, or a microwell plate.

11. 11. The method of claim 10, wherein the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in inverted pyramidal microwells.

12. 12. The method of claim 11 , wherein 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. be.

13. 6. The method of any one of claims 1 to 5, wherein step (a) comprises culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in a feeder cell (FC) medium, and optionally passage the mammalian pluripotent stem cells and the extraembryonic stem cells in the feeder cell medium at least twice.

14. 14. The method of claim 13, wherein the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the FC medium for about 2 days.

15. 14. The method of claim 13, wherein step (a) comprises culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in in vitro culture (IVC) medium, optionally after culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the FC medium.

16. 16. The method of claim 15, wherein the mammalian pluripotent stem cells and the extraembryonic stem cells are cultured in the IVC medium for about 2 days.

17. 14. The method of claim 13, wherein the FC medium and the IVC medium comprise a basal culture medium.

18. 18. The method of claim 17, wherein the basal culture medium comprises Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), non-human serum or its serum substitute, a reducing agent, an antibiotic, L-glutamine or an analog thereof, or any combination thereof.

19. 20. The method of claim 18, wherein the non-human serum or serum substitute thereof comprises fetal bovine serum, bovine serum albumin, KnockOut™ serum substitute, or any combination thereof.

20. 20. The method of claim 18, wherein the reducing agent comprises beta-mercaptoethanol (BME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof.

21. 19. The method of claim 18, wherein the antibiotic comprises penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof.

22. 14. The method of claim 13, wherein the FC medium comprises sodium pyruvate.

23. 14. The method of claim 13, wherein the FC medium comprises a non-essential amino acid.

24. 14. The method of claim 13, wherein the FC medium comprises DMEM, fetal bovine serum, sodium pyruvate, GlutaMax, MEM non-essential amino acids, 2-mercaptoethanol, penicillin and / or streptomycin, or any combination thereof.

25. 14. The method of claim 13, wherein the FC medium comprises DMEM, about 15% fetal bovine serum, about 1 mM sodium pyruvate, about 2 mM GlutaMax, about 1% MEM non-essential amino acids, about 0.1 mM 2-mercaptoethanol, about 1% penicillin and / or streptomycin, or any combination thereof.

26. 14. The method of claim 13, wherein the FC medium further comprises an anticoagulant, optionally heparin, a fibroblast growth factor (FGF), optionally FGF2 and / or FGF4, or any combination thereof.

27. 16. The method of claim 15, wherein the IVC medium comprises: a) insulin, an insulin analog, or an insulin receptor agonist; b) an estrogen, an estrogen analog, or an estrogen receptor agonist; and c) Progesterone, a progesterone analog, or a progesterone receptor agonist.

28. 28. The method of claim 27, wherein the insulin receptor agonist is selected from IGF-I, IGF-II, analogs thereof, or any combination thereof.

29. 28. The method of claim 27, wherein the estrogen receptor agonist is selected from β-estradiol, estrone, estriol, and estetrol, or analogs thereof.

30. 16. The method of claim 15, wherein the IVC medium comprises transferrin, sodium selenide, ethanolamine, or analogs thereof.

31. 16. The method of claim 15, wherein the IVC medium does not contain sodium pyruvate.

32. 16. The method of claim 15, wherein the IVC medium comprises DMEM / F12, fetal bovine serum, GlutaMax, ITS-X, β-estradiol, progesterone, N-acetyl-L-cysteine, penicillin and / or streptomycin, or any combination thereof.

33. 16. The method of claim 15, wherein the IVC medium comprises DMEM / F12, about 20% fetal bovine serum, about 2 mM GlutaMax, about 1× ITS-X, about 8 nM β-estradiol, about 200 ng / ml progesterone, about 25 μM N-acetyl-L-cysteine, about 1% penicillin and / or streptomycin, or any combination thereof.

34. 6. The method of claim 1, wherein culturing the mammalian pluripotent stem cells and the extraembryonic stem cells in the first culture medium comprises increasing a serum concentration, optionally increasing the serum concentration from about 20% to about 30%.

35. 6. The method of any one of claims 1 to 5, wherein step (a) comprises transferring the mammalian pluripotent stem cells and the extraembryonic stem cells from one substrate to another.

36. 6. The method of any one of claims 1 to 5, wherein the post-implantation embryo structure is cultured in the second culture medium for up to 3 days.

37. 6. The method of any one of claims 1 to 5, wherein step (b) is from embryonic day E5.5 to E8.

0.

38. 6. The method according to any one of claims 1 to 5, wherein the neutralizing embryo structure resembles a natural embryo structure at E8.

0.

39. 6. The method of any one of claims 1 to 5, wherein the neutralizing embryo structure is cultured in a substrate, optionally comprising a dish, a U-plate, a flask, or a microwell plate.

40. 6. The method of any one of claims 1 to 5, wherein the second culture medium is a post-implantation culture medium capable of supporting the development of an embryo outside the uterus.

41. 6. The method of claim 1, wherein the second culture medium comprises DMEM, non-human serum, human umbilical cord serum, L-glutamine or an analog thereof, an antibiotic, or any combination thereof.

42. 41. The method of claim 40, wherein the non-human serum comprises rat serum and / or bovine serum.

43. 6. The method of claim 1, wherein the second culture medium comprises bicarbonate.

44. 6. The method of claim 1, wherein the second culture medium comprises HEPES.

45. 42. The method of claim 41, wherein the non-human serum is rat serum.

46. 6. The method of claim 1, wherein the second culture medium comprises DMEM, rat serum, human umbilical cord serum, GlutaMax, penicillin and / or streptomycin, HEPES, or any combination thereof.

47. 6. The method of claim 1, wherein the second culture medium comprises about 25% DMEM, about 50% rat serum, and about 25% human umbilical cord serum.

48. 6. The method of claim 1, wherein step (b) comprises adding glucose to the second culture medium.

49. 49. The method of claim 48, wherein step (b) comprises adding at least 3 mg / ml of glucose to the second culture medium.

50. 49. The method of claim 48, wherein the glucose is added at least once, optionally on day 3 of culture.

51. 6. The method of any one of claims 1 to 5, wherein step (b) comprises culturing the post-implantation embryonic structure in a medium containing about 1 mg / ml glucose for 2 days, and culturing the post-implantation embryonic structure in a medium containing about 3 mg / ml glucose for 1 day.

52. 6. The method of any one of claims 1 to 5, wherein step (c) is from embryonic day E8.0 to at least E8.

5.

53. 6. The method of any one of claims 1 to 5, wherein the synthetic embryo resembles a natural embryo structure at E8.

5.

54. 6. The method of any one of claims 1 to 5, wherein the synthetic embryo resembles a natural embryo structure at E9.

0.

55. 6. The method of any one of claims 1 to 5, wherein the neutralizing embryo structure is cultured under dynamic conditions in the second culture medium.

56. 56. The method of claim 55, wherein the second culture medium in step (c) comprises at least 30% non-human serum.

57. 6. The method of claim 1, wherein step (b) comprises adding at least 3 mg / ml of glucose to the second culture medium, optionally at least 3.5 mg / ml of glucose.

58. 6. The method of any one of claims 1 to 5, wherein the dynamic conditions include suspension agitation, optionally rotation.

59. 59. The method of claim 58, wherein step (c) is carried out in a rolling bottle.

60. 60. The method of claim 59, wherein each roller bottle contains two synthetic embryoid bodies in about 3 ml of medium.

61. 6. The method of claim 1, wherein the dynamic conditions include supplying a plurality of gases to the culture chamber.

62. 6. The method of any one of claims 1 to 5, wherein the dynamic conditions include a gas pressure of about 0.5 to about 3 pounds per square inch (psi), optionally about 0.5 to 1 psi, optionally about 0.5 psi.

63. 6. The method of any one of claims 1 to 5, wherein the dynamic conditions include supplying a constant concentration of oxygen to the culture chamber.

64. 6. The method of any one of claims 1 to 5, wherein the dynamic conditions include supplying increasing concentrations of oxygen to the culture chamber.

65. 6. The method of any one of claims 1 to 5, wherein the culture chamber has an atmosphere containing an increasing oxygen concentration from about 5% to about 25%, optionally from about 5% to about 13%, optionally from about 13% to about 18%, optionally from about 18% to about 21%.

66. 6. The method of any one of claims 1 to 5, wherein the synthetic embryo has an established brain region, a neural tube, a beating heart, and / or a gut.

67. 6. The method of any one of claims 1 to 5, wherein the synthetic embryo comprises developing somites and primordial germ cells.

68. 6. The method of any one of claims 1 to 5, wherein the method does not include any in vivo steps.

69. 6. The method of any one of claims 1 to 5, wherein in culturing steps (a), (b), and (c), none of the mammalian pluripotent stem cells, the extraembryonic stem cells, the post-implantation embryonic structure, and the neutralizing embryo structure are present in an in vivo environment, and optionally, wherein the in vivo environment comprises a tissue, an organ, an organism, or a combination thereof.

70. 6. The method of any one of claims 1 to 5, wherein the synthetic embryo is a mouse embryo.

71. A synthetic embryo obtained by the method of any one of claims 1 to 5.

72. 6. An in vitro culture (IVC) medium for generating a synthetic embryo in vitro according to the method of any one of claims 1 to 5, wherein said medium comprises: a basal culture medium containing at least 20% non-human serum; Insulin, an insulin analog, or an insulin receptor agonist; an estrogen, an estrogen analog, or an estrogen receptor agonist; and Progesterone, a progesterone analog, or a progesterone receptor agonist.

73. 73. The IVC medium of claim 72, wherein the basal culture medium comprises a reducing agent, an antibiotic, or a combination thereof.

74. 73. The IVC medium of claim 72, wherein the non-human serum comprises fetal bovine serum, bovine serum albumin, or both.

75. A method for investigating mechanisms involved in embryonic development, said method comprising the method of any one of claims 1 to 5.

76. A method for identifying a compound useful for treating a disease, said method comprising contacting a synthetic embryo obtainable by an in vitro method according to any one of claims 1 to 5 with said compound.

77. 6. A synthetic embryo produced according to the method of any one of claims 1 to 5, wherein the synthetic embryo is implanted into a subject for use in a method for diagnosing or treating a disease or disorder in the subject.

78. 78. The synthetic embryo of claim 77, wherein said pluripotent stem cells and said at least one extraembryonic stem cell are obtained from said subject.

79. 10. A method for elucidating the role of a gene in embryonic development, the method comprising the steps of obtaining pluripotent stem cells and / or extraembryonic stem cells in which the gene has been modified or knocked out, and culturing the pluripotent stem cells and extraembryonic stem cells using the in vitro method of any one of claims 1 to 5.

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