Stem cell-derived embryo formation induced by differences in adhesion and tension

Abstract

Disclosed herein are methods and compositions for use in producing synthetic embryos. In some embodiments, the methods include culturing stem cells that overexpress at least one cadherin.

Description

Priority claim

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 403,685, filed September 2, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Nos. HD100456 and HD104575 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] [Reference to sequence listing] This application is submitted with an electronic Sequence Listing. The Sequence Listing is provided as a file entitled 30KJ-302456-US_Sequence Listing, created on August 29, 2023, and is 13,333 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Technical Field]

[0004] The present disclosure relates generally to the fields of cell culture and embryogenesis. [Background technology]

[0005] Trophectoderm stem (TS) cells, extraembryonic endoderm (XEN) cells, and embryonic stem (ES) cells are derived from the polar trophectoderm (TE), primitive endoderm (PE), and epiblast (EPI) of preimplantation embryos, respectively. Remarkably, these stem cells can be used to construct synthetic embryos (so-called ETX embryos) in vitro that, unlike other models, recapitulate various aspects of early mammalian embryonic development. For example, blastoid cells resemble preimplantation embryos but have limited potential for postimplantation development. Gastruloid cells can mimic aspects of gastrulation but lack the anterior-most structures of postimplantation embryos. In contrast, ETX embryos resemble postimplantation embryos and recapitulate both gene expression patterns and cell movements typical of gastrulation. ETX embryos develop into egg cylinders from a random combination of ES, TS, and XEN cells. Surprisingly, ES cells form the EPI compartment, TS cells form the ExE compartment, and XEN cells form an enveloping VE-like layer.

[0006] The self-organization of many cellular systems relies on the formation of distinct cell-cell contacts, which in turn rely on differential adhesion via cadherin molecules and / or differential cortical tension due to reorganization of the actin cortical network. Indeed, by elucidating the fundamental principles of self-organization, synthetic genetic programs controlling the expression of distinct cadherins can direct the formation of custom multicellular structures. However, the principles operating in the self-assembly of embryonic structures in vitro have not previously been explored. Understanding the principles by which genetic information is translated into physical processes to achieve the morphogenetic transitions that form embryos is necessary. Our ability to generate more complex stem cell embryo-like models or organoids is needed. Summary of the Invention [Problem to be solved by the invention]

[0007] Disclosed herein are methods for producing a synthetic embryo in vitro. In some embodiments, the method includes: (a) providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); (b) providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); (c) providing a plurality of extraembryonic (XEN) cells; and (d) contacting the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture; the plurality of engineered ESCs and their derivatives, the plurality of engineered TSCs and their derivatives, and the plurality of XEN cells and their derivatives organize to form a synthetic embryo, the synthetic embryo comprising one TS-derived compartment and one ES-derived compartment, and covered by an outer XEN-derived monolayer. The method may include (e) about 3 days after the contacting step (d), replacing the first medium with a second medium.

[0008] In some embodiments, the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of XEN cells organize into a multicellular aggregate structure within about 12-24 hours from the contacting step (d). In some embodiments, the multicellular aggregate structure develops into a multicellular aggregate structure comprising one TS-derived compartment and one ES-derived compartment at least partially covered by an outer XEN-derived monolayer at about 30% efficiency about 12 hours after the contacting step (d). In some embodiments, the multicellular aggregate structure develops into a synthetic embryo at about 40% efficiency at least 3 days after the contacting step (d).

[0009] In some embodiments, the synthetic embryo develops a single internal cavity at about 90% efficiency. In some embodiments, the single internal cavity develops between 4 and 5 days after the contacting step (d). In some embodiments, the multicellular aggregate structure develops into a synthetic embryo containing a single internal cavity at about 40% efficiency. In some embodiments, the single internal cavity develops between 4 and 5 days after the contacting step (d). In some embodiments, the multicellular aggregate structure develops into a synthetic embryo containing a laminin-containing basement membrane at about 78% efficiency. In some embodiments, the laminin-containing basement membrane develops between 4 and 5 days after the contacting step (d).

[0010] In some embodiments, the synthetic embryo has a length of at least 200 μm about 72 hours after the contacting step (d). In some embodiments, the synthetic embryo has a length of about 200 μm to about 500 μm about 72 hours after the contacting step (d). In some embodiments, the synthetic embryo has a length of at least 4×10 about 72 hours after the contacting step (d). 3 μm 2 In some embodiments, the synthetic embryos have a size of about 6 x 10 about 72 hours after the contacting step (d). 3 μm 2 ~About 10×10 3 μm 2 It has a size of

[0011] In some embodiments, the TS-derived compartment comprises cells expressing at least one TS cell marker. In some embodiments, the at least one TS cell marker comprises Tfap2C, EOMES, or both. In some embodiments, the ES-derived compartment comprises cells expressing at least one ES cell marker. In some embodiments, the at least one ES cell marker comprises Oct4. In some embodiments, the XEN-derived monolayer comprises cells expressing at least one XEN cell marker. In some embodiments, the at least one XEN cell marker comprises Gata4, Gata6, or both. In some embodiments, the synthetic embryo resembles an egg cylinder structure about 3 days after the contacting step (d). In some embodiments, the synthetic embryo resembles a post-implantation embryo structure about 4-5 days after the contacting step (d).

[0012] In some embodiments, providing the plurality of engineered ESCs comprises: (i) providing an expression construct comprising a nucleic acid encoding E-cadherin operably linked to at least one expression control element that enables gene expression in mammalian cells; and (ii) introducing the expression construct into ESCs in a manner that enables expression of the introduced construct in at least one of the ESCs, thereby generating at least one engineered ESC. In some embodiments, providing the plurality of engineered ESCs comprises culturing the at least one engineered ESC of (ii).

[0013] In some embodiments, providing the plurality of engineered TSCs comprises: (i) providing an expression construct comprising a nucleic acid encoding P-cadherin operably linked to at least one expression control element enabling gene expression in mammalian cells; and (ii) introducing the expression construct into TSCs in a manner enabling expression of the introduced construct in at least one of the TSCs, thereby generating at least one engineered TSC. In some embodiments, providing the plurality of engineered TSCs comprises culturing at least one engineered TSC of (ii).

[0014] In some embodiments, the expression control element comprises a promoter, an enhancer, a 5' untranslated region, a 3' untranslated region, or any combination thereof. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a constitutive or inducible promoter. In some embodiments, at least a portion of the plurality of engineered ESCs overexpress E-cadherin relative to wild-type ESCs. In some embodiments, at least a portion of the plurality of engineered TSCs overexpress P-cadherin relative to wild-type TSCs. In some embodiments, the plurality of XEN cells are wild-type XEN cells. In some embodiments, none of the plurality of XEN cells has been engineered to overexpress E-cadherin, P-cadherin, or K-cadherin.

[0015] In some embodiments, the plurality of engineered ESCs comprises at least 5,000 ESCs. In some embodiments, the plurality of engineered ESCs comprises 6,000-7,000 ESCs. In some embodiments, the plurality of engineered TSCs comprises at least 10,000 TSCs. In some embodiments, the plurality of engineered TSCs comprises 15,000-19,000 TSCs. In some embodiments, the plurality of XEN cells comprises at least 5,000 XEN cells. In some embodiments, the plurality of XEN cells comprises 5,000-6,000 XEN cells. In some embodiments, the ESCs, TSCs and / or XEN cells are derived from a natural mammalian embryo. In some embodiments, the natural mammalian embryo is a mouse or human natural embryo.

[0016] In some embodiments, the co-culture is carried out in an inverted pyramidal microwell. In some embodiments, the inverted pyramidal microwell is about 400 μm or about 800 μm in size. In some embodiments, the inverted pyramidal microwell is about 400 μm or about 800 μm in diameter. In some embodiments, the first culture medium in step (d) comprises a ROCK inhibitor. The method may include removing the ROCK inhibitor after about 24 hours of co-culture in the first culture medium.

[0017] In some embodiments, the first culture medium and the second culture medium each comprise a basal culture medium. In some embodiments, the basal culture medium comprises Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Roswell Park Memorial Institute (RPMI) Medium 1640, Neurobasal®, Neurobasal® A, Connaught Medical Research Laboratories 1066 (CMRL-1066), or any combination thereof. In some embodiments, the first culture medium and the second culture medium each comprise non-human serum or a serum substitute thereof, a reducing agent, and an antibiotic. In some embodiments, the non-human serum or serum substitute comprises fetal bovine serum, bovine serum albumin, KnockOut™ serum substitute, or any combination thereof. In some embodiments, the reducing agent comprises β-mercaptoethanol (2-ME), 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. In some embodiments, the first culture medium and the second culture medium each comprise N2 supplement, B27 supplement, insulin-transferrin-selenium-ethanolamine (ITS-X), GlutaMax™, non-essential amino acids, ascorbic acid, sodium pyruvate, or any combination thereof. In some embodiments, the first culture medium comprises DMEM, FBS, GlutaMax™, 2-ME, non-essential amino acids, sodium pyruvate, HEPES, and penicillin-streptomycin. In some embodiments, the first culture medium comprises a ROCK inhibitor. In some embodiments, the first culture medium comprises DMEM, 12.5% ​​FBS, 2 mM GlutaMax™, 0.1 mM 2-ME, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.02 M HEPES, 1% penicillin-streptomycin, and 7.5 nM ROCK inhibitor.In some embodiments, the first culture medium comprises DMEM, 12.5% ​​FBS, 2 mM GlutaMax™, 0.1 mM 2-ME, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.02 M HEPES, and 1% penicillin-streptomycin. In some embodiments, the first culture medium does not contain a ROCK inhibitor. In some embodiments, the second culture medium comprises DMEM / F12, FBS, GlutaMax™, penicillin-streptomycin, ITS-X, β-estradiol, progesterone, and N-acetyl-L-cysteine. The second culture medium may contain DMEM / F12, 20% FBS, 2 mM GlutaMax™, 1% penicillin-streptomycin, 1× ITS-X, 8 nM β-estradiol, 200 ng / mL progesterone, and 25 mM N-acetyl-L-cysteine.

[0018] The method may include (f) replacing the second culture medium with a third culture medium about one day after step (e). In some embodiments, the third culture medium comprises DMEM / F12, KnockOut™ serum replacement, GlutaMax™, penicillin-streptomycin, ITS-X, β-estradiol, progesterone, and N-acetyl-L-cysteine. In some embodiments, the third culture medium comprises DMEM / F12, 30% KnockOut™ serum replacement, 2 mM GlutaMax™, 1% penicillin-streptomycin, 1× ITS-X, 8 nM β-estradiol, 200 ng / mL progesterone, and 25 mM N-acetyl-L-cysteine.

[0019] The present disclosure includes differentiated cells obtained by any of the methods disclosed herein. The present disclosure includes methods for determining the effect of a test agent on embryonic development. In some embodiments, the method includes: a) providing a synthetic embryo produced by any of the methods provided 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 includes 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 include producing a synthetic embryo by any of the methods disclosed herein. The method may include contacting the synthetic embryo with the test agent before step (d). The method may include determining the subsequent effect on the formation of the synthetic embryo. In some embodiments, the effect on the formation of multicellular aggregate structures, egg cylinder structures, and / or post-implantation structures is determined. The method may include recording one or more images of the synthetic embryo. The present disclosure includes methods for investigating mechanisms involved in embryogenesis. Disclosed herein is a method for identifying a compound useful for treating a disease. In some embodiments, the method comprises contacting a compound with a synthetic embryo or differentiated cell obtained by any of the methods provided herein. Disclosed herein is a method for diagnosing or treating a disease or disorder in a subject. In some embodiments, the method comprises using a synthetic embryo or differentiated cell obtained by the methods provided herein, or any combination thereof. Also provided is a method for elucidating the role of a gene in embryonic development. In some embodiments, the method comprises obtaining a gene-modified or knocked-out ESC, TSC, and / or XEN cell, and culturing the cell to obtain a plurality of cells for use in the method for generating a synthetic embryo disclosed herein.

[0020] The patent or application file contains at least one color drawing. Copies of the patent or patent application publication with such color drawing will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0021] [Figure 1]Figure 1A is a non-limiting, exemplary schematic diagram related to the differences in the cadherin coding between ETX and natural embryos. It illustrates the self-organization and morphological transitions in natural and stem cell-derived (ETX) embryos. The epiblast (EPI) of natural embryos and ES cells of ETX embryos, as well as the trophectoderm (TE) and TS cells of ETX embryos are depicted. Also depicted are primitive endoderm (PE) and visceral endoderm (VE) in natural embryos, and XEN cells in ETX embryos. From E6.0 to E6.5, a mesoderm layer underlying the outer XEN or endodermal (VE)-derived layer is shown. ExE, extraembryonic ectoderm. Figure 1B illustrates non-limiting, exemplary data related to the differences in the cadherin coding between ETX and natural embryos, showing a comparison of average scRNA-seq read counts between ES and TS cells. Data points to the left (right) of the dashed line represent transcripts that are more than two-fold enriched in TS (ES) cells. The center dots of the dashed lines indicate equally expressed genes. Figure 1C shows non-limiting, exemplary data related to differences in the cadherin coding in ETX and natural embryos, including non-limiting, exemplary data showing a comparison of average scRNA-seq read counts between XEN and ES cells. Data points to the left (right) of the dashed line indicate transcripts that are more than two-fold enriched in ES (XEN) cells. The center dots of the dashed lines indicate equally expressed genes. In Figures 1B and 1C, transcripts related to cadherins and protocadherins are highlighted (excluding Tfap2c and Oct4). Figure 1D shows non-limiting, exemplary data related to differences in the cadherin coding in ETX and natural embryos, including exemplary violin plots showing gene expression of Cdh1 (top), Cdh3 (middle), and Cdh6 (bottom) from scRNA-seq in natural and ETX embryos at different stages. NE45, NE55, and NE65 represent natural embryos collected at days 4.5, 5.5, and 6.5. ETX4, ETX5, and ETX6 represent ETX embryos collected at days 4, 5, and 6. Figure 1E shows a non-limiting, exemplary schematic relating to differences in cadherin coding in ETX and natural embryos, and an exemplary schematic of chimeric aggregation.Cadherin OE ES cells expressing H2B-rFP were mated with wild-type embryos at the 8-cell stage. Contribution to EPI, PE, TE, or excluded cells was assessed at E4.5 and indicated in the figure. Figure 1F shows a non-limiting, exemplary schematic diagram relating to differences in the cadherin code between ETX and natural embryos, and shows non-limiting, exemplary images of chimeras stained with RFP, Sox17, and DNA (DAPI). Scale bar, 50 μm. The enlarged image shows the area indicated by the dashed box on the left (scale bar, 10 μm). Experiments were repeated three times. WT, wild-type. Figure 1G shows non-limiting, exemplary data relating to differences in the cadherin code between ETX and natural embryos, and, similar to Figure 1E, shows non-limiting, exemplary data regarding the percentage of cells contributing to EPI, PE, TE, or excluded cells in chimeras. Data are presented as violin plots. Each dot corresponds to an embryo. n = 32 embryos of wild-type ES chimeras (3365 cells total), n = 16 embryos of Cdh1 OE ES chimeras (1787 cells total), n = 13 embryos of Cdh3 OE ES chimeras (1574 cells total), n = 16 embryos of Cdh6 OE ES chimeras (1894 cells total). Statistical significance was determined by one-way ANOVA and multiple comparison tests. Numerical data are available as source data. [Figure 2]Figure 2A shows non-limiting, exemplary data on differences in adhesion force in ETX embryos and is an exemplary schematic diagram illustrating the measurement of cell-cell adhesion force by AFM. Figure 2B shows non-limiting, exemplary data on differences in adhesion force in ETX embryos and is a diagram depicting exemplary force-distance curves obtained according to the procedure depicted in Figure 2A, allowing for quantification of maximum adhesion force (Fmax). Figure 2C shows non-limiting, exemplary data on differences in adhesion force in ETX embryos and is an exemplary chart showing Fmax for the indicated homotypic and heterotypic adhesions between three different cell types. Experiments were performed independently three times. Total measured cell pairs: n = 60 (ES-ES), n = 177 (TS-TS), n = 101 (XEN-XEN), n = 124 (ES-TS), n = 148 (XEN-TS), n = 134 (XEN-ES). Statistical significance was determined by one-way analysis of variance and multiple comparison tests. In the box plots in Figure 2C, the line within the box indicates the median, and the error bars indicate the minimum and maximum values. The edges of the box indicate the lower and upper quartile values. Figure 2D shows non-limiting, exemplary data on differences in adhesion force in ETX embryos. It shows an exemplary schematic diagram of a weakly and strongly adherent cell pair in force equilibrium. θ is the contact angle of the two adherent cells. Figure 2E shows non-limiting, exemplary data on differences in adhesion force in ETX embryos. It shows exemplary data on the distribution of contact angle measurements across all cell-cell contacts. Total cell pairs measured: n = 31 (ES-ES), n = 38 (TS-TS), n = 30 (XEN-XEN), n = 32 (TS-ES), n = 36 (XEN-TS), n = 29 (XEN-ES). N = 3 for all conditions. Statistical significance was determined by one-way analysis of variance and multiple comparison tests. In the box plots in Figure 2E, the line within the box indicates the median, and the error bars indicate the minimum and maximum values. The edges of the box indicate the lower and upper quartiles. Figure 2F shows non-limiting, exemplary data on differences in adhesion forces in ETX embryos, including exemplary data on adhesion forces between cells and different cadherins. Left: Schematic showing measurement of cell-cadherin adhesion forces by AFM. Right: Quantification of results.n = 42 (ES-E-cadherin), n = 35 (ES-P-cadherin), n = 41 (TS-E-cadherin), n = 37 (TS-P-cadherin). N = 3 for all conditions. Statistical significance was determined by unpaired, two-tailed Student's t-test. In the boxplots in Figure 2F, the line within the box indicates the median, and the error bars indicate the minimum and maximum values. The edges of the box indicate the lower and upper quartiles. Figure 2G shows non-limiting, exemplary data regarding differences in adhesive strength in ETX embryos. It shows an exemplary graph of Fmax for homotypic adhesion between three different cell types after downregulation of Cdh1 or Cdh3. n = 60 (WTES-ES), n = 18 (Cdh1 KD ES-ES), n = 19 (Cdh3 KD ES-ES), n = 177 (wild-type TS-TS), n = 20 (Cdh1 KD TS-TS), n = 20 (Cdh3 KD TS-TS), n = 101 (wild-type XEN-XEN), n = 19 (Cdh1 KD XEN-XEN), n = 19 (Cdh3 KD XEN-XEN). N = 3 for all conditions. Statistical significance was determined by one-way ANOVA and multiple comparison tests. In the box plots in Figure 2G, the line within the box indicates the median, and the error bars indicate the minimum and maximum values. The edges of the box indicate the lower and upper quartiles. Figure 2H shows non-limiting, exemplary data on adhesion force differences in ETX embryos, including an exemplary heatmap of the adhesion parameter matrix generated by sampling measured AFM adhesion forces and parameterizing CPM. Figure 2I shows non-limiting, exemplary data on adhesion force differences in ETX embryos, illustrating the bootstrap procedure for inferring the distribution of conformations under CPM (N = 498). The schematic represents all possible screened conformations, showing that ETX-like conformations are the most representative. Conformations observed at frequencies <5% are grouped together. MCS, Markov chain step. [Figure 3]Figure 3A shows non-limiting, exemplary data demonstrating that differences in cadherin coding regulate self-organization in ETX embryos, showing representative images of ETX embryos assembled at different times. Scale bar, 50 μm. ES, Tfap2c; XEN, Gata4; TS, Oct4. The experiment was repeated four times. Figure 3B shows non-limiting, exemplary data demonstrating that differences in cadherin coding regulate self-organization in ETX embryos, showing the diversity of self-assembled structures collected on day 3. Scale bar, 100 μm. Staining is the same as in Figure 3A. The experiment was repeated four times. Figure 3C shows non-limiting, exemplary data demonstrating that differences in cadherin coding regulate self-organization in ETX embryos, showing representative, non-limiting images of correctly and incorrectly sorted ETX structures after 3 days. The inset schematic shows an example of sorting results. Scale bar, 100 μm. The experiment was repeated four times. Figure 3D shows non-limiting, exemplary data demonstrating that differences in cadherin coding regulate self-organization in ETX embryos. A pie chart depicts the percentage of correctly and incorrectly sorted ETX structures at day 3. The 4,000 structures analyzed included three different stem cell types. Four independent experiments were performed. Figure 3E shows non-limiting, exemplary data demonstrating that differences in cadherin coding regulate self-organization in ETX embryos. Representative images are shown for cell sorting of Cdh1 or Cdh6 KD or OE XEN cells in combination with wild-type ES and TS cells. Wild-type XEN cells served as a control. Scale bar, 100 μm. Staining is the same as in Figure 3A. Experiments were repeated three times. Figure 3F shows non-limiting, exemplary data demonstrating that differences in cadherin coding control self-organization in ETX embryos, showing graphs quantifying ETX structures formed by XEN cells overexpressing (OE) Cdh1 or Cdh6, or KD of either Cdh1 or Cdh6, compared with well-sorted or missorted XEN cells.Total number of structures: n = 470 (WT XEN), n = 282 (Cdh1 KD XEN), n = 519 (Cdh6 KD XEN), n = 326 (Cdh1 OE XEN), n = 281 (Cdh6 OE XEN). N = 3. Data are shown as mean ± SD. Statistical significance was determined by one-way ANOVA with multiple comparisons. Figure 3G shows non-limiting, exemplary data demonstrating that differences in cadherin coding control self-organization in ETX embryos. Figure 3G also shows non-limiting, exemplary data for cadherin KD in ES and TS cells. Left: Representative images of ETX structures in Cdh1 and Cdh3 KD ES and TS cells. Scale bar: 100 μm. Right: Quantification showing successfully sorted and incorrectly sorted ETX embryos under the indicated conditions. Total number of structures: n = 4186 (control), n = 2940 (Cdh1 KD ES), n = 2471 (Cdh3 KD ES), n = 2407 (Cdh1 KD TS), n = 2151 (Cdh3 KD TS). N = 3. Data are shown as mean ± SD. Statistical significance was determined by one-way analysis of variance and multiple comparisons. Figure 3H shows non-limiting, exemplary data demonstrating that differences in cadherin coding control self-organization in ETX embryos. Figure 3H shows non-limiting, exemplary data on the formation of Cdh1 OE ES cells and Cdh3 OE TS cells and synthetic embryos. Left: Representative images of ETX structures formed by combining Cdh1 OE ES cells with Cdh3 OE TS cells and wild-type XEN cells. ES cells, TS cells, and XEN cells are labeled as in Figure 3A. Middle, Magnified view showing successfully sorted ETX constructs, as indicated by the white arrow to the left. Scale bar, 100 μm. Right, Quantification of successfully sorted ETX constructs; n = 3451 (control) and n = 2348 (Cdh1 and Cdh3 OE) constructs were selected from five independent experiments. Data are shown as mean ± SD. Statistical significance was determined by unpaired, two-tailed Student's t-test. [Figure 4]Figure 4A shows non-limiting, exemplary data demonstrating that proper self-organization is required for proper morphogenesis. It depicts the time course of assembly of ETX embryos stained for E-cadherin (monochrome, see bottom panel), Oct4(ES), and Gata4(XEN). The bottom image is a magnified version of the top image, showing E-cadherin staining around the nascent cavity, as indicated by the dashed line around the cavity. The dashed line also indicates the boundary between the ES and XEN compartments. Scale bar, 5 µm. Figure 4B shows non-limiting, exemplary data demonstrating that proper self-organization is required for proper morphogenesis. It shows representative images demonstrating Oct4(ES), Gata4(XEN), E-cadherin (monochrome, right panel), and DAPI (gray) staining in day 4 cadherin-OE ETX constructs formed by combining E-cadherin-OE ES cells with P-cadherin-OE TS cells and wild-type XEN cells. ETX constructs formed by combining wild-type cells served as a control. Scale bar, 100 μm. Figure 4C shows non-limiting, exemplary data demonstrating that proper self-organization is necessary for proper morphogenesis. It shows a graph comparing and quantifying the formation of junctional cavities in cadherin-OE and control ETX constructs. n = 361 (control group) and n = 253 (cadherin-OE group). N = 5 for each condition. Data are shown as mean ± SD. Statistical significance was determined by unpaired, two-tailed Student's t-test. Figure 4D shows non-limiting, exemplary data demonstrating that proper self-organization is necessary for proper morphogenesis. It shows representative images showing Oct4(ES), Gata4(XEN), laminin (monochrome, right panel), and DAPI nuclear staining in day 4 cadherin-OE ETX constructs formed by combining E-cadherin-OE ES cells with P-cadherin-OE TS cells and wild-type XEN cells. ETX constructs formed by combining wild-type cells served as controls. Scale bar, 100 μm.Figure 4E shows non-limiting, exemplary data demonstrating that proper self-organization is required for proper morphogenesis, and graphically illustrates the quantification of structures containing continuous or discontinuous laminin. N=3. Data are presented as mean ± SD. Statistical significance was determined by unpaired, two-tailed Student's t-test. Figure 4F shows non-limiting, exemplary data demonstrating that proper self-organization is required for proper morphogenesis, and illustrates a schematic diagram of the self-organization principle in stem cell-derived ETX embryos as disclosed herein. Differential expression of E-, K-, and P-cadherin allows for the sorting of ES (epiblast-like), XEN (VE-like), and TS (TE-like) stem cells. Wild-type ES cells with low E-cadherin expression and wild-type TS cells with low P-cadherin expression exhibited poor overall sorting efficiency. This can be overcome by overexpressing E-cadherin in ES cells and P-cadherin in TS cells to increase the efficiency of ETX embryo formation. Proper morphogenesis, including cavity formation, basement membrane formation (shown as a layer between the outer PE / XEN-derived layer and the inner EPI / ES- and TE / TS-derived compartments), and symmetry breaking, is only observed in well-sorted structures. [Figure 5]Figure 5A shows non-limiting, exemplary data related to the differences in cadherin coding between ETX- and natural embryos. It displays a UMAP dimensionality reduction model showing the expression profiles of Cdh1, Cdh3, and Cdh6 in different clusters, as indicated by the dashed lines. Each dot represents a single cell shaded by sample type. Figure 5B shows non-limiting, exemplary data related to the differences in cadherin coding between ETX- and natural embryos. It displays a heatmap showing the average expression of cadherin- and protocadherin-related genes revealed by scRNA-seq in natural embryos collected at days 4.5, 5.5, and 6.5 post-fertilization (NE, n = 50) and in successfully sorted ETX embryos (n = 50) cultured at days 4, 5, and 6. Figure 5C shows non-limiting, exemplary data related to the differences in cadherin coding between ETX- and natural embryos. It displays data on cadherin expression. The images show colonies of cultured ES and TS cells stained to reveal E-cadherin and P-cadherin. The graphs show quantification values ​​indicating the average intensity (AU) of E-cadherin or P-cadherin at cell-cell junctions. 20 colonies were selected from three separate experiments and quantified. The scale bar represents 100 μm. Data are shown as mean ± SD. Statistics were calculated using an unpaired, two-tailed Student's t-test. Figure 5D shows non-limiting, exemplary data related to the difference in cadherin coding between ETX- and natural embryos. The images show natural embryos (E5.5) and ETX embryos (day 4) stained to reveal E-cadherin and P-cadherin. The enlarged insets show E- or P-cadherin staining in the ExE and EPI compartments of natural embryos and the TS and ES compartments of ETX embryos. The graph shows the quantification values, which represent the average intensity (AU) of E-cadherin or P-cadherin at cell-cell junctions. Quantification was performed using n = 20 ETX embryos and n = 19 natural embryos. Data are shown as mean ± SD. Statistics were calculated using an unpaired, two-tailed Student's t-test.Scale bars represent 100 μm (main image) and 20 μm (inset). Figure 5E shows non-limiting, exemplary data related to differences in cadherin coding between ETX-embryos and natural embryos. Representative images of E4.5 chimeras (8-cell embryos aggregated with Cdh6 OE ES) stained for RFP, Sox17, and DAPI are shown. Experiments were repeated three times. Scale bars represent 50 μm. The zoomed image shows the area indicated by the dashed line (scale bar represents 10 μm). Figure 5F shows non-limiting, exemplary data related to differences in cadherin coding between ETX-embryos and natural embryos. Representative images of E4.5 chimeras (8-cell embryos aggregated with Cdh3 OE ES) stained for RFP, Sox17, and DAPI are shown. Experiments were repeated three times. Scale bars represent 50 μm. The zoomed image shows the area indicated by the dashed line (scale bar represents 10 μm). [Figure 6]Figure 6A shows non-limiting, exemplary data related to differences in adhesion forces in ETX embryos. Representative images of doublets of homotypic and heterotypic cell pairs (Gata4, Tfap2c, Oct4, F-actin) are shown. Experiments were repeated three times. The scale bar is 10 μm. Figure 6B shows non-limiting, exemplary data related to differences in adhesion forces in ETX embryos. It demonstrates the use of the ImSAnE "Unrolling" algorithm to project 3D E-cadherin staining stacks onto a 2D plane. Cell contact angles were quantified using a built-in correction method. Geometric observations, as well as general distortions in projections, can be accurately quantified. The scale bar is 100 μm. Figure 6C shows non-limiting, exemplary data related to differences in adhesion forces in ETX embryos. It shows a graph of cell-cell contact angle measurements based on the ImSAnE method in day 4 ETX embryos and E5.5 native embryos. All measured cell pairs in ETX embryos: ES-ES: n=24; TS-TS: n=15; XEN-XEN: n=16; ES-TS: n=24; XEN-TS: n=19; XEN-ES: n=16; XENi-XENi: n=16. All measured cell pairs in natural embryos: EPI-EPI: n=20; TE-TE: n=17; VE-VE: n=22; EPI-TE: n=24; EPI-VE: n=16; TE-VE: n=19; VEi-VEi: n=24. Data are shown as box plots, with black lines within boxes representing medians and error bars representing minimum to maximum values. Statistics were calculated by one-way analysis of variance with multiple comparison tests. Figure 6D shows non-limiting, exemplary data related to differences in adhesion strength in ETX embryos. A magnified view of the boundary region of a day 4 ETX embryo stained for E-cadherin shows homophilic contacts highlighted in blue (TS-TS, e.g., bottom right panel), red (ES-ES, e.g., top of the image in the top left panel), and purple (XEN-XEN). The heterophilic boundary interface is highlighted in yellow (see the top two panels). The angles formed at the tricellular junctions between different types are shown: EX, TX, ET, angles between heterophilic contacts (ES-XEN, TS-XEN, ES-TS), and EE, TT, XX, angles between homophilic contacts (ES-ES, TS-TS, XEN-XEN). XXi shows the contact angle of XEN cells at the cell-culture medium interface.Experiments were repeated six times. Scale bars represent 20 μm. Figure 6E shows non-limiting, exemplary data related to differences in adhesive strength in ETX embryos. It shows graphs of E-cadherin and P-cadherin mRNA expression in cells after downregulation of E- or P-cadherin by RNAi; scrambled siRNA was used as a control. P-cadherin mRNA expression in ES cells after P-cadherin overexpression. N=4 for all conditions. Data are presented as mean ± SD. Statistics were calculated using an unpaired, two-tailed Student's t-test. [Figure 7]Figure 7A shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in the cadherin cord and cortical tension. The time course of the formation of correctly sorted ETX embryos after seeding is shown. 0.5 h: 0 / 515 constructs; 12 h: 79 / 1292 constructs; 24 h: 160 / 1074 constructs; 48 h: 134 / 888 constructs; 72 h: 93 / 702 constructs. N=3 per condition. Data are shown as mean ± SD. Statistics were calculated using unpaired, two-tailed Student's t-test. Figure 7B shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by live cell imaging and tracking. H2B-RFP-XEN, H2B-CFP-ES, and EGFP-TS were overlaid with Imaris cell tracking spheres. Figure 7C shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in cadherin cords and cortical tension. It shows a graph quantifying the mobility of various cell types during self-organization. Data are presented as mean ± SD at different time points. Figure 7D shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in cadherin cords and cortical tension. It shows a bar graph depicting the average mobility of different cell types during self-organization at different time ranges after cell seeding. Data are presented as mean ± SEM. For quantification, 12 structures were imaged from three independent experiments. Statistics were calculated using an unpaired, two-tailed Student's t-test. Figure 7E shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in cadherin cords and cortical tension. It shows exemplary images of structures made from a small number of XEN cells (control) and a large number of XEN cells stained on days 1 and 3. Experiments were repeated three times. Scale bar, 10 μm. Figure 7F shows non-limiting, exemplary data relating to differences in cadherin cords and cortical tension controlling self-organization in ETX embryos, showing a schematic representation of the morphological transitions with low versus high numbers of XEN cells.Figure 7G shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in cadherin cords and cortical tension. It shows a graph of cortical stiffness measurements of the indicated cell types before and after treatment with blebbistatin (Bleb). Total number of cells measured in each condition: ES: n=58; ES+Bleb: n=34; TS: n=68; TS+Bleb: n=31; XEN: n=68; XEN+Bleb: n=35. Data are shown as mean ± SD. Statistics were calculated using ANOVA and multiple comparison tests. Figure 7H shows non-limiting, exemplary data relating to the regulation of self-organization in ETX embryos by differences in cadherin cords and cortical tension. It shows data from an experiment in which day 3 successfully selected ETX embryos were cultured with either blebbistatin, cytochalasin D, or DMSO (control) for 24 hours during the consolidation phase and immunostained to reveal the indicated markers. Quantification shows the percentage of disorganized ETX structures. n = 84 (Bleb-treated), n = 83 (Cyto D-treated), n = 75 (control), N = 3 for each condition. Data are shown as mean ± SD. Statistics were calculated using unpaired, two-tailed Student's t-test. Scale bar, 100 μm. Figure 7I shows non-limiting, exemplary data relating differences in cadherin cords and cortical tension to regulate self-organization in ETX embryos, showing CPM modeling demonstrating the effect of XEN cell stiffness (λP) on externalization efficiency (N = 474). Sorting efficiency calculated at each time point was plotted as a heatmap and overlaid with contour lines (dotted lines). [Figure 8]Figure 8A shows non-limiting, exemplary data related to cadherin heterogeneity within the same cell population in ETX embryos. A pie chart shows the different ETX embryos missorted under the indicated conditions. n = 4186 (control), n = 2940 (Cdh1-KD ES), n = 2471 (Cdh3-KD ES), n = 2407 (Cdh1-KD TS), and n = 2151 (Cdh3-KD TS) constructs were collected from three independent experiments for quantification. Figure 8B shows non-limiting, exemplary data related to cadherin heterogeneity within the same cell population in ETX embryos. It shows exemplary images of immunostaining of ES cells (top) and TS cells (bottom) to reveal E-cadherin and P-cadherin, respectively. Nuclei were stained with DAPI. The scale bar represents 100 µm. The zoomed image shows the area indicated by the dashed line. The experiment was repeated five times. Figure 8C shows non-limiting, exemplary data related to cadherin heterogeneity within the same cell population in ETX embryos, showing flow cytometry analysis of E-cadherin in wild-type ES cells, E-cadherin knockdown ES cells, and E-cadherin overexpressing ES cells (top). Flow cytometry analysis of P-cadherin in wild-type TS cells, P-cadherin knockdown TS cells, and P-cadherin overexpressing TS cells (bottom). CV (coefficient of variation) values ​​are shown relative to the peak value of the plot. Figure 8D shows non-limiting, exemplary data related to cadherin heterogeneity within the same cell population in ETX embryos, showing the indicated FACS profiles. Top: FACS profiles of E-cadherin in E-cadherin KD, WT, and E-cadherin OE ES cells. Bottom: FACS profiles of P-cadherin in P-cadherin KD, WT, and P-cadherin OE TS cells. [Figure 9]Figure 9A shows non-limiting, exemplary data demonstrating that cadherin heterogeneity affects cell positioning in ETX embryos. It shows a schematic diagram and representative images of day 4 ETX embryos constructed from TS cells overexpressing (OE) or knocking down (KD) the indicated cadherins. The experiment was repeated six times. The scale bar represents 100 μm. P-cadherin-overexpressing TS cells and E-cadherin-overexpressing ES cells were prestained with Hoechst to distinguish them from cadherin-knockdown cells (see bottom right panel). The scale bar represents 40 μm in the zoomed panel. Figure 9B shows non-limiting, exemplary data demonstrating that cadherin heterogeneity affects cell positioning in ETX embryos. It shows a schematic diagram and representative images of day 4 ETX embryos constructed from ES cells overexpressing (OE) or knocking down (KD) the indicated cadherins. The experiment was repeated six times. The scale bar represents 100 μm. P-cadherin-overexpressing TS cells and E-cadherin-overexpressing ES cells were prestained with Hoechst to distinguish them from cadherin-knockdown cells (see bottom right panel). The scale bar represents 40 μm in the zoomed panels. Figure 9C shows non-limiting, exemplary data demonstrating that cadherin heterogeneity affects cell positioning in ETX embryos. It shows exemplary images of constructs made from E-cadherin-OE-ES cells, P-cadherin-OE-TS cells, and XEN cells stained at different time points to reveal ES cells (Oct4), TS cells (Tfap2c), and XEN cells (Gata4). The scale bar represents 100 μm. Figure 9D shows non-limiting, exemplary data demonstrating that cadherin heterogeneity affects cell positioning in ETX embryos. It shows a graph quantifying the time course of formation of correctly sorted ETX embryos after seeding. Control: 12h: 24 / 332 constructs; 24h: 83 / 531 constructs; 48h: 71 / 448 constructs; 72h: 51 / 378 constructs. Cadherin OE: 12h: 80 / 276 constructs, 24h: 139 / 385 constructs, 48h: 136 / 374 constructs, 72h: 151 / 455 constructs. N = 3 for all conditions. Data are shown as mean ± SD.Statistics were calculated by unpaired two-tailed Student's t-test. P values ​​indicate significance between control and cadherin OE ETX at the same time point. [Figure 10]Figure 10A provides non-limiting, exemplary data demonstrating that proper cell sorting and self-organization are necessary for proper morphogenesis. The figures show a comparison and quantification of cavity formation in constructs containing missorted ES or TS cells. Good sorted constructs: n=73; missorted ES constructs: n=103; missorted TS constructs: n=109. Missorted XEN constructs: n=57. N=3 for each condition. Scale bar, 100 μm. Statistics calculated by unpaired, two-tailed Student's t-test. Figure 10B provides non-limiting, exemplary data demonstrating that proper cell sorting and self-organization are necessary for proper morphogenesis. The figures show a comparison and quantification of cavity formation in constructs containing missorted XEN cells. Good sorted constructs: n=73; missorted ES constructs: n=103; missorted TS constructs: n=109. Missorted XEN constructs: n=57. N=3 for each condition. Scale bar, 100 μm. Statistics calculated by unpaired, two-tailed Student's t-test. Figure 10C provides non-limiting, exemplary data demonstrating that proper cell sorting and self-organization are necessary for proper morphogenesis. The average lengths of cadherin-OE ETX and control ETX structures are shown at different time points. 20-30 structures were collected at each time point. Data are shown as mean ± SD. Statistics calculated by unpaired, two-tailed Student's t-test. P values ​​indicate significant differences between cadherin-OE and control ETX structures at the same time point. Figure 10D provides non-limiting, exemplary data demonstrating that proper cell sorting and self-organization are necessary for proper morphogenesis. The internal cavity sizes of cadherin-OE ETX and control ETX structures are shown at different time points. 20-30 structures were collected at each time point. Data are shown as mean ± SD. Statistics calculated by unpaired, two-tailed Student's t-test. P values ​​indicate significant differences between cadherin-OE and control ETX structures at the same time point. Figure 10E provides non-limiting, exemplary data demonstrating that correct cell sorting and self-organization are necessary for proper morphogenesis, showing the comparison and quantification of basement membrane formation in constructs containing well-sorted and missorted XEN.Successfully sorted constructs: n = 84; incorrectly sorted XEN constructs: n = 74. N = 3 for each condition. Data are shown as mean ± SD. Statistics were calculated by unpaired, two-tailed Student's t-test. Scale bar, 100 μm. Figure 10F provides non-limiting, exemplary data demonstrating that correct cell sorting and self-organization are necessary for proper morphogenesis. It shows a schematic image demonstrating that natural and ETX embryos use different pathways to form postimplantation embryos. In ETX embryos, lineage-specific stem cells bypass the blastocyst structure and directly build postimplantation embryos. [Figure 11] FIG. 11 shows the time course of development of synthetic embryos formed from wild-type ES cells, TS cells and XEN cells. DETAILED DESCRIPTION OF THE INVENTION

[0022] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like elements unless 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 herein. It will be readily understood that aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated and made part of the disclosure herein.

[0023] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety for relevant art.

[0024] Disclosed herein is a method for producing a synthetic embryo in vitro. In some embodiments, the method includes: (a) providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); (b) providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); (c) providing a plurality of extraembryonic (XEN) cells; and (d) contacting the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture; the plurality of engineered ESCs and their derivatives, the plurality of engineered TSCs and their derivatives, and the plurality of XEN cells and their derivatives organize to form a synthetic embryo, the synthetic embryo comprising one TS-derived compartment and one ES-derived compartment, and covered by an outer XEN-derived monolayer.

[0025] Disclosed herein are methods for investigating mechanisms involved in embryogenesis. Disclosed herein are methods for identifying compounds useful for treating disease. In some embodiments, the methods include contacting a synthetic embryo or a differentiated cell obtained by any of the methods provided herein with a compound. Disclosed herein are methods for diagnosing or treating a disease or disorder in a subject. In some embodiments, the methods include using a synthetic embryo or a differentiated cell obtained by the methods provided herein, or any combination thereof. Also provided are methods for elucidating the role of genes in embryonic development. In some embodiments, the methods include obtaining ESCs, TSCs, and / or XEN cells in which genes have been modified or knocked out, and culturing the cells to obtain a plurality of cells for use in the methods for generating synthetic embryos disclosed herein.

[0026] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, 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 the purposes of this disclosure, the following terms are defined below.

[0027] As used herein, the term "about" when referring to measurable values ​​such as compound amount, dose, time, and temperature is meant to encompass variations of 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount.

[0028] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as neurons. A differentiated cell is one that occupies a more specialized ("committed") position within a cellular lineage. When applied to the differentiation process, the term "committed" refers to a cell that, under normal circumstances, will continue to differentiate into a specific cell type or subset of cell types and has progressed along the differentiation pathway to a 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 cell's inheritance, i.e., what cells it originates from and what cells it can give rise to. The lineage of a cell places the cell within the genetic scheme of development and differentiation. As used herein, a "lineage-specific marker" refers to a feature specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of uncommitted cells into a lineage of interest.

[0029] As used herein, "marker," "lineage marker," or "lineage-specific marker" can refer to a nucleic acid molecule or polypeptide molecule that is differentially expressed in cells of interest. Differential expression can mean increased levels of a positive marker and decreased levels of a negative marker compared to undifferentiated cells. Detectable levels of the marker nucleic acid or polypeptide are sufficiently high or low in cells of interest compared to other cells so that cells of interest can be identified and distinguished from other cells using any of a variety of methods known in the art. In some embodiments, the marker can be enriched. The term "enriched," as used herein, shall have its ordinary meaning and may also refer to a statistically significant increase in the level of a gene product (e.g., mRNA and / or protein) in one condition compared to another (e.g., in one cell layer compared to another).

[0030] As used herein, the term "concentration" shall have its ordinary meaning and may also refer to (a) mass concentration, molar concentration, volume concentration, mass fraction, mole fraction, or volume fraction, or (b) the ratio of the mass or volume of one component in a mixture or solution to the mass or volume of another component in the mixture or solution (e.g., ng / mL). In some embodiments, concentration may refer to the ratio of activity units per volume (e.g., U / mL).

[0031] As used herein, the term "analog" refers to a compound that may be structurally related to a related molecule. As used herein, the term "agonist" may refer to a compound that may not be structurally related to a related molecule. For example, an agonist can activate a related receptor by changing the conformation of the receptor. In any case, these terms are used herein to refer to compounds or molecules that can mimic, reproduce, or otherwise generally replace the specific biological activity of a related molecule.

[0032] As used herein, the terms "culture medium" and "culture medium" can be used interchangeably and refer to a liquid substance used to support the growth and development of stem cells and embryos. The culture medium used in accordance with some embodiments of the present invention can be an aqueous culture 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.

[0033] Method for producing synthetic embryos Previous systems for constructing synthetic embryos from large numbers of stem cells are inefficient. Only approximately 10–15% of synthetic embryos self-assembled from three constituent stem cell lines (ES, TS, and XEN / or induced XEN by Gata4 / 6 or Sox17 induction in ES) possess the correct topology of a 5-day-old natural embryo: distinct EPI and TE compartments completely surrounded by a VE. The mechanisms underlying this success rate are of interest, for example, for optimizing the efficiency of constructing correct synthetic embryos and for discovering the fundamental principles of embryo self-assembly and how they fail in synthetic and natural embryos.

[0034] As disclosed herein, differences in cadherin expression and tension between different types of stem cells can be exploited to optimize the efficiency of self-organization. By modulating the expression of adhesion and tension-regulating molecules or transcription factors that define stem cell identity, the efficiency of synthetic embryo formation can be increased from 10-15% to 35-45%. This disclosure reveals the genetic and biophysical principles underlying the proper formation of synthetic postimplantation embryos in preparation for gastrulation and neurulation. In some embodiments, this strategy can also be applied to increase the efficiency of formation of other types of organoids. Those skilled in the art will appreciate the potential applications and uses of the disclosed methods, including some non-limiting examples provided below.

[0035] Without being bound by any particular theory, embryonic construction is controlled by instructions inherent in the embryo (e.g., "self-assembly"). As described herein, self-organization of many cellular systems relies on the formation of distinct cell-cell contacts, which in turn rely on differential adhesion via cadherin molecules and / or differential cortical tension due to rearrangements of the actin cortical network. Indeed, synthetic genetic programs controlling the expression of distinct cadherins, which have revealed fundamental principles of self-organization, can direct the formation of custom multicellular structures. However, the principles operating in the self-assembly of embryonic structures in vitro have not previously been explored.

[0036] In some embodiments, given that self-organization is a major bottleneck in the construction of efficient multicellular embryos and organoids, applying knowledge of this stage (e.g., using the methods disclosed herein) to construct more robust embryonic models of early development or different types of organoids offers the potential to transform research in regenerative medicine and drug discovery, particularly research into creating stem cell models for early human development.

[0037] Furthermore, as an improvement over previous screens in organoids, the stem cell embryo methodology provided herein can be applied toward multiple translational goals, including, but not limited to, improving the efficiency of generating other types of organoids and identifying genetic and pharmacological modulators of regulatory mechanisms of embryonic and organ development.

[0038] In some embodiments, a comprehensive understanding of the mechanisms of cell sorting, tissue segregation, and morphogenesis during this critical developmental stage can shed light on pathological changes that lead to embryonic lethality and congenital malformations. Furthermore, because many of these processes are impaired in diseases such as cancer and fibrosis, the cellular mechanisms elucidated using the disclosed methods may be relevant to disease biology.

[0039] In some embodiments, identifying the principles of self-organization in natural and stem cell mammalian embryos will have important translational value by helping to uncover regulatory pathways in natural development that allow mammalian embryos to "correct their mistakes."

[0040] In some embodiments, knowledge gained from studying mammalian (e.g., mouse) stem cell embryo models will lead to the successful construction of corresponding human stem cell embryo models by modulating differential cadherin expression and cortical tension between different cell types. Such experimental models will be valuable in studying mechanisms relevant to early human development and disease. The tractable nature of these in vitro model systems will enable detailed investigation of many aspects of human embryonic development.

[0041] Disclosed herein are methods for producing a synthetic embryo in vitro. In some embodiments, the method includes: (a) providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); (b) providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); (c) providing a plurality of extraembryonic (XEN) cells; and (d) contacting the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture; wherein the plurality of engineered ESCs and their derivatives, the plurality of engineered TSCs and their derivatives, and the plurality of XEN cells and their derivatives organize to form a synthetic embryo, the synthetic embryo comprising one TS-derived compartment and one ES-derived compartment, and covered by an outer XEN-derived monolayer.

[0042] Cadherin Cadherins (named for "calcium-dependent adhesion") are cell adhesion molecules that are important for the formation of adherens junctions that connect cells together. Cadherins are a type 1 transmembrane protein that require calcium (Ca) for their function. 2+ ) ions, hence the name. Cell-cell adhesion is mediated by the extracellular cadherin domain, while the intracellular cytoplasmic tail binds many adaptor and signaling proteins. The cadherin superfamily includes classical cadherins, desmogleins, desmocollins, and protocadherins, and these proteins function in a variety of processes, including but not limited to cell adhesion and morphogenesis.

[0043] The Cdh1 gene encodes E-cadherin, a classical cadherin of the cadheric superfamily. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed to generate the mature glycoprotein. This calcium-dependent cell-cell adhesion protein contains five extracellular cadherin repeats, a transmembrane domain, and a highly conserved cytoplasmic tail. Mutations in this gene are associated with gastric, breast, colorectal, thyroid, and ovarian cancers. Loss of function of this gene is thought to contribute to cancer progression by increasing proliferation, invasion, and / or metastasis. The ectodomain of this protein mediates bacterial adhesion to mammalian cells, while the cytoplasmic domain is required for internalization. This gene is located in a gene cluster with other members of the cadherin family on chromosome 16. Its NCBI Gene ID is 999, its Ensemble ID is ENSG00000039068, and its UniProt ID is P12830.

[0044] The Cdh3 gene encodes P-cadherin, a classical cadherin of the cadherin superfamily. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed to generate the mature glycoprotein. This calcium-dependent cell-cell adhesion protein contains five extracellular cadherin repeats, a transmembrane domain, and a highly conserved cytoplasmic tail. This gene is located in a gene cluster on the long arm of chromosome 16 and is involved in loss of heterozygosity events in breast and prostate cancer. Furthermore, aberrant expression of this protein is observed in cervical adenocarcinoma. Mutations in this gene are associated with hypotrichosis and ectodermal dysplasia, ectrodactyly, and macular dystrophy syndrome with juvenile macular dystrophy (EEMS). NCBI gene ID is 1001. Ensemble ID is ENSG00000062038, and UniProt ID is P22223.

[0045] The Cdh6 gene encodes K-cadherin, a type II cadherin that may play a role in kidney development and the formation of the endometrium and placenta. Decreased expression of this gene may be associated with tumor growth and metastasis. Its NCBI gene ID is 1004, its Ensemble ID is ENSG00000113361, and its UniProt ID is P55285.

[0046] Stem cells, embryonic development, mammalian development In some embodiments, the methods disclosed herein do not include an in vivo step. In some embodiments, none of the ESCs, TSCs, XEN cells, multicellular aggregates, and synthetic embryos are present in an in vivo environment during any of the culture steps disclosed herein. The in vivo environment may include a tissue, an organ, an organism, or a combination thereof.

[0047] Disclosed herein is a method for producing a synthetic embryo in vitro. In some embodiments, the method includes: (a) providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); (b) providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); (c) providing a plurality of extraembryonic (XEN) cells; and (d) contacting the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture; wherein the plurality of engineered ESCs and their derivatives, the plurality of engineered TSCs and their derivatives, and the plurality of XEN cells and their derivatives organize to form a synthetic embryo, the synthetic embryo comprising one TS-derived compartment and one ES-derived compartment, and covered by an outer XEN-derived monolayer. The method may include (e) about 3 days after the contacting step (d), replacing the first medium with a second medium.

[0048] Mammalian embryonic development shares some characteristics with all species, but it will be appreciated that different mammalian species develop in different ways and at different rates. Generally, however, the fertilized egg undergoes several division steps (passing through the 2-cell, 4-cell, and 8-cell stages) before compacting to form a solid ball of cells called a morula, within which cells continue to divide. Eventually, the inner cells of the morula become the inner cell mass, and the outer cells become the trophectoderm. The morula then develops into a blastocyst, which is surrounded by trophectoderm and contains a fluid-filled vesicle with an inner cell mass at one end.

[0049] As used herein, the term "embryo" refers to a mammalian organism from the single-cell stage. The embryos described herein are produced by in vitro culture from stem cells under appropriate conditions and resemble natural embryos of corresponding stages generated in vivo, such as having similar morphology, length, weight, cell type composition, and expression of developmental marker genes. Embryonic developmental stages can be defined by the development of specific structures and can be used to define equivalent stages in the development of other species. In some embodiments, embryonic developmental stages can be defined according to the Carnegie Stages of Development, a standardized system used to provide a unified developmental chronology of vertebrate embryos.

[0050] [Table 1]

[0051] In some embodiments, the mammalian embryos produced herein are mouse embryos. Theiler established the numbered stages of mouse development. The earliest stages applicable to (C57BLxCBA)F1 mice are listed in the "emouse digital atlas" (www.emouseatlas.org) as shown in Table 2 below.

[0052] [Table 2-1] [Table 2-2]

[0053] The developmental stage of the synthetic embryo produced herein can be defined according to its embryonic day. 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 its counterpart in vivo (in the fallopian tube or uterus) at a specified day after fertilization, with E0 being considered a fertilized egg.

[0054] In some embodiments, the methods and compositions described herein allow for culture up to or through Theiler developmental 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 developmental stages (a), 5(b), 5(c), 6, 7, 8, 9, and beyond, and corresponding developmental stages in other species (e.g., post-implantation stages). In some embodiments, the synthetic embryos produced herein can reach at least E4, E4.5, E5, E5.5, E6, E6.5, and beyond (e.g., post-implantation stages).

[0055] The methods and compositions described herein can be applied to embryos derived from any suitable mammalian species, such as primates, including humans, apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, pigs, hamsters); cats; dogs; lagomorphs (e.g., rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or livestock mammals. The methods and compositions described herein can be applied to embryos derived from any non-human mammal, including, but not limited to, those described above. Thus, any of the embodiments of the culture medium defined herein may be capable of supporting the development of non-human mammalian embryos on a substrate from pre-implantation to post-implantation developmental stages.

[0056] The term "preimplantation stage" can be used herein to refer to a developmental stage earlier than Theiler's developmental stage 7, Carnegie's developmental stage 5(a), and corresponding stages in other species. As used herein, the term "postimplantation stage" can refer to a developmental stage later than Theiler's developmental stage 7, Carnegie's developmental stage 5(a), and corresponding stages in other species. The "postimplantation stage" can be determined by detecting upregulation of one or more genes by the embryo.

[0057] As used herein, the term "stem cell" refers to a cell that can differentiate into one or more differentiated cell types. Stem cells may be totipotent cells. Stem cells may be pluripotent cells. Totipotent stem cells typically have the capacity to differentiate into all cell types. Totipotent stem cells typically are derived from embryos. As used herein, the term "progenitor cell" refers to a cell that is committed to a specific cell lineage and produces a specific, limited range of differentiated cell types through a series of cell divisions. An example of a progenitor cell is a myoblast, which has the capacity to differentiate into only one type of cell, but is not itself fully mature or fully differentiated.

[0058] Examples of stem cells include: pluripotent embryonic stem (ES) cells derived from fertilized eggs or cloned embryos; epiblast stem cells; trophoblast stem cells; extraembryonic endoderm (XEN) stem cells; somatic stem cells and pluripotent stem cells present in tissues in vivo, such as hepatic stem cells, dermal stem cells, and germline stem cells that form the basis of each tissue; pluripotent stem cells derived from germline stem cells; pluripotent stem cells obtained by nuclear reprogramming of somatic cells; totipotent stem cells and non-totipotent stem cells. Partially committed stem cells, such as progenitor cells, can also be cultured using the media described herein and according to the methods described herein.

[0059] The methods and compositions described herein can be applied to stem cells derived from any suitable mammalian species, such as primates, including humans, apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (e.g., rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or livestock mammals. The methods and compositions described herein can be applied to stem cells derived from any non-human mammal, including, but not limited to, those described above. In some embodiments, the non-human mammal is a rodent.

[0060] As used herein, the term "pluripotent stem cells" (PSCs) can refer to cells that can differentiate into several different terminally differentiated cell types. Pluripotent stem cells can be derived from a variety of tissues or organ systems, including, but not limited to, blood, nerve, cardiac and skeletal muscle, skin, intestine, bone, kidney, liver, pancreas, and thymus. In some embodiments, PSCs can be cultured in vitro and have the potential to differentiate into all cells except the placenta. Pluripotent stem cells can 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).

[0061] PSCs can be obtained from fertilized eggs, cloned embryos, germline stem cells, or stem cells in tissues. Also included are cells with pluripotency similar to that of embryonic stem cells, artificially conferred by introducing several different genes into somatic cells (also called induced pluripotent stem cells or iPS cells). Induced pluripotent stem cells can be derived from any suitable source (e.g., hair follicles, skin cells, fibroblasts, etc.). Pluripotent stem cells can be prepared by methods known in the art. Any of the stem cells defined herein may be derived from diseased or non-diseased tissue. Stem cells can be derived from any suitable mammalian species, such as primates, including humans, apes (e.g., gorillas, chimpanzees, or orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, and hamsters); cats; dogs; lagomorphs (including rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or livestock mammals. The presently disclosed methods can be applied to stem cells derived from any non-human mammal, including but not limited to those mentioned above.

[0062] In some embodiments, the PSC cells disclosed herein are mammalian embryonic stem cells (ESCs). As used herein, the term "embryonic stem cells" (ES cells) refers to pluripotent stem cells derived from the inner cell mass of the blastocyst, an early preimplantation embryo. Such cells are expected to express genes involved in the naive pluripotency network (e.g., Oct4 / Nanog, Sox2, Klf4, etc.). Such cells may also have Oct4 proximal enhancer activity. They may contribute to all embryonic tissues of the chimera. ES cells may be derived from mammalian embryos, from iPS cells, or from suitable cell lines. Non-limiting examples of the stem cells include embryonic stem cells of mammals and the like established by culturing early preimplantation embryos, embryonic stem cells established by culturing early embryos prepared by nuclear transfer of somatic cell nuclei, induced pluripotent stem cells (iPS cells) established by introducing several different transcription factors into somatic cells, and pluripotent stem cells prepared by modifying genes on the chromosomes of embryonic stem cells or iPS cells using genetic engineering techniques. More specifically, embryonic stem cells include embryonic stem cells established from the inner cell mass that constitutes the early embryo, embryonic stem cells established from primordial germ cells, cells isolated from a pluripotent cell population of an early preimplantation embryo (e.g., primitive ectoderm), and cells obtained by culturing these cells.

[0063] As will be appreciated by those skilled in the art, ES cells can be obtained from stem cell banks such as the UK Stem Cell Bank. The Jackson Laboratory in the United States (which provides Jax mice) also stores and obtains mouse ES cells, which are available for purchase. Preferably, the ES cells are obtained or obtainable by methods that do not involve the destruction of human or non-human animal embryos.

[0064] As used herein, the term "trophoblast stem cells" (TS) refers to stem cells derived from the trophoblast lineage of the embryo. Trophoblast stem cells are preferably not extraembryonic cells derived from the two cell types that are precursors of the human placenta: cytotrophoblasts and syncytiotrophoblasts. These cells can be derived from late preimplantation stages (E4.5) or early postimplantation stages (E5.5), but the resulting cell lineage is equivalent to the stem cell compartment present in the extraembryonic ectoderm of the mouse egg cylinder after implantation. Transcription factors such as Elf5, Eomes, and Tfap2C signal this lineage. TS cells are also considered to be cells that are precursors to differentiated cells of the placenta. In mice, TS cells can be derived from either the polar trophectoderm of the blastocyst or the product of the extraembryonic ectoderm that develops from the polar trophectoderm after implantation.

[0065] As used herein, the term "extraembryonic endodermal stem cells" (XEN stem cells) refers to stem cells derived from the extraembryonic endoderm of an embryo (e.g., a mouse embryo). Extraembryonic endoderm is a derivative of hypoblast cells that typically migrate to the blastocyst cavity (beginning on day 8 of human embryonic development), line the blastocyst cavity, and give rise to the primitive and definitive yolk sac. The extraembryonic endoderm fills the remaining cavity of the blastocyst. In some embodiments, XEN stem cells as used herein include inducible XEN stem cells that can express GATA transcription factors upon induction (e.g., by doxycycline treatment). In some embodiments, XEN cells are also derived from ESCs by overexpression of PrE-specific genes, GATA transcription factors (e.g., Gata4 / 6), or Sox J 7, or treatment with growth factors. In some embodiments, XEN stem cells as used herein are inducible XEN stem cells that can express GATA4 upon induction.

[0066] synthetic embryos Disclosed herein is a method for producing a synthetic embryo in vitro. In some embodiments, the method includes: (a) providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); (b) providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); (c) providing a plurality of extraembryonic (XEN) cells; and (d) contacting the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture; wherein the plurality of engineered ESCs and their derivatives, the plurality of engineered TSCs and their derivatives, and the plurality of XEN cells and their derivatives organize to form a synthetic embryo, the synthetic embryo comprising one TS-derived compartment and one ES-derived compartment, and covered by an outer XEN-derived monolayer. The method may include (e) about 3 days after the contacting step (d), replacing the first medium with a second medium. The method may include (f) about 1 day after step (e), replacing the second medium with a third medium.

[0067] In some embodiments, the methods disclosed herein advantageously increase the probability and / or frequency of generating "well-sorted" composite embryos. As used herein, the term "well-sorted" is given its ordinary meaning and refers to a multicellular aggregate or composite embryo containing one TS-derived compartment and one ES-derived compartment, partially or completely covered by an outer XEN-derived monolayer. See also, e.g., Figures 1A and 3A.

[0068] In some embodiments, the plurality of engineered ESCs, the plurality of engineered TSCs, and the plurality of XEN cells organize into a multicellular aggregate structure within about 12-24 hours (e.g., about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, or a number or range between any two of these values) from the contacting step. In some embodiments, the multicellular aggregate structure develops into a multicellular aggregate structure comprising one TS-derived compartment and one ES-derived compartment, at least partially covered by an outer XEN-derived monolayer, with about 30% efficiency (e.g., "successfully sorted") about 12 hours after the contacting step. In some embodiments, the multicellular aggregate structures are at least about 30% (e.g., 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%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 14 , 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%, or a numerical value or range between any two of these values). In some embodiments, the multicellular aggregate structure develops into a synthetic embryo (e.g., a "successfully selected" synthetic embryo) with about 40% efficiency at least 3 days after the contacting step.In some embodiments, the multicellular aggregate structures comprise at least about 40% (e.g., 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%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 14 , 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%, or a number or range between any two of these values.

[0069] In some embodiments, the synthetic embryo (e.g., a "good selection" synthetic embryo) develops a single internal cavity with an efficiency of about 90%. In some embodiments, the synthetic embryo (e.g., a "good selection" synthetic embryo) develops a single internal cavity with an efficiency of at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values). In some embodiments, the single internal cavity develops 4-5 days after the contacting step.

[0070] In some embodiments, the multicellular aggregate structures develop into synthetic embryos (e.g., "good selected" synthetic embryos) and contain a single internal cavity with an efficiency of about 40%. In some embodiments, the multicellular aggregate structures develop into synthetic embryos (e.g., "good selected" synthetic embryos) and contain a single internal cavity with an efficiency of at least about 40% (e.g., 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%. %, 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%, or a numerical value or range between any two of these values), into a synthetic embryo (e.g., a "good selected" synthetic embryo) containing a single internal cavity. In some embodiments, a single internal cavity develops 4-5 days after the contacting step.

[0071] In some embodiments, the multicellular aggregate structures develop into synthetic embryos (e.g., "good selection" synthetic embryos) comprising a laminin-containing basement membrane with an efficiency of about 78%. In some embodiments, the multicellular aggregate structures develop into synthetic embryos (e.g., "good selection" synthetic embryos) comprising a laminin-containing basement membrane with an efficiency of at least about 78% (e.g., 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range between any two of these values). In some embodiments, the laminin-containing basement membrane develops 4-5 days after the contacting step.

[0072] In some embodiments, the synthetic embryo (e.g., a "good selection" synthetic embryo) has a length of at least 200 μm about 72 hours after the contacting step. In some embodiments, the synthetic embryo (e.g., a "good selection" synthetic embryo) has a length of about 200 μm to about 500 μm (e.g., about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, or a number or range between any two of these values) about 72 hours after the contacting step. In some embodiments, the synthetic embryo has a length of at least 4×10 about 72 hours after the contacting step. 3 μm 2 In some embodiments, the synthetic embryos (e.g., "good selection" synthetic embryos) have a size of about 6 x 10 about 72 hours after the contacting step. 3 μm 2 ~About 10×10 3 μm 2 (For example, about 6 × 10 3 μm 2 , 6.5×10 3 μm 2 , 7×10 3 μm 2 , 7.5×10 3 μm 2 , 8×10 3 μm 2 , 8.5×10 3 μm 2 , 9×10 3 μm 2 , 9.5×10 3 μm 2 , 10×10 3 μm 2 , or a number or range between any two of these values).

[0073] The TS-derived compartment may contain cells expressing at least one TS cell marker. The at least one TS cell marker may include Tfap2C, EOMES, or both. The ES-derived compartment may contain cells expressing at least one ES cell marker. The at least one ES cell marker may include Oct4. The XEN-derived monolayer may contain cells expressing at least one XEN cell marker. The at least one XEN cell marker may include Gata4, Gata6, or both. In some embodiments, about 3 days after the contacting step, the synthetic embryo resembles an egg cylinder structure. In some embodiments, about 4-5 days after the contacting step, the synthetic embryo resembles a post-implantation embryo structure (see also Table 2).

[0074] Provided herein are methods, compositions, and culture media for modeling mammalian embryonic development by culturing stem cells, including pluripotent stem cells (e.g., embryonic stem cells) and extraembryonic stem cells. The methods, compositions, and culture media disclosed herein are capable of producing synthetic embryos through various developmental stages. In some embodiments, synthetic embryos produced using the methods and compositions described herein can reach post-implantation (e.g., post-implantation, early gastrulation). In some embodiments, synthetic embryos produced herein can reach early gastrulation. In some embodiments, synthetic embryos produced herein can reach late gastrulation. In some embodiments, synthetic embryos produced herein can reach early neurulation. In some embodiments, synthetic embryos produced herein can reach late neurulation. In some embodiments, synthetic embryos produced herein can reach early organogenesis or later.

[0075] Synthetic embryos produced using the methods and culture media described herein can include post-implantation embryos, e.g., pre-implantation gastrulation embryo structures. As used herein, the term "post-implantation pre-gastrulation" in the context of mammalian embryos (e.g., mouse embryos) refers to embryos after the implantation blastocyst stage and before early gastrulation, characterized by an egg cylinder shape before symmetry is broken. Post-implantation pre-gastrulation embryos can be defined as Theiler developmental stages TS7-TS8 (see also Table 2). In some embodiments, post-implantation pre-gastrulation stages refer to E4.5-6.5, optionally E4.5-6, optionally E5-6.5, and optionally E5-5.5. In some embodiments, post-implantation pre-gastrulation stages refer to E5.5.

[0076] The embryonic stage of synthetic embryos generated using the methods and culture media disclosed herein can be assessed by comparing them with their in vivo counterparts 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 as further described in the Examples section. In some embodiments, the post-implantation pre-gastrulation embryonic structure resembles the E5.5 natural embryonic structure. In some embodiments, the post-implantation pre-gastrulation embryonic structure and the E5.5 natural embryonic structure have similar morphology, cell type composition, and gene expression characteristics. In some embodiments, post-implantation, pre-gastrulation embryonic structures comprise a hollowed-out epithelial embryonic stem (ES) cell and trophoblast stem (TS) cell compartment surrounded by a VE-like layer.

[0077] In some embodiments, synthetic embryos produced using the methods and culture media described herein comprise post-implantation embryos, e.g., early-gastrulation embryo structures. As used herein, the term "gastrulation" in the context of embryos refers to embryos after the expanded blastocyst stage and before the somitogenesis stage, characterized by the formation of a primitive streak and an epithelial-mesenchymal transition, which form the three germ layers. The gastrulation process is generally considered to be the process by which a bilayered germinal disc transforms into a tripartite disc, as endoderm and mesoderm emerge between the ectoderm and endoderm. As used herein, the term "early gastrulation" in the context of mammalian embryos (e.g., mouse embryos) refers to embryos after pre-gastrulation and before late gastrulation, characterized by an egg cylinder shape with a posterior primitive streak. Early-gastrulation embryos can be defined as Theiler's developmental stages TS8-TS10 (see Table 2). In some embodiments, early gastrulation refers to E5-7.75, optionally E5-6.5, optionally E6.25-7.25, optionally E6.5-7.75, optionally E6.5-7.5. In some embodiments, early gastrulation refers to E6.5-E7.

[0078] In some embodiments, the early gastrulation embryonic structure comprises a primitive amniotic cavity (resulting from the merging of the ES cell and TS cell compartment cavities), a fully migrated AVE (the boundary between the ES cell and TS cell compartments), and gastrulation, which can be manifested by epithelial-mesenchymal transition and the formation of a cell layer between the ES cell and VE-like layers.

[0079] In some embodiments, culturing the synthetic embryo from post-implantation pre-gastrulation to early gastrulation is achieved by culturing the embryo structure in a culture medium for an appropriate period of time. In some embodiments, culturing the embryo structure from post-implantation pre-gastrulation to early gastrulation is performed for at least 1 day (e.g., 1 day, 2 days, or 3 days). In some embodiments, culturing is from E5.5 to E6.6.

[0080] In some embodiments, gastrulation embryo structures produced using the methods and culture media described herein resemble naturally occurring gastrulation embryos. In some embodiments, synthetic and naturally occurring gastrulation embryos have similar morphology, cell type composition, and gene expression characteristics. In some embodiments, culturing post-implantation pre-gastrulation embryo structures to their early gastrulation stage is continued to allow the post-implantation embryo structures to develop to late gastrulation or complete gastrulation. As used herein, the term "late gastrulation" in the context of mammalian embryos (e.g., mouse embryos) refers to embryos after early gastrulation and before the early somite stage, characterized by an egg-cylindrical embryo with differentiated, defined endoderm, mesoderm, and ectoderm layers. Late gastrulation embryos can be defined as Theiler's developmental stages TS10-TS11 (see also 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 synthetic embryos produced using the methods and culture conditions described herein are mammalian embryos. In some embodiments, the mammalian embryos are non-human embryos, such as mouse embryos or rabbit embryos. In some embodiments, the mammalian embryos are human embryos.

[0082] The embryonic stage of the synthetic embryos described herein can be assessed in comparison with in vivo or natural embryonic counterparts of the same developmental stage by multiple methods, including, but not limited to, morphology, length, body mass, weight, expression of developmental marker genes using specific antibodies or primers, and transcriptional profiling, as further described herein below and in the Examples section. Morphological assessment of embryonic development can be performed according to the Carnegie developmental stages (also see Table 2; Developmental Stages of the Human Embryo (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 embryonic days.

[0083] In some embodiments, one or more developmental markers described herein can be used to assess the developmental stage of a synthetic embryonic structure. Numerous methods exist in the art for detecting the presence, absence, or amount of marker gene products (e.g., mRNA and / or protein), as well as their localization or subcellular localization (e.g., nuclear and / or cytoplasmic) in embryonic structures. Marker expression can be assessed by any of a wide variety of well-known methods for detecting the expression of transcribed molecules or proteins. Non-limiting examples of such methods include immunological methods for detecting secreted proteins, cell surface proteins, cytoplasmic proteins, 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.

[0084] In some embodiments, the activity of a particular gene is characterized by measurements of gene transcripts (e.g., mRNA), by measurements of the amount of translated protein, or by measurements of gene product activity. Marker expression can be monitored in a variety of ways, including detection of mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of gene expression levels (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or can involve qualitative assessment of gene expression levels, particularly comparison with control levels. The type of level detected will be clear from the context.

[0085] In another embodiment, detecting or determining the expression level of a marker and its functionally similar homologs (including fragments thereof or genetic modifications (e.g., in their regulatory or promoter regions)) comprises detecting or determining RNA levels for the marker of interest. In some embodiments, one or more cells can be obtained from a synthetic embryonic structure and RNA is isolated from the cells. In some embodiments, RNA is obtained from a single cell. For example, cells can be isolated from a tissue sample by laser capture microdissection (LCM). This technique allows cells to be isolated from tissue fragments, including stained tissue sections, thereby ensuring that the desired cell is isolated. It is also possible to obtain cells, for example, from synthetic embryonic cells, 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.).

[0086] For example, when isolating RNA from synthetic embryos at various developmental stages and / or from the cells that make up the synthetic embryos, it may be important to prevent further changes in gene expression after removing the tissue or cells from the subject. Expression levels are known to change rapidly after perturbations, such as heat shock or activation with reagents such as lipopolysaccharide (LPS). Furthermore, RNA in tissues and cells can be rapidly degraded. Therefore, in some embodiments, tissues and cells obtained from a subject are flash-frozen as soon as possible.

[0087] RNA can be extracted from cells by various methods, such as guanidium thiocyanate lysis followed by CsCl centrifugation. Methods for obtaining RNA from single cells are also known to those skilled in the art. RNA samples can then be enriched for specific species. In some embodiments, poly(A)+ RNA is isolated from the RNA sample. Such purification typically utilizes poly(A) tails on mRNA. In particular, as described above, poly(T) oligonucleotides can be immobilized on a solid support to serve as affinity ligands for mRNA. Commercially available kits for this purpose include the MessageMaker kit (Life Technologies, Grand Island, NY). In some embodiments, the RNA population is enriched for a marker sequence. Enrichment can be achieved, for example, by primer-directed cDNA synthesis or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription.

[0088] Regardless of whether specific species or sequences are enriched, the RNA population can be further amplified.As defined herein, "amplification process" increases the copy number of polynucleotide (e.g., RNA).For example, when RNA is mRNA, amplification process such as RT-PCR can be used to amplify mRNA so that signal is detectable or detection is enhanced.This type of amplification process is particularly useful when the size or volume of biological sample, tissue sample or tumor sample is small.

[0089] Various amplification and detection methods can be used.For example, reverse transcription of mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, as described in U.S. Patent No. 5,322,770, using a single enzyme for both steps, or reverse transcription of mRNA into 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 method.Real-time PCR can also be used. Other known amplification methods that can be used herein include the so-called "NASBA" or "3 SR" technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (described in G.T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; target-mediated amplification as described in PCT Publication WO 9322461; PCR; ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077). (1988)); self-sustained sequence replication (SSR) (see, e.g., Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)); and transcriptional amplification (see, e.g., Kwoh et al., Proc. 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 suitable for use in the disclosed methods include Northern analysis, RNase protection assays (RPA), microarrays, and PCR-based techniques such as quantitative PCR and differential display PCR. For example, in Northern blotting, an RNA preparation is run on a denaturing agarose gel and transferred to a suitable support, such as activated cellulose, nitrocellulose, or a glass or nylon membrane. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed, and analyzed by autoradiography.

[0090] Visualization by in situ hybridization can also be employed, in which radiolabeled antisense RNA probes are hybridized to thin sections of the sample, washed, cleaved with RNase, and exposed to a sensitive emulsion for autoradiography. The sample can be stained with hematoxylin to reveal the histological composition of the sample, and the developed emulsion is revealed by dark-field imaging with appropriate light filters. Non-radioactive labels, such as digoxigenin, can also be used. In some embodiments, the probe is labeled with a fluorescent moiety.

[0091] Alternatively, mRNA expression can be detected on a DNA array, chip, or microarray. The labeled nucleic acid of a test sample obtained from a subject can be hybridized to a solid surface containing marker DNA. A positive hybridization signal is obtained in a sample containing marker transcripts. Methods for preparing DNA arrays and their use are well known in the art (see, for example, U.S. Patent Nos. 66186796; 6379897; 6664377; 6451536; 548257; U.S. Patent Application Publication No. 2003 / 0157485). SAGE (Serial Analysis of Gene Expression) can also be performed (see, for example, U.S. Patent Application Publication No. 20030215858). In some embodiments, next-generation sequencing (e.g., RNA-seq) can be used to analyze the total mRNA expression from one (e.g., single-cell RNA-seq) or multiple cells. The nucleic acid target molecule labeled with barcode (e.g., origin-specific barcode) can be sequenced by barcode to generate a single read and / or contig that includes both the sequence of the target molecule and the barcode or a part thereof.Exemplary next-generation sequencing technologies include, among others, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing and nanopore sequencing.Methods for constructing sequencing libraries are known in the art.

[0092] Single-cell sequencing can be high-throughput single-cell RNA sequencing. In certain embodiments, single-cell sequencing is low-cost, high-throughput single-cell RNA sequencing. Without being bound by any particular theory, single-cell RNA sequencing can efficiently and cost-effectively sequence thousands to tens of thousands of single cells. In certain embodiments, single-cell RNA sequencing involves pairing single cells in droplets with oligonucleotides for reverse transcription, where the oligonucleotides are configured to provide a cell-origin-specific barcode that uniquely identifies transcripts from each cell and a unique molecular identifier (UMI) that uniquely identifies each transcript. In certain embodiments, single-cell RNA sequencing involves pairing single cells in droplets with single oligonucleotide-coated microparticle beads 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 embodiments of the present disclosure, unbiased classification of cells in a biological sample comprises 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).

[0093] The activity or level of a lineage marker protein can be detected and / or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those skilled in the art. Any method known in the art for detecting a polypeptide can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, conjugate-ligand assay, immunohistochemistry, agglutination, complement assay, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography.

[0094] The following are non-limiting examples of techniques that can be used to detect marker proteins based on the present disclosure and according to the preference of the skilled artisan. 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, and labeled protein A or anti-immunoglobulin ( 125 The expression of the marker protein is then assayed with a secondary immunoreagent such as iodine (I, horseradish peroxidase, alkaline phosphatase, or an appropriate label, including a fluorescent dye). Chromatographic detection can also be used.

[0125] Immunohistochemistry can be used to detect the expression of a marker protein. An appropriate antibody is contacted, for example, with a thin layer of cells, washed, and then contacted with a second, labeled antibody. The label can be a fluorescent marker, peroxidase, an enzyme such as avidin, or a radiolabel. The assay is scored visually using a microscope. Anti-marker protein antibodies, such as intrabodies, can also be used for imaging purposes, for example, to detect the presence of the marker protein in cells or embryos. Suitable labels include radioisotopes, iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112Fluorescent labels include biotin, technetium (mTc), fluorescein, rhodamine, and other fluorescent labels. Antibodies that can be used to detect marker proteins include any antibody, natural or synthetic, full-length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the marker protein to be detected. The phrase "specifically binds" refers, for example, to an antibody binding to an epitope, antigen, or antigenic determinant in such a way that binding can be displaced or competed for by a second preparation of the same or similar epitope, antigen, or antigenic determinant. An antibody may preferentially bind to a marker protein compared to other proteins, such as related proteins.

[0095] Antibodies are commercially available or can be prepared according to methods known in the art. Antibodies and their derivatives that can be used include polyclonal or monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, primatized (CDR-grafted) antibodies, veneered or single-chain antibodies, and functional fragments of antibodies, i.e., marker protein-binding fragments. For example, antibody fragments capable of binding to a marker protein or a portion thereof, including, but not limited to, Fv, Fab, Fab', and F(ab')2 fragments, can be used. Such fragments can be produced by enzymatic cleavage or recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the required substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in various truncated forms using antibody genes with one or more stop codons introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain, and hinge region of the heavy chain. In some embodiments, agents that specifically bind to marker proteins other than antibodies, such as peptides, are used. Peptides that specifically bind to marker proteins can be identified by any means known in the art. For example, peptides that specifically bind to marker proteins can be screened using a peptide phage display library.

[0096] nucleic acid The methods described herein involve providing engineered cells (eg, ESCs or TSCs) that overexpress at least one cadherin.

[0097] Providing a plurality of engineered ESCs may include: (i) providing an expression construct comprising a nucleic acid encoding E-cadherin operably linked to at least one expression control element that enables gene expression in mammalian cells; and (ii) introducing the expression construct into ESCs in a manner that enables expression of the introduced construct in at least one of the ESCs, thereby producing at least one engineered ESC. Providing a plurality of engineered ESCs may include culturing at least one engineered ESC of (ii).

[0098] Providing a plurality of engineered TSCs may include: (i) providing an expression construct comprising a nucleic acid encoding P-cadherin operably linked to at least one expression control element that allows gene expression in mammalian cells; and (ii) introducing the expression construct into TSCs in a manner that allows expression of the introduced construct in at least one of the TSCs, thereby producing at least one engineered TSC. Providing a plurality of engineered TSCs may include culturing at least one engineered TSC of (ii).

[0099] The expression control element may include a promoter, an enhancer, a 5' untranslated region, a 3' untranslated region, or any combination thereof. The promoter may be a ubiquitous promoter. The promoter may be a constitutive promoter or an inducible promoter.

[0100] In some embodiments, at least a portion of the plurality of engineered ESCs overexpress E-cadherin compared to wild-type ESCs. In some embodiments, at least a portion of the plurality of engineered TSCs overexpress P-cadherin compared to wild-type TSCs. The plurality of XEN cells can be wild-type XEN cells. In some embodiments, none of the plurality of XEN cells has been engineered to overexpress E-cadherin, P-cadherin, and K-cadherin.

[0101] In some embodiments, the engineered ESC or plurality of engineered ESCs overexpress E-cadherin (e.g., compared to wild-type) by at least about 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between these values). In some embodiments, the engineered TSC or plurality of engineered TSCs overexpress P-cadherin (e.g., compared to wild-type) by at least about 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 90-fold, 100-fold, or a number or range between these values). In some embodiments, overexpression is determined by any method known in the art, including, for example, quantitative PCR, fluorescence-activated cell sorting (FACS), or Western blot. For example, any of the methods described above for detecting lineage markers can also be used.

[0102] As used herein, "genetic construct" or "construct" shall be given their ordinary meaning and may refer to a nucleic acid comprising a nucleotide sequence encoding a gene product (e.g., RNA and / or protein). The nucleic acid may include at least one regulatory element (e.g., an expression control element) for expression. The nucleic acid may comprise a vector, such as a viral vector. In some embodiments, the vector may include an adenovirus vector, an adeno-associated virus vector, an Epstein-Barr virus vector, a herpesvirus vector, an attenuated HIV vector, a retrovirus vector, a vaccinia virus vector, or any combination thereof. In some embodiments, the vector may include an RNA virus vector. In some embodiments, the vector may be derived from one or more negative-strand RNA viruses of the Mononegavirales order. In some embodiments, the vector may be a rabies virus vector. Many such vectors useful for the introduction of exogenous genes into mammalian cells are available. Vectors may be episomal, e.g., viral vectors such as plasmids, cytomegalovirus, and adenovirus, or retroviral vectors such as MMLV, HIV-1, and ALV, which are integrated into the cellular genome by homologous recombination or random integration. In some embodiments, a combination of a retrovirus and an appropriate packaging cell line may also be used, in which case the capsid protein functions to infect target cells. Retroviral vectors may be "defective," i.e., unable to produce viral proteins necessary for productive infection. Replication of the vector may require propagation in a packaging cell line. The term "vector," as used herein, refers to a nucleic acid construct designed for delivery into a host cell or transfer between different host cells. As used herein, vectors may be viral or non-viral. The term "vector" encompasses any genetic element capable of replication and introducing genetic sequences into cells when associated with appropriate control elements.Vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, viral particles, and the like.

[0103] In addition to viral vectors, various additional tools have been developed that can be used to integrate foreign genes into cells. One such method that can be used to integrate a polynucleotide encoding a target gene into cells involves the use of transposons. Transposons are polynucleotides that encode a transposase enzyme and contain a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered into a cell, expression of the transposase gene begins, producing an active enzyme that excises the gene of interest from the transposon. This activity is mediated by the transposase's site-specific recognition of the transposon's excision sites. In some cases, these excision sites are terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the mammalian cell genome by transposase-catalyzed cleavage of similar excision sites present in the cell's nuclear genome. This allows the gene of interest to be inserted into the complementary excision site in the excised nuclear DNA, followed by covalent phosphodiester bonds linking the gene of interest to the DNA of the mammalian cell genome, completing the integration process. In some cases, transposon is retrotransposon, and the gene encoding target gene is first transcribed into RNA product, and then reverse transcribed into DNA before being integrated into mammalian cell genome.Exemplary transposon systems include piggybac transposon (for example, as described in detail in International Publication No. 2010 / 085699) and sleeping beauty transposon (for example, as described in detail in US Patent Application Publication No. 2005 / 0112764), the disclosure of each of which is incorporated herein by reference.

[0104] As used herein, the term "expression vector" or "construct" refers to a vector that directs the expression of an RNA or polypeptide (e.g., E-cadherin) contained therein from a nucleic acid sequence linked to a transcriptional regulatory sequence on the vector. This sequence may be heterologous to the cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and protein, and optionally, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. A "gene product" includes RNA transcribed from a gene and a polypeptide resulting from translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0105] Integrating vectors contain delivered RNA / DNA that is permanently integrated into the host cell chromosome. Non-integrating vectors remain episomal, meaning that the nucleic acid contained within them is not integrated into the host cell chromosome. Examples of integrating vectors include retroviral vectors, lentiviral vectors, hybrid adenoviral vectors, and herpes simplex virus vectors. An example of a non-integrating vector is a non-integrating viral vector. Non-integrating viral vectors do not integrate their genomes into host DNA, eliminating the risks posed by integrating retroviruses. One example is the Epstein-Barr oriP / nuclear antigen 1 (EBNAl) vector, which is capable of limited autonomous replication and is known to function in mammalian cells. Because it contains two elements derived from the Epstein-Barr virus, oriP and EBNAl, the binding of the EBNAl protein to the viral replicon region oriP allows the plasmid to remain episomal for a relatively long period of time in mammalian cells. This special feature of oriP / EBNAl vectors makes them ideal for generating integration-free iPSCs. Other non-integrating viral vectors are adenovirus vectors and adeno-associated virus (AAV) vectors. Other non-integrating viral vectors contemplated by the present invention are single-stranded, negative-sense RNA viral vectors, such as Sendai virus vectors and rabies virus vectors. Another example of a non-integrating vector is a minicircle vector. A minicircle vector is a circularized vector in which the plasmid backbone is released, leaving only the eukaryotic promoter and cDNA to be expressed. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element derived from a virus and has the ability to be packaged into a viral vector particle. Viral vectors can contain nucleic acids encoding the polypeptides described herein in place of non-essential viral genes. The vectors and / or particles can be used to introduce nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0106] In some embodiments, the vector may contain regulatory sequences that, for example, enable translation of multiple proteins from a single mRNA. Non-limiting examples of such regulatory sequences include an internal ribosome entry site (IRES) and a 2A self-processing sequence. In some embodiments, the 2A sequence is a 2A peptide site (F2A sequence) from foot-and-mouth disease virus. In some embodiments, the F2A sequence has a canonical furin cleavage site. In some embodiments, the vector also contains regulatory control elements known to those skilled in the art that can affect expression of the RNA and / or protein products encoded by the polynucleotide in desired cells of the subject. In some embodiments, functionally, expression of the polynucleotide can be at least partially controlled by operably linked regulatory elements such that the elements regulate transcription of the polynucleotide, transport, processing, and stability of the RNA encoded by the polynucleotide, and, optionally, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' to the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' to the transcribed sequence or within the transcribed sequence. Another example of a regulatory element is a recognition sequence for a microRNA. Other examples of regulatory elements are introns and the splice donor and splice acceptor sequences that regulate the splicing of said introns. Other examples of regulatory elements are transcription termination signals and / or polyadenylation sequences.

[0107] Expression control elements and promoters include those that are active in specific tissues or cell types, and are referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are typically active in specific cells or tissues (e.g., the liver, brain, central nervous system, spinal cord, eye, retina, or lung). Expression control elements are typically active in specific cell, tissue, or organ types because they are recognized by transcriptional activator proteins or other transcriptional regulators specific to these cells, tissues, or organs.

[0108] Expression control elements also include ubiquitous or promiscuous promoters / enhancers that can drive expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in a variety of mammalian cell types; promoter / enhancer sequences of ubiquitously or promiscuous expressed mammalian genes, such as, but not limited to, beta-actin, ubiquitin, or EF1α; or synthetic elements that do not occur in nature.

[0109] Expression control elements can also confer expression in a regulatable manner, i.e., in such a way that expression of an operably linked polynucleotide can be increased or decreased by a signal or stimulus. Regulatable elements that increase expression of an operably linked polynucleotide in response to a signal or stimulus are also referred to as "inducible elements" (i.e., induced by the signal). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. Regulatable elements that decrease expression of an operably linked polynucleotide in response to a signal or stimulus are referred to as "repressive elements" (i.e., the signal decreases expression such that when the signal is removed or absent, expression increases). Typically, the amount of increase or decrease effected by such elements is proportional to the amount of signal or stimulus present: the greater the amount of signal or stimulus, the greater the increase or decrease in expression.

[0110] The promoter can promote ubiquitous or tissue-specific expression of an operably linked nucleic acid (e.g., engineered nucleic acid) sequence from any species, including humans. In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters, known to those skilled in the art, include TDH3, PGK1, PKC1, TDH2, PYK1, TPI1, AT1, CMV, EF1α, SV40, PGK1 (human or mouse), Ubc, human β-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6 (see, for example, the Addgene website: blog.addgene.org / plasmids-101-the-promoter-region).

[0111] Non-limiting examples of ubiquitous promoters include tetracycline-responsive promoters (under relevant conditions), CMV (EF1α), SV40 promoter, PGK1, Ubc, CAG, human β-actin gene promoter, RSV promoter, EFS promoter, and promoters containing an upstream activating sequence (UAS). In certain embodiments, the promoter is a mammalian promoter.

[0112] In some embodiments, the promoters of the present disclosure are suitable for use in AAV vectors. See, for example, U.S. Patent Application Publication No. 2018 / 0155789 (incorporated herein by reference in its entirety for this purpose).

[0113] Non-limiting examples of constitutive promoters include CP1, CMV, EF1α, SV40, PGK1, Ubc, human β-actin, β-tubulin, CAG, Ac5, Rosa26 promoter, COL1A1 promoter, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, U6, red opsin promoter (red promoter), rhodopsin promoter (rho promoter), cone arrestin promoter (car promoter), and rhodopsin kinase promoter (rk promoter). In some examples, the constitutive promoter is the Rosa26 promoter. In some examples, the constitutive promoter is the COL1A1 promoter.

[0114] An "inducible promoter" is characterized by initiating or enhancing transcriptional activity in the presence of, upon influence by, or contact with an inducing agent. The inducing agent can be an endogenous or usually exogenous condition, compound, drug, or protein that contacts an engineered nucleic acid (e.g., an engineered nucleic acid) in such a manner that it is active to induce transcriptional activity from the inducible promoter. In certain embodiments, the inducing agent is a tetracycline-sensitive protein (e.g., tTA or rtTA, a TetR family regulator). Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters, and tetracycline repressor protein (TetR, e.g., SEQ ID NO: 26) or TetRKRAB, e.g., SEQ ID NO: 27), other tetracycline-responsive promoter systems containing tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA), and tetracycline operator sequence (tetO) and reverse tetracycline transactivator fusion protein (rtTA), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from yeast, mouse, and human metallothionein (a protein that binds and sequesters metal ions) genes), pathogenicity-regulated promoters (e.g., promoters induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters.Non-limiting examples of inducible systems using light-regulated promoters are described in Wang et al., Nat. Methods. 2012 Feb. 12; 9(3):266-9. Additional non-limiting examples of inducible promoters include mifepristone-responsive promoters (e.g., GAL4-E1b promoters) and coumamycin-responsive promoters. For example, see Zhao et al., Hum Gene Ther. 2003 Nov. 20; 14(17):1619-29.

[0115] As used herein, a "reverse tetracycline transactivator" ("rtTA") is an inducing agent that can bind to a TRE promoter (e.g., a TRE3G, TRE2, or P-tight promoter) in the presence of tetracycline (e.g., doxycycline) and drive expression of a transgene operably linked to the TRE promoter. rtTAs generally contain a mutant tetracycline repressor DNA-binding protein (TetR) and a transcription activation domain (see, e.g., Gossen et al., Science. 1995 Jun. 23; 268(5218):1766-9 and any of the transactivation domains listed therein). The mutant TetR domain is capable of binding to a TRE promoter when bound to tetracycline. See, e.g., U.S. Provisional Application No. 62 / 738,894, entitled MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, filed September 28, 2018, attorney docket number H0824.70300US00, which is incorporated herein by reference in its entirety.

[0116] Physical methods for introducing polynucleotide into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection and electroporation.Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art.See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY).A preferred method for introducing polynucleotide into cells is calcium phosphate transfection.

[0117] Chemical means for introducing polynucleotides into cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is liposomes (e.g., artificial membrane vesicles). Other cutting-edge targeted delivery methods for nucleic acids are available, such as the delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0118] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for the introduction of nucleic acids into cells. In some embodiments, the nucleic acid may be bound to a lipid. The lipid-bound nucleic acid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule bound to both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, dispersed in a solution containing lipids, contained in or complexed with micelles, or otherwise bound to a lipid. The lipid, lipid / DNA, or lipid / expression vector-bound composition is not limited to a particular structure in solution.

[0119] The nucleic acids described herein can be introduced into cells using any of a number of different methods, for example, commercially available methods including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Massachusetts) or Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated gene transfer, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0120] In some embodiments, non-viral methods can be used to deliver the nucleic acids described herein to cells. In some embodiments, non-viral methods include the use of transposons (also called transposable elements). In some embodiments, a transposon is a piece of DNA that can insert itself into a certain location in a genome, for example, a piece of DNA that can self-replicate and insert its copy into a genome, or a piece of DNA that can be spliced ​​from a longer nucleic acid and inserted into another location in a genome. For example, a transposon comprises a DNA sequence made of inverted repeats flanking a gene for transposition. Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty transposon system (SBTS) and the piggyBac (PB) transposon system. In some embodiments, the engineered products described herein are produced by combining gene insertion using SBTS with gene editing using nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator effector nucleases (TALENs), CRISPR / Cas systems, or engineered meganucleases re-engineered homing endonucleases).

[0121] culture In some embodiments of the methods disclosed herein, the method includes: providing a plurality of engineered embryonic stem cells (ESCs), wherein at least a portion of the plurality of engineered ESCs overexpress E-cadherin (Cdh1); providing a plurality of engineered trophoblast stem cells (TSCs), wherein at least a portion of the plurality of engineered TSCs overexpress P-cadherin (Cdh3); and providing a plurality of extraembryonic (XEN) cells.

[0122] The plurality of engineered ESCs may comprise at least 5,000 ESCs. The plurality of engineered ESCs may comprise 6,000-7,000 ESCs (e.g., about 6,000, about 6,100, about 6,200, about 6,300, about 6,400, about 6,500, about 6,600, about 6,700, about 6,800, about 6,900, about 7,000, or a number or range between any two of these values). The plurality of engineered TSCs may comprise at least 10,000 TSCs. The plurality of engineered TSCs can comprise 15,000 to 19,000 TSCs (e.g., about 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, or a number or range between any two of these values). The plurality of XEN cells can comprise at least 5,000 XEN cells. The plurality of XEN cells can comprise 5,000 to 6,000 XENs (e.g., about 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, or a number or range between any two of these values). The ESCs, TSCs, and / or XEN cells can be derived from natural mammalian embryos. The natural mammalian embryo may be a mouse or a human natural embryo.

[0123] Co-culture can be performed using a physical substrate (e.g., a culture dish or plate). In some embodiments, the culture plate includes one or more wells or microwells. Co-culture can be performed in an inverted pyramidal microwell. The inverted pyramidal microwell can be about 400 μm or about 800 μm in size (or, for example, about 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, or more). The inverted pyramidal microwell can be about 400 μm or about 800 μm in diameter (or, for example, about 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, or more).

[0124] The methods disclosed herein can be applied to any suitable 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, or 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.

[0125] Culture medium Provided herein are culture media (e.g., first, second, and third culture media) for producing the synthetic embryos of the present disclosure. In some embodiments, the method includes contacting a plurality of engineered ESCs, a plurality of engineered TSCs, and a plurality of extraembryonic (XEN) cells with a first culture medium to form a co-culture. The method may include replacing the first culture medium with a second culture medium about 3 days after the contacting step. The first culture medium and the second culture medium may each comprise a basal culture medium. The basal culture medium may include water, salts, amino acids, a carbon source, vitamins, lipids, and a buffering agent. Suitable carbon sources can be determined by those skilled in the art from compounds such as glucose, sucrose, sorbitol, galactose, mannose, fructose, mannitol, maltodextrin, trehalose dihydrate, and cyclodextrin. Basal media are commercially available, for example, under the trade names Advanced DMEM / F12 (Gibco, 12634-010) and CMRL-1066 (Invitrogen or Sigma). Basal culture media may include Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Roswell Memorial Research Institute (RPMI) Medium 1640, Neurobasal®, Neurobasal® A, Connaught Medical Laboratory 1066 (CMRL-1066), or any combination thereof. Basal culture media may include Dulbecco's Modified Eagle's Medium (DMEM), DMEM Nutrient Mixture 12 (DMEM / F12), Roswell Memorial Research Institute (RPMI) Medium 1640, Neurobasal®, Neurobasal® A, Connaught Medical Laboratory 1066 (CMRL-1066), or any combination thereof.

[0126] The first and second culture media may each contain non-human serum or a serum substitute thereof, a reducing agent, and an antibiotic. The non-human serum or serum substitute may include fetal bovine serum, bovine serum albumin, KnockOut™ serum substitute, or any combination thereof. The reducing agent may include β-mercaptoethanol (2-ME), N-acetyl-L-cysteine, dithiothreitol (DTT), or any combination thereof. The antibiotic may include penicillin-streptomycin, amphotericin B, ampicillin, erythromycin, gentamicin, kanamycin, neomycin, nystatin, polymyxin B, tetracycline, thiabendazole, tylosin, or any combination thereof. The first culture medium and the second culture medium may each contain N2 supplement, B27 supplement, insulin-transferrin-selenium-ethanolamine (ITS-X), GlutaMax™, non-essential amino acids, ascorbic acid, sodium pyruvate, or any combination thereof.

[0127] Each component of the culture medium described herein can be present in an amount suitable for the culture medium to support the self-assembly of stem cells into synthetic embryonic structures. Prior to co-culturing as described herein, the stem cells described herein, e.g., a plurality of engineered ES cells, can be individually cultured in a suitable culture medium, e.g., as described in U.S. Patent Application Publication No. 2022 / 0308041 and PCT Publication No. 2023 / 114754 (the contents of which are incorporated herein by reference in their entireties).

[0128] In some embodiments, the amount of one or more components in a solution or medium of the present disclosure may constitute 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 numerical value or range between any two of these values) of volume / medium volume (% v / v), volume / medium weight (% w / v), or weight / medium weight (% w / w).

[0129] Sodium pyruvate can be contained in the culture medium at a concentration of about 0.05 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.25 mM to about 5 mM, or about 0.5 mM to about 2.5 mM, for example, about 1 mM.

[0130] The culture medium of the present disclosure may contain an amino acid selected from the group consisting of L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. Non-essential amino acids may be contained in the culture medium, for example, glycine (about 1 mg / mL to about 25 mg / mL or about 5 mg / mL to about 10 mg / mL, e.g., 7.5 mg / mL), L-alanine (about 1 mg / mL to about 25 mg / mL or about 5 mg / mL to about 10 mg / mL, e.g., about 9 mg / mL), L-asparagine (about 5 mg / mL to about 30 mg / mL or about 10 mg / mL to about 15 mg / mL, e.g., 13.2 mg / mL), L-aspartic acid (about 5 mg / mL to about 30 mg / mL or about 10 mg / mL to about 15 mg / mL, e.g., 13.2 mg / mL), L-aspartic acid (about 5 mg / mL to about 30 mg / mL or about 15 mg / mL to about 15 mg / mL, e.g., 13.2 mg / mL), L-asparagine (about 5 mg / mL to about 30 mg / mL or about 15 ... The active ingredient in the composition of the present invention includes L-glutamic acid (about 5 mg / mL to about 50 mg / mL or about 10 mg / mL to about 20 mg / mL, for example, about 15 mg / mL), L-proline (about 5 mg / mL to about 30 mg / mL or about 10 mg / mL to about 15 mg / mL, for example, about 11 mg / mL), and / or L-serine (about 5 mg / mL to about 30 mg / mL or about 10 mg / mL to about 15 mg / mL, for example, 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. Penicillin may be contained 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, for example, about 25 units / mL. Streptomycin may be contained 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, or 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.

[0131] The culture medium may be serum-free, substantially serum-free, or essentially serum-free. The culture medium may contain a serum replacement medium. Such serum replacement media are commercially available under the trade names KSR (KnockOut™ Serum Replacement, Invitrogen, 10828-010) and N2B27 (e.g., Invitrogen, ME100137L1). The serum replacement medium may be present in the culture medium at about 5% to about 60%, about 10% to about 50%, about 15% to about 45%, or about 20% to about 40%, e.g., about 30%. In some embodiments, the in vitro culture medium is serum-free or substantially serum-free and contains 30% serum replacement. The concentration or amount of one or more components in the solution or medium may vary. For example, the concentration or amount of non-human serum or its serum replacement, reducing agent, and / or antibiotic can be adjusted as needed by those skilled in the art.

[0132] In some embodiments, the amount of non-human serum or serum substitute thereof may constitute 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 numerical value or range between any two of these values) of volume per medium volume (% v / v), volume per medium weight (% w / v), or weight per medium weight (% w / w). In some embodiments, the amount of antibiotic may comprise 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 / medium volume (% v / v), volume / medium weight (% w / v), or weight / medium weight (% w / w). For example, the amount of reducing agent may vary. For example, in some embodiments, the concentration of reducing agent in the composition may be 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). When percentages are given for drugs, ingredients and compounds, they can be % w / w, % w / v or % v / v of the total formulation unless otherwise specified.

[0133] The first culture medium may contain DMEM, FBS, GlutaMax™, 2-ME, non-essential amino acids, sodium pyruvate, HEPES, and penicillin-streptomycin. The first culture medium may contain a ROCK inhibitor. The first culture medium may contain DMEM, 12.5% ​​FBS, 2 mM GlutaMax™, 0.1 mM 2-ME, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.02 M HEPES, 1% penicillin-streptomycin, and 7.5 nM ROCK inhibitor. The first culture medium may contain DMEM, 12.5% ​​FBS, 2 mM GlutaMax™, 0.1 mM 2-ME, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.02 M HEPES, and 1% penicillin-streptomycin.

[0134] The first culture medium in step (d) may contain a ROCK inhibitor. In some embodiments, the first culture medium does not contain a ROCK inhibitor. The method may include removing the ROCK inhibitor after about 24 hours of co-culture in the first culture medium. Rho-associated protein kinase (e.g., ROCK) inhibitors include N-[(1S)-2-hydroxy-1-phenylethyl]-N'-[4-(4-pyridinyl)phenyl]-urea (AS 1892802), fasudil hydrochloride (HA), and the like. 1077), -[3-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide (GSK269962), 4-[4-(trifluoromethyl)phenyl]-N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide (GSK 429286), (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H 1152 dihydrochloride), (S)-(+)-4-glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (glycyl-H 1152 dihydrochloride), N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 dihydrochloride), (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-Benzodioxin-2-carboxamide dihydrochloride (SR 3677 dihydrochloride) and trans-4-[(R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (thiazovivin), 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-1 (B1), Rock-1 (C-19), Rock-1 (H-11), Rock-1 (G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2 (D-11), Rock-2 (N-19), Rock-2 (H-85), Rock-2 (30-J); a commercially available Rock antibody from Santa Cruz Biotechnology 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-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA (R): Rock-2 siRNA (h): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433, Rock-2 siRNA (R): sc-108088, but are not limited to these. In some embodiments, the ROCK inhibitor comprises Y-27632.

[0135] The first culture medium may contain, for example, 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 mM, or a number or range between any two of these values). In some embodiments, the first culture medium comprises a ROCK inhibitor at a concentration of about 7.5 nM. In some embodiments, the first culture medium contains about 1 nM to about 100 nM (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, In some embodiments, the first culture medium comprises about 7.5 nM Y-27632.

[0136] In some embodiments, the second and / or third culture medium each contain one or more hormones or their analogs. The term "analog" is used herein to refer to a biologically active analog of any of the components of the culture medium. Such analogs may be natural or synthetic.

[0137] The specific biologically active ligands and compounds used in the medium defined herein, such as insulin, progesterone, activin, etc., are used for illustrative purposes.However, those skilled in the art will easily recognize that analogs of such ligands and compounds can also be used as substitutes, as long as they retain the relevant biological activity.Those skilled in the art will be able to identify other biologically active compounds suitable for use as substitutes in a routine manner.For example, these may be naturally occurring compounds, or may be compounds that can be produced by synthetic or semi-synthetic methods.

[0138] As used herein, the term "analog" can refer to a compound that may be structurally related to a related molecule. As used herein, the term "agonist" can refer to a compound that may not be structurally related to a related molecule. For example, an agonist can activate a related receptor by changing the conformation of the receptor. In either case, however, these terms are used herein to refer to compounds or molecules that can mimic, reproduce, or otherwise generally replace the specific biological activity of a related molecule.

[0139] The amount of one or more hormones present in the culture medium can vary. For example, in some embodiments, the second and / or third culture medium contains a hormone 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / mL, 1 μg / mL, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL, 1 mg / mL, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / mL or a number or range between any two of these values) of one or more hormones (e.g., progesterone) and / or one or more growth factors (e.g., insulin or insulin-like growth factor).In some embodiments, the second and / or third culture medium contains about 0.5 nM to about 1 mM (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900 nM, 0.5 mM, 1 mM or any two numbers or ranges between these values) of a hormone and / or insulin or insulin-like growth factor.

[0140] The second culture medium may contain DMEM / F12, FBS, GlutaMax™, penicillin-streptomycin, ITS-X, β-estradiol, progesterone, and N-acetyl-L-cysteine. The second culture medium may contain DMEM / F12, 20% FBS, 2 mM GlutaMax™, 1% penicillin-streptomycin, 1× ITS-X, 8 nM β-estradiol, 200 ng / mL progesterone, and 25 mM N-acetyl-L-cysteine.

[0141] The method includes: approximately one day after replacing the first culture medium with the second culture medium, replacing the second culture medium with a third culture medium. The third culture medium may include DMEM / F12, KnockOut™ serum replacement, GlutaMax™, penicillin-streptomycin, ITS-X, β-estradiol, progesterone, and N-acetyl-L-cysteine. The third culture medium may include DMEM / F12, 30% KnockOut™ serum replacement, 2 mM GlutaMax™, 1% penicillin-streptomycin, 1× ITS-X, 8 nM β-estradiol, 200 ng / mL progesterone, and 25 mM N-acetyl-L-cysteine.

[0142] Applications of the synthetic embryos provided herein As will be appreciated by those skilled in the art, the synthetic embryos of the present disclosure can be applied in multiple ways, some exemplary applications of which are further described below.

[0143] Provided herein is a differentiated cell obtained by any of the methods disclosed herein. The method may include: removing one or more cells from a synthetic embryo; and culturing the one or more cells to produce a differentiated cell. In some embodiments, the differentiated cell is selected from the group including exocrine epithelial cells, hormone-secreting cells, cells of the internal organ system, cells of the nervous system, metabolic and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells, and stromal cells.

[0144] Differentiated cells can be produced using the compositions and methods of the present disclosure. Examples of differentiated cells include cells derived primarily from endoderm, cells derived primarily from ectoderm, cells derived primarily from mesoderm, and cells derived primarily from germline. Cells derived primarily from endoderm include exocrine epithelial cells and hormone-secreting cells. Exocrine epithelial cells include salivary gland cells, von Ebner's gland cells of the tongue, mammary gland cells, lacrimal gland cells, ear canal gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, Molar gland cells of the eyelid, sebaceous gland cells, Bowman's gland cells of the nose, Brunner's gland cells of the duodenum, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, Litre's gland cells, endometrial cells, isolated goblet cells of the respiratory and digestive tract, gastric mucosal cells, gastric gland enzyme cells, gastric gland oxygenogen cells, gastric gland acid-secreting cells, pancreatic acinar cells, Paneth cells of the small intestine, type II pneumocytes of the lung, and Clara cells of the lung. Hormone-secreting cells include anterior pituitary cells, pituitary intermediate cells, magnocellular neurosecretory cells, intestinal and respiratory cells, thyroid cells, parathyroid cells, adrenal cells, testicular Leydig cells, theca interna cells of ovarian follicles, luteal cells, juxtaglomerular cells, renal macula densa cells, perirenal cells, and renal mesangial cells.

[0145] Cells derived primarily from the ectoderm include cells of the integumentary and nervous systems. Cells of the integumentary system include keratinizing epithelial cells (e.g., epidermal keratinocytes, epidermal basal cells, fingernail and toenail keratinocytes, nail bed basal cells, medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular root sheath cells, Huxley's layer root sheath cells, Henle's layer root sheath cells, outer root sheath cells, and hair matrix cells), moist stratified barrier epithelial cells (e.g., surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, and urothelial cells). Cells of the nervous system include sensory transduction cells (inner auditory hair cells of the organ of Corti, outer auditory hair cells of the organ of Corti, basal cells of the olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of the epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor cells of the retina of the eye, proprioceptive primary sensory neurons, tactile primary sensory neurons, type I carotid body cells, type II carotid body cells, type I hair cells of the vestibular system of the ear, type II hair cells of the vestibular system of the ear, and type I taste bud cells), autonomic neurons (cholinergic neurons, adrenergic neurons), These include neurons in the olfactory system (such as olfactory vesicles and peptidergic neurons), sensory and peripheral neural support cells (such as inner column cells of Corti, outer column cells of Corti, inner phalangeal cells of Corti, outer phalangeal cells of Corti, border cells of Corti, Hensen's cells of Corti, vestibular organ support cells, taste bud support cells, olfactory epithelium support cells, Schwann cells, satellite glial cells, and enteric glial cells), central nervous system neurons and glial cells (such as astrocytes, neurons, oligodendrocytes, and spindle neurons), and lens cells (such as anterior lens epithelial cells and crystallin-containing lens fiber cells).

[0146] Cells derived from the mesoderm include metabolic and storage cells, barrier function cells, extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells, and interstitial cells. Metabolic and storage cells include hepatocytes, adipocytes, and hepatic adipocytes. Barrier function cells (lung, intestine, exocrine glands, and urogenital tract) include renal cells (renal parietal cells, renal glomerular podocytes, renal proximal tubule brush border cells, Henle's loop thin segment cells, renal distal tubule cells, renal collecting duct cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gallbladder epithelial cells, efferent duct aciliated cells, epididymal principal cells, and epididymal basal cells). Extracellular matrix cells include ameloblasts, epithelial cells of the vestibular system of the ear, epithelial cells of the interdental organ of Corti, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular fibroblasts, other non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts / cementocytes, odontoblasts / odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts / osteocytes, osteoprogenitor cells, hyalocytes of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, hepatic stellate cells, and pancreatic stellate cells. Contractile cells include skeletal muscle cells, including red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nuclear bag cells, and muscle spindle nuclear chain cells, satellite cells, cardiac muscle cells, including conventional cardiomyocytes, nodular cardiomyocytes, and Purkinje fiber cells, smooth muscle cells, myoepithelial cells of the iris, and myoepithelial cells of exocrine glands. Blood and immune system cells include erythrocytes, megakaryocytes, monocytes, connective tissue macrophages, Langerhans cells, osteoclasts, dendritic cells, microglial cells, neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, hybridoma cells, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, and committed progenitor cells of the blood and immune system. Germ cells include oogonia / oocytes, spermatids, spermatocytes, spermatogonia, and sperm. Nurse cells include follicle cells, Sertoli cells, and thymic epithelial cells, and interstitial cells include interstitial kidney cells.

[0147] The disclosed compositions and methods can be used to generate differentiated cells from any suitable mammalian species, such as: primates, including humans, apes (e.g., gorillas, chimpanzees, orangutans), Old World monkeys, and New World monkeys; rodents (e.g., mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (including rabbits); cattle; sheep; goats; horses; pigs; and any other domestic, agricultural, laboratory, or livestock mammals. The presently disclosed compositions and methods can be used to generate differentiated cells from any non-human mammal, including, but not limited to, those described above.

[0148] In some embodiments, provided are differentiated cells obtained by the in vitro methods described herein. Provided are synthetic embryos produced by the in vitro methods disclosed herein. Disclosed herein are compositions for producing synthetic embryos.

[0149] The synthetic embryos disclosed herein may have a variety of uses, including, for example, use in studying the mechanisms of embryonic development and in treating diseases or disorders in subjects.

[0150] Disclosed herein is a method for determining the effect of a test agent on embryonic development. In some embodiments, the method includes: a) providing a synthetic embryo produced by any of the methods provided 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, determining can include 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.

[0151] The method may include producing a synthetic embryo by any method disclosed herein. The method may include contacting the synthetic embryo with a test agent prior to step (d). The method may include determining the subsequent effect on the formation of the synthetic embryo. In some embodiments, the effect on the formation of a multicellular aggregate structure, an egg cylinder structure, and / or a post-implantation structure is determined.

[0152] The method can include recording multiple images of the synthetic embryo. The multiple images can be recorded over a predetermined period of time, thus illustrating, for example, the development of a multicellular aggregate into a synthetic embryo. The imaging device can include a microscope device, a suitable recording device, and optionally an image processing device.

[0153] Typically, fluorescent markers, such as fluorescent dyes or fluorescent marker proteins, are used to image embryonic development. Such markers can be added to the culture system. For example, fluorescent dyes can be added to visualize specific molecules or cellular structures. For example, DAPI can be used to stain DNA, and MitoTracker (Invitrogen) can be used to stain mitochondria. Additionally or alternatively, synthetic embryos can endogenously produce such fluorescent markers, for example, by including one or more cells expressing a fluorescent marker protein. Such cells may be genetically modified to confer the ability to express such marker proteins. Therefore, fluorescent imaging devices are particularly suitable for the described method. Imaging devices can include fluorescent microscopes, such as confocal microscopes, including, but not limited to, wide-field, scanning, and spinning disk confocal microscopes, as well as light-sheet microscopes.

[0154] Confocal microscopy images a single point on a specimen at any given time, but by scanning different points within the specimen in a regular raster, two- or three-dimensional images can be generated, providing image data that can be assembled into two- or three-dimensional images. For example, scanning a specimen in a single plane can generate a two-dimensional image of a slice through the specimen. Multiple, or "stacked," such two-dimensional images can be combined to obtain a three-dimensional image. Spinning disk confocal microscopy offers additional advantages over confocal laser scanning microscopy. Furthermore, light sheet microscopy can also provide better imaging of embryonic development.

[0155] Disclosed herein are methods for identifying compounds useful for treating disease. In some embodiments, the methods include contacting a synthetic embryo or differentiated cell obtained by any of the methods provided herein with a compound.

[0156] Disclosed herein are methods for diagnosing or treating a disease or disorder in a subject. In some embodiments, the methods include the use of synthetic embryos or differentiated cells, or any combination thereof, obtained by the methods provided herein. Also provided are methods for elucidating the role of genes in embryonic development. In some embodiments, the methods include obtaining ESCs, TSCs, and / or XEN cells in which genes have been modified or knocked out, and culturing the cells to obtain a plurality of cells for use in the methods for producing synthetic embryos disclosed herein. [Example]

[0157] 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.

[0158] Example 1 Self-assembly of stem cell-derived synthetic embryos by utilizing cadherin cords and cortical tension Mammalian embryos differentiate sequentially into trophectoderm and inner cell mass, the latter of which differentiates into primitive endoderm and epiblast. Cells derived from three lineages—trophoblast stem cells (TS), extraembryonic endoderm cells (XEN), and embryonic stem cells (ES)—can self-assemble into synthetic embryos. Methods are needed to better understand the mechanisms controlling these events. Disclosed herein is a stem cell-specific cadherin code that drives synthetic embryo development. The XEN cell cadherin code allows XEN cells to sort into a layer below ES cells, recapitulating the preimplantation sorting of epiblast and primitive endoderm. The TS cell cadherin code allows TS cells to sort out of ES cells, resembling the postimplantation accumulation of extraembryonic ectoderm onto the epiblast. While differential cadherin expression drives initial cell sorting, cortical tension solidifies tissue organization. By optimizing the expression of the cadherin code in different stem cell lines, the frequency of correctly formed synthetic embryos was tripled. Thus, by utilizing the cadherin code from different stages of development, lineage-specific stem cells can bypass preimplantation structures and directly construct postimplantation embryos.

[0159] Cadherins and protocadherins regulate cell adhesion in many different systems. Cells expressing different types and levels of cadherins exhibit differences in cell-cell adhesion and sorting. Furthermore, synthetic genetic programs in which distinct cell-cell contacts dictate differential cadherin expression can induce self-organization and sequential assembly into multidomain structures.

[0160] To determine the role of cadherins in the self-assembly of ETX embryos, we analyzed single-cell RNA sequencing (scRNA-seq) data to examine cadherin expression in the components of ETX embryos: embryonic stem (ES) cell lines, trophoblast (TS) cell lines, and extraembryonic endoderm (XEN) cell lines (Figure 1A). We found that E-cadherin (Cdh1) messenger RNA (mRNA) was equally abundant in ES and TS cells, whereas P-cadherin (Cdh3) was expressed only in TS cells and K-cadherin (Cdh6) was expressed primarily in XEN cells (Figure 1B-C). The differential expression of cadherins in ES, TS, and XEN cells suggested a role for cadherins in driving the self-assembly of ETX embryos.

[0161] Next, we examined the expression of these three cadherins in cells dissociated at successive stages from either ETX or natural embryos (Figure 1A). The ES / epiblast, TS / trophectoderm, and XEN / primitive endoderm lineages, defined by the expression of their respective markers, showed similar cadherin expression dynamics in ETX and natural embryos (Figure 1D and Figure 5A). In natural embryos, E-cadherin was expressed in all lineages from E4.5 to E6.5; P-cadherin expression was elevated only in postimplantation trophectoderm (E5.5 and E6.5); and K-cadherin expression was elevated only in preimplantation primitive endoderm (E4.5), which is sorted below the epiblast (Figure 1D and Figure 5B). The corresponding proteins, as well as mRNAs, were confirmed to be differentially expressed in ES or TS colonies (Figure 5C), day 4 ETX embryos, and E5.5 natural embryos (Figure 5D). Thus, XEN cells most closely resemble E4.5 primitive endoderm cells of the preimplantation embryo, whereas TS cells resemble extraembryonic ectoderm cells of the postimplantation embryo.

[0162] ES cells readily form chimeras with 8-cell embryos and are sorted to the epiblast lineage. Because E-cadherin and K-cadherin are differentially expressed in the epiblast and primitive endoderm of native preimplantation embryos, we next investigated whether overexpression of these cadherins in ES cells (OE) affected subsequent sorting of chimeras into blastocysts (Figure 1E). Wild-type ES cells (n = 32 embryos) and ES cells overexpressing E-cadherin (Cdh1 OE) contributed only to the epiblast of chimeras (n = 16 embryos) (Figure 1F-G). In contrast, ES cells overexpressing K-cadherin (Cdh6 OE) frequently contributed to the primitive endoderm (n = 16 embryos) (Figure 1F-G and Figure 5E). These data are consistent with K-cadherin promoting the localization of primitive endoderm and E-cadherin promoting the localization of epiblast.

[0163] P-cadherin-overexpressing ES cells (Cdh3 OE) were excluded from preimplantation embryos (n=13 embryos) and sorted outside the trophectoderm (Figures 1F-G and 5F). This is consistent with low P-cadherin expression across all blastocyst lineages and high P-cadherin expression in the trophectoderm only in spontaneous postimplantation embryos.

[0164] To assess whether differences in adhesive forces are involved in the self-assembly of ETX embryos, we measured the cell-cell adhesion of ES, TS, and XEN cells in vitro using atomic force microscopy (AFM) (Figure 2A-B). The mean adhesive forces between ES-ES cell couples (1.94 ± 0.54 nN) and TS-TS cell couples (2.20 ± 0.85 nN) were significantly higher than those between XEN-XEN cell couples (0.55 ± 0.11 nN) or ES-TS cell couples (0.57 ± 0.36 nN), indicating a tendency for ES and TS cells to form homotypic associations. Furthermore, the adhesion force between XEN-ES cell couples (0.83 ± 0.96 nN) was greater than that between XEN-XEN cell couples (0.55 ± 0.11 nN) or XEN-TS cell couples (0.46 ± 0.24 nN), indicating that XEN cells have the highest affinity for ES cells (Figure 2C). Adhesion forces were calculated from the contact angles between the cells (Figure 2D). The contact angles at ES-ES, TS-TS, and XEN-ES junctions were greater than those between ES-TS, XEN-XEN, and TS-XEN cells (Figure 2E and Figure 6A), consistent with AFM measurements.

[0165] To compare cell-cell contact angles in ETX and native embryos, we employed the Imaging Surface Analysis Environment (ImSAnE) algorithm to extract focal planes from three-dimensional (3D) stacks of E-cadherin-stained day 4 ETX and E5.5 native embryos and unfold these into 2D projections (Figure 6B). Consistent with cell contact angle measurements in double stem cells, homotypic contact angles were larger than heterotypic contact angles in ETX and native embryos (Figure 6C). We also noted that contact angles between XEN-XEN and VE-VE cells on the surfaces of ETX and native embryos were close to 180° (Figure 6C-D), indicating a smooth boundary interface and reflecting high relative tension along the interface after self-organization. Together, these measurements indicate differences in cadherin expression and adhesion between the stem cells that create ETX embryos and the lineages that compose ETX embryos.

[0166] To investigate the potential relationship between differences in cadherin expression and differences in adhesion, we measured the adhesion force between ES cells and TS cells and immobilized E-cadherin (Cdh1) or P-cadherin (Cdh3) substrates (Figure 2F). ES cells exhibited higher adhesion forces to immobilized E-cadherin (2.13 ± 0.83 nN) than to P-cadherin (1.07 ± 0.54 nN), whereas TS cells exhibited comparable adhesion forces to both E-cadherin (2.02 ± 0.89 nN) and P-cadherin (2.41 ± 0.86 nN). We then knocked down cadherins in stem cells using RNA interference. Knockdown of E-cadherin reduced ES-ES cell adhesion by 4-fold. KD of P- or E-cadherin similarly reduced TS-TS adhesion, suggesting that downregulation of a single cadherin is sufficient to reduce TS-TS adhesion below a critical threshold (Figure 2G and Figure 6E). Removal of E- or P-cadherin from XEN cells did not affect homotypic adhesion (Figure 2G). Thus, E-cadherin is required for homotypic adhesion of ES cells, and both E- and P-cadherin are required for homotypic adhesion of TS cells.

[0167] To assess whether the measured adhesion forces were sufficient to create ETX embryos, we resampled the AFM adhesion force measurements to provide parameters to simulate assembly using the Cellular Potts Model (CPM) (Figure 2H). This analysis showed that among the many possible sorting configurations in the three cell types, ETX-like structures were most favored (Figure 2I) (see modeling below).

[0168] Next, we determined how the observed cadherin code affects the efficiency of ETX embryo development. Single-cell suspensions of ES, TS, and XEN cells seeded in microwell plates assembled into numerous structures, of which 15.4% formed ETX structures that recapitulated postimplantation embryo morphogenesis (Figure 3A and Figure S1). In contrast, 38.2% of structures had two or more ES compartments, 30.8% had two or more TS compartments, and 12.8% mislocalized XEN cells or lacked the outer XEN layer (referred to as incorrectly sorted ETX structures) (Figure 3B-Figure 3D). The percentage of correctly sorted ETX embryos stagnated at 15% after the first day of culture (Figure 7A). Thus, the three cell types undergo a sorting phase within the first 24 hours of seeding before integrating into compartments. We hypothesized that due to their low mobility, cells can no longer be sorted during the integration phase. To test this, we performed time-lapse microscopy to track cell mobility during ETX embryogenesis (Fig. 7B), revealing that all cell types are mobile during cell sorting and relatively immobile during tissue integration (Fig. 7C-D).

[0169] XEN cell sorting followed a specific biphasic pattern. XEN cells were efficiently sorted; over 90% of XEN cells formed a monolayer, first enveloping ES cells and then spreading over TS cells. Knockout of E-cadherin (Cdh1) or K-cadherin (Cdh6), which are coexpressed in XEN cells, reduced the frequency of ETX embryos with a continuous XEN layer (Figure 3E-F). However, XEN cells with Cdh1 or Cdh6 OEs were often missorted within these compartments (Figure 3E-F). Thus, an optimal balance of E-cadherin and K-cadherin contributes to the proper sorting of XEN cells in ETX embryos.

[0170] Differences in adhesion cannot fully explain the ability of XEN cells to envelop the TS layer. While not bound by any particular theory, this is because the adhesion force between ES cells and XEN cells is greater than that between ES cells and TS cells, and XEN cells were found between the ES and TS compartments in approximately 10% of the CPM simulations that sampled these data (Figure 2I). The discrepancy between the predicted interface hierarchy for the sorted configuration in ETX embryos and the measured adhesion force difference led to the hypothesis that the number of XEN cells used to create ETX embryos was low and insufficient to envelop all ES cells during the sorting stage. To test this, we seeded 5–10 XEN cells on ES and TS cells and allowed the nascent structures to settle on days 1 and 3. The results showed that initially, a small number of XEN cells enveloped only the ES cells; subsequently, TS cells enveloped the entire structure (Figure 7E–7F). When approximately 10 ES cells were seeded per construct, the XEN cells completely covered the ES cells, thereby excluding the TS cells (FIGS. 7E-7F), consistent with measurements of differential adhesion.

[0171] Previous studies have reported the role of cortical stiffness in cell sorting, particularly cell exteriorization, prompting us to examine whether cortical tension influences the ability of XEN cells to form an outer monolayer. Indeed, AFM measurements showed that XEN cells exhibited lower cortical stiffness than TS or ES cells (Figure 7G). To determine whether the difference in cortical stiffness between ETX embryos, a distinct stem cell type, is due to differences in actomyosin activity, as in other systems, we measured cortical stiffness in the presence of blebbistatin (a myosin inhibitor). Blebbistatin reduced the cortical stiffness of both ES and TS cells to the same level as XEN (Figure 7H). Furthermore, when successfully sorted ETX embryos were treated with either blebbistatin or cytochalasin D (an actin depolymerizing agent) for 24 h on day 3 (once the primary sorting step was completed), they failed to maintain efficient sorting compared to control ETX embryos (Figure 7H). Furthermore, when successfully sorted ETX embryos were treated with either blebbistatin or cytochalasin D for 24 h on day 3, once the primary cell sorting step was complete, over 80% and over 85% of the blebbistatin- and cytochalasin D-treated structures, respectively, failed to remain sorted compared with 18% of control ETX embryos ( Figure 7H ).

[0172] To further investigate the role of cortical stiffness in XEN cell externalization, we used CPM, which allows for independent modulation of cortical stiffness. Lower stiffness increased both cell sorting efficiency and the rate of XEN cell externalization (Figure 7I), indicating that XEN cell stiffness is important for this event. Together, these data indicate that cortical stiffness, in addition to differential expression of different cadherins, plays a role in stem cell self-assembly into ETX embryos.

[0173] Next, we investigated the function of cadherin coding regions in ES and TS cells during ETX embryogenesis. Knockout of P-cadherin (Cdh3) in TS cells, but not in ES cells, resulted in mislocalization of TS and disrupted ETX embryogenesis. Similarly, knockout of E-cadherin (Cdh1) in ES cells, but not in TS cells, inhibited ETX embryogenesis. ETX embryogenesis still occurred after E-cadherin depletion from TS cells (Figure 3G and Figure 8A), indicating that differential P-cadherin expression between ES and TS cells is sufficient to drive their selection. It was noted that E-cadherin and P-cadherin showed different expression levels in individual wild-type ES and TS cells, respectively (Figure 8B-Figure 8D). A subset of wild-type stem cells with low cadherin expression appears to impair ETX embryogenesis. Indeed, when wild-type ES cells and XEN cells were combined with either the P-cadherin OE or P-cadherin KD subsets of TS cells, P-cadherin KD TS cells were mislocalized to the ES compartment. Similarly, when wild-type XEN cells and TS cells were combined with either the E-cadherin OE or E-cadherin KD subsets of ES cells, mislocalization of E-cadherin KD ES cells was observed in the TS compartment (Figure 9A-B). Thus, ES and TS cell populations with low expression of E-cadherin and P-cadherin, respectively, are impaired in sorting in ETX embryos. Strikingly, when E-cadherin OE ES cells and P-cadherin OE TS cells were mixed with wild-type XEN cells, the efficiency of ETX embryo development increased nearly threefold, from approximately 15% with wild-type stem cells to approximately 42% with OE cells (Figure 3H). A time course of sorting of E-cadherin OE ES cells, P-cadherin OE TS cells, and XEN cells revealed that approximately 30% of these structures were successfully sorted 12 hours after cell plating, compared with 6.8% of wild-type structures (Figures 9C-9D).

[0174] Thus, as suggested by our simulations, sorting rates increase after cadherin OE. Taken together, these results indicate that variations in E-cadherin expression in ES cells and P-cadherin expression in TS cells limit the efficiency of ETX embryo development.

[0175] Because both lumen formation and basement membrane formation are required for implantation-stage embryo morphogenesis, we next determined whether the self-organization of cadherin-enhanced ETX embryos also improved these events. By day 2, ETX embryos generated from wild-type stem cells formed a central lumen in the ES compartment, equivalent to the lumen of the epiblast rosette at implantation. By day 3, numerous lumens, equivalent to the multiple lumens of the E5.5 extraembryonic ectoderm, developed in the TS compartment. These lumens then merged into a single cavity between days 4 and 5, similar to spontaneous development by E6.0 (Figure 4A). Such a single cavity formed in over 90% of properly sorted ETX embryos, but in less than 5% of ETX constructs containing incorrectly sorted ES and TS cells (Figure 10A). Furthermore, a cavity was completely absent in ETX constructs containing incorrectly sorted XEN cells (Figure 10B). Importantly, proper sorting and amniotic cavity formation were observed in only 9% of constructs constructed from wild-type cells, but in 40% of constructs constructed from E-cadherin OE ES cells, P-cadherin OE TS cells, and wild-type XEN cells (Figure 4B-4C). Furthermore, the constructs and cavities formed from cadherin OE cells were longer than those in ETX embryos constructed from wild-type cells after 3 days of culture (Figure 10C-10D). Taken together, these results demonstrate that E-cadherin OE and P-cadherin OE in ES and TS cells, respectively, promote cavity formation in ETX embryos.

[0176] Lumen formation requires signaling from the basement membrane produced by the visceral endoderm. Accordingly, we found that ETX constructs with missorted XEN cells lacking a cavity also failed to establish a basement membrane (Figure 10E). A continuous laminin-containing basement membrane was detected in 78% of constructs made from E-cadherin OE ES cells, P-cadherin OE TS cells, and XEN cells (Figure 4D), but only in 45% of constructs made from wild-type ES cells, TS cells, and XEN cells (Figure 4E). Thus, increased expression of E-cadherin and P-cadherin in ES and TS cells increases the successful formation of a basement membrane, lumen, and nucleus in correctly sorted ETX embryos (Figure 4F).

[0177] The methods disclosed herein shed light on the self-assembly of stem cells into synthetic embryos. As described herein, this requires a cadherin code that sorts ES and TS cells into distinct compartments through strong homotypic interactions. In contrast, heterotypic interactions enable XEN cells to first surround ES cells and then TS cells. XEN cells possess cadherin codes resembling those of preimplantation primitive endoderm, yet acquire the ability to support postimplantation synthetic morphogenesis (Figure 10F). While not bound by any particular theory, these differences between natural and ETX embryos highlight the clear shared use of rules between biological development and bioengineering. The construction of synthetic embryos exploits these codes in a distinct way: XEN cells are sorted into a layer below ES cells using the preimplantation code of primitive endoderm, whereas TS cells are sorted as a cluster above ES cells using the postimplantation code of extraembryonic ectoderm.

[0178] The outcome of cell sorting has previously been modeled by considering cell-specific differences in interfacial energy, which maximizes the most energetically favorable cell interface. Differences in interfacial energy are thought to reflect differences in adhesion, with cadherins being the best-characterized effectors, as proposed by the differential adhesion hypothesis (DAH). Accordingly, we demonstrate herein that cell sorting in ETX embryos is driven by an increase in cadherin-mediated homotypic interactions relative to heterotypic interactions. The later development of the differential interfacial tension hypothesis (DITH), which proposes a role for differences in cortical tension in sorting, resonates with our findings regarding the externalization of XEN cells during self-assembly. Together, our findings support a balance between adhesion and tension (DAH vs. DITH) as in biophysical models of cell sorting. However, in some embodiments, we highlight that incomplete ES-TS sorting still results in local order, and that global sorting is necessary to fully recapitulate natural morphogenesis. DAH and DITH only account for the local sorting of ES and TS cells to form homotypic clusters, even in missorted structures. For complete sorting, ETX embryos must escape from locally correct neighborhoods within globally incorrect patterns and explore alternative conformations. If cells remain at a minimum before sorting is complete, the structure remains missorted.

[0179] The importance of cell-type-specific cadherin codes is demonstrated by the finding that established wild-type stem cell lines exhibit heterogeneous cadherin expression, with some subsets below the threshold required to support proper sorting. Increasing E-cadherin expression in ES cells and P-cadherin expression in TS cells significantly improves the efficiency of ETX embryonic development (Figure 4F). This highlights a broader challenge in synthetic biology: characterizing and ameliorating heterogeneity in stem cell lines. While this factor remains largely undefined, it has been reported. In some embodiments, such heterogeneity can affect the distribution of cell-cell adhesions within the same cell population, potentially disrupting the hierarchy of interactions necessary to drive the self-organization of other organoid structures. Therefore, the self-organization principles described herein may overcome these challenges and provide a means to enhance the efficiency of organoid formation of various types by modulating interactions and the physical properties of cells.

[0180] method cell culture All cells were cultured at 37°C in 20% O2 and 5% CO2 and passaged when they reached 70% confluency. Cells were tested weekly for mycoplasma contamination by PCR.

[0181] ES cells were cultured on 0.1% gelatin-coated plates in N2B27 medium with 1 μM MEK inhibitor PD0325901, 3 μM GSK3 inhibitor CHIR99021, and 10 ng / mL leukemia inhibitory factor. N2B27 medium contained a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) / F12 (21331-020; Thermo Fisher Scientific) and Neurobasal-A (10888-022; Thermo Fisher Scientific) medium supplemented with 0.5% vol / vol N2 (17502048; Thermo Fisher Scientific), 1% vol / vol B27 (10889-038; Thermo Fisher Scientific), 100 μM β-mercaptoethanol (31350-010; Thermo Fisher Scientific), 1% vol / vol penicillin-streptomycin mixture (15140122; Thermo Fisher Scientific), and 1% vol / vol GlutaMAX (35050-061; Thermo Fisher Scientific).

[0182] TS cells (wild-type) were cultured on CF1 mouse embryonic fibroblasts (MEFs) treated with mitomycin C (M4287; Sigma-Aldrich) in TSF4H medium containing RPMI 1640 (M3817; Sigma-Aldrich) supplemented with 20% fetal bovine serum (FBS; 35-010-CV; Thermo Fisher Scientific), 2 mM l-glutamine, 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate, 1% penicillin-streptomycin (M7167; Sigma-Aldrich), 25 ng ml-1 FGF4 (5846-F4; R&D Systems), and 1 μg / ml heparin (H3149; Sigma-Aldrich).

[0183] TS cells (Cdh3 OE) were cultured on laminin-coated plates in TX medium with 50 ng / mL IL11 (50117-MNCE; Sino Biological), 50 ng / mL activin (Qk001-ActA-100; Qkine), 25 ng / mL Bmp7 (PeproTech; 120-03P-10 μg), 5 nM lysophosphatidic acid (Santa Cruz Biotechnology; sc-201053), and 200 nM 8Br-cAMP (B 007-500; BIOLOG Life Science Institute). TX medium was prepared by mixing DMEM and F12 medium (21331-020; Thermo Fisher Scientific) in a 1:1 ratio and supplemented with 19.4 μg / mL insulin (342106; Sigma-Aldrich), 64 μg / mL l-ascorbic acid (A4403; Sigma-Aldrich), 14 ng / mL sodium selenite (S5261; Sigma-Aldrich), 543 μg / mL sodium bicarbonate (S5761; Sigma-Aldrich), 10.7 μg / mL holo-transferrin (T4132; Sigma-Aldrich), 1% penicillin-streptomycin (M7167; Sigma-Aldrich), 25 ng / mL FGF4 (5846-F4; R&D Systems), and 2 ng / mL TGF-β1 (100-21 Cdh3 OE TS cells were cultured on MEFs in TSF4H medium after selection with 1 μg / mL heparin (H3149; Sigma-Aldrich) and 1 μg / mL heparin (C; PeproTech).

[0184] XEN cells were cultured on gelatin-coated plates in 70% MEF-conditioned Integrative Growth Medium (IDG) (c-IDG) medium, which contained DMEM (21969; Gibco) supplemented with 12.5% ​​FBS (35-010-CV; Thermo Fisher Scientific), 2 mM GlutaMax (35050-038; Gibco), 0.1 mM 2-mercaptoethanol (31350-010; Gibco), 0.1 mM non-essential amino acids (11140-035; Gibco), 1 mM sodium pyruvate (11360-039; Gibco), 0.02 M HEPES (15630080; Gibco), and 1% penicillin-streptomycin (15140122; Gibco).

[0185] MEF cells were cultured on gelatin-coated plates in DMEM medium (41966; Thermo Fisher Scientific) supplemented with 15% FBS (35-010-CV; Thermo Fisher Scientific), penicillin-streptomycin (15140122; Thermo Fisher Scientific), GlutaMAX (35050061; Thermo Fisher Scientific), MEM non-essential amino acids (11140035; Thermo Fisher Scientific), sodium pyruvate (11360070; Thermo Fisher Scientific), and 100 μM β-mercaptoethanol (31350-010; Thermo Fisher Scientific). mouse embryo

[0186] mouse embryo Mice were housed in accordance with national and international guidelines. All experiments were regulated by the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 after ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. Experiments were approved by the Home Office. Animals were inspected daily, and those showing health concerns were culled by cervical dislocation. Six-week-old female CD-1 mice were used for all animal experiments. All experimental mice were pathogen-free, housed under a 12-hour light / 12-hour dark cycle, and had unlimited access to water and food. The temperature in the facility was controlled and maintained at 21°C.

[0187] Cell line generation Experiments were performed using mouse E14 wild-type ES cells, Cdh1, Cdh6 OE ES cells were generated from E14 wild-type ES cells, Cdh3 OE TS cells from wild-type TS cells, and Cdh1 and Cdh6 OE XEN cells from wild-type XEN cells (see below).

[0188] To generate cadherin OE ES, TS, or XEN cell lines, 0.5 μg of the super piggyBac transposase expression vector (PB210PA-1; System Biosciences) and 2 μg of Cdh1-pHygro, Cdh3-pHygro, or Cdh6-pHygro plasmid were cotransfected into cells using Lipofectamine. Cells were passaged 24 hours after transfection and then subjected to selection in medium containing 50 μg ml-1 hygromycin (10687010; Thermo Fisher Scientific) for 1 week. A similar method was used to generate stable nuclear reporter H2B-RFP XEN cell lines.

[0189] Cloning Cloning was performed using Gateway technology (Thermo Fisher Scientific). The desired fragments (Cdh1, Cdh3, or Cdh6) were amplified by PCR to introduce an attB site. These fragments were cloned into the pDONR221 vector (a gift from J. Silva) using BP clonase II (11789020; Thermo Fisher Scientific). The desired fragments (Cdh1, Cdh3, or Cdh6) were subcloned into the pHygro vector, which contains a hygromycin resistance cassette, for expression in stem cells. Recombination reactions were performed using LR Clonase II (11791100; Thermo Fisher Scientific).

[0190] PiggyBac-based expression plasmids for Cdh1, Cdh3, or Cdh6 were generated by PCR amplification of the respective genes in pENTR-Cdh1 (49776; Addgene), Cdh3 (Myc-DDK tagged) (MR227345; OriGene Technologies), or Cdh6 (mouse tagged ORF clone) (MG222740; OriGene Technologies) using the oligos shown in Table 5.

[0191] Small interfering RNA Cells were transfected with 25 nM small interfering RNA (siRNA) directed against Cdh1 (1027418-SI00946631), Cdh3 (1027418-SI02666440), or Cdh6 (1027418-SI00946967) (Qiagen) siRNA, or control scrambled siRNA (Qiagen), using Lipofectamine 3000 transfection reagent according to the manufacturer's instructions. Cells were harvested 72 hours post-transfection and assayed by quantitative PCR (qPCR).

[0192] Flow cytometry analysis Single ES and TS cell suspensions were harvested, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100 and 0.1% glycine for 30 minutes at room temperature. The cells were then incubated with anti-E-cadherin antibody (1:200; 13-1900; Thermo Fisher Scientific) or anti-P-cadherin antibody (1:100; sc-1501; Santa Cruz Biotechnology) in blocking buffer (phosphate-buffered saline (PBS) containing 10% FBS + Tween 20) (PBST) overnight at 4°C. After washing twice with PBST, the cells were incubated with secondary antibodies (1:500 dilution) in blocking buffer for 1-2 hours at room temperature. The cells were then analyzed by quantitative flow cytometry (BD Biosciences), and E- and P-cadherin intensity profiles were plotted using FlowJo software (version 10.7.1) (https: / / www.flowjo.com).

[0193] Double cell experiments To measure cell-cell contact angles, 1200 dissociated ES, TS, or XEN cells were mixed in pairs and seeded onto AggreWell plates (34411; STEMCELL Technologies) pretreated with washing solution (07010; STEMCELL Technologies). The cells were centrifuged at 100 g for 3 minutes. After incubation at 37°C for 1 hour, the cells were harvested and fixed for immunostaining.

[0194] Preparation of cadherin-coated surfaces To measure adhesion forces between cells and cadherin-coated surfaces, gold-coated glass coverslips (AU.0100; Platypus) were briefly cleaned in argon plasma for 30 seconds and then functionalized by immersion in a thiol solution (P50757; Sigma-Aldrich) for 16 hours. Excess thiol was then removed by rinsing with EDTA buffer (15575-020; Thermo Fisher Scientific). The coverslips were then incubated with 10 μg / mL recombinant E-cadherin (8875-EC-050; R&D Systems) or P-cadherin (761-MP-050; R&D Systems) for 12 hours at 4°C. Before force measurements, the cadherin-coated surfaces were washed with HEPES (15630106; Thermo Fisher Scientific) and activated by incubation in the same buffer for 30 minutes.

[0195] Adhesion force measurement Cell-cell or cell-cadherin adhesion forces were measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica). Tipless silicon nitride cantilevers were V-shaped and had a nominal spring constant (60 pN nm-1; NP-0; Veeco Instruments). The atomic force microscope cantilevers were plasma-cleaned before functionalization with concanavalin A. The system was calibrated before each experiment by measuring the deflection sensitivity of the glass surface in cell-free medium at 37 °C, allowing the cantilever spring constant to be determined in situ. Before loading the sample, the sample stage movement was calibrated using NanoScope software (version 6.13). Before measurements, cells were dissociated with TrypLE (12604013; Thermo Fisher Scientific) and resuspended in HEPES-buffered cell culture medium (15630056; Thermo Fisher Scientific). A cell suspension was loaded into the sample chamber of an atomic force microscope (AFM), and a single cell was captured by pressing the cantilever against the cell with a contact force of 500 pN for 1 minute. The cell was then lifted from the surface and allowed to firmly adhere to the cantilever for 5 minutes. To measure cell-cell adhesion, the captured cell was lowered into contact with another single cell cultured on a gelatin-coated glass-bottom Petri dish (FD35; WPI).

[0196] To measure cell-cadherin adhesion force, a captured cell was lowered and brought into contact with a cadherin-coated coverslip. The approach and retraction speeds were kept constant at 10 μm / s, and the contact force was 2 nN. Three force curves were acquired for each cell. The captured cell was allowed to recover for 3 min between different adhesion force measurement cycles and then adhered to the surface at different positions. Direct observation confirmed that the probing cell remained on the cantilever before and after measurements. The maximum cell adhesion force and the step height of a single burst force were extracted from the retrace curves using JPK IP software.

[0197] Cell cortex stiffness measurement Cell stiffness was measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica) as previously described. Cell stiffness was measured using the point-and-shoot method (NanoScope software; Bruker). All cells were maintained in CO2-independent cell culture medium during the measurements.

[0198] Fluorescent 10 μm polystyrene beads (Invitrogen) were attached to silicon nitride cantilevers with a nominal spring constant of 0.06 N m (NP-S type D; Bruker). Indentations were performed using the single-force option with a total indentation depth of 50–100 nm. PUNIAS software was used to obtain cell stiffness values ​​from the force curves. Multiple force-displacement curves (five separate locations) were fitted to the Hertzian model to calculate the stiffness (Young's modulus) of the cell cortex.

[0199] Generation of stem cell-derived ETX embryos Approximately 6,000–7,000 ES cells, 15,000–19,000 TS cells, and 5,000–6,000 XEN cells were added dropwise to an AggreWell plate (34411; STEMCELL Technologies) with 1,200 microwells per well. The microwells were treated with washing solution (07010; STEMCELL Technologies). The cells were centrifuged at 100 g for 3 minutes. 1.5 mL of c-IDG medium containing 7.5 nM ROCK inhibitor (72304; STEMCELL Technologies) was added dropwise to each well. The next day (Day 1), 1 mL of medium was gently removed from each well and replaced with 1 mL of fresh c-IDG medium without ROCK inhibitor. This step was repeated once to completely remove the ROCK inhibitor. On Day 2, 1 mL of c-IDG medium was replaced with 1 mL of fresh medium. On Day 3, the medium was replaced with IVC1 medium. IVC1 culture medium contained advanced DMEM / F12 (21331-020; Gibco) supplemented with 20% (vol / vol) FBS, 2 mM GlutaMax, 1% vol / vol penicillin-streptomycin, 1× ITS-X (51500-056; Thermo Fisher Scientific), 8 nM β-estradiol, 200 ng ml−1 progesterone, and 25 mM N-acetyl-l-cysteine.

[0200] Immunofluorescence Native embryos, stem cell-derived constructs, or stem cells were fixed in 4% paraformaldehyde (15710; Electron Microscopy Sciences) for 20–30 min at room temperature, washed twice with PBST (containing 0.05% Tween 20), and permeabilized in 0.3% Triton X-100 and 0.1% glycine for 30 min at room temperature. Primary antibody incubation was performed overnight at 4°C in blocking buffer (PBST containing 10% FBS). The following day, samples were washed twice with PBST and then incubated with secondary antibody (1:500) in blocking buffer for 1–2 h at room temperature. Embryos were transferred to a drop of PBST in an oil-filled optical plate prior to confocal imaging.

[0201] The following primary antibodies were used: Tfap2c (1:200; AF5059; R&D Systems), Brachyury (1:200; AF2085; R&D Systems), Gata4 (1:500; 36966; Cell Signaling Technology), laminin (1:500; L9393; Sigma-Aldrich), Oct4 (1:500; sc-5279; Santa Cruz Biotechnology), E-cadherin (1:200; 13-1900; Thermo Fisher Scientific), and P-cadherin (1:100; sc-1501 (Santa Cruz Biotechnology) or MS-1741 (Fisher Scientific)). The following secondary antibodies from Thermo Fisher Scientific were used: Alexa Fluor 488 donkey anti-mouse (1:500; A-21202), Alexa Fluor 488 donkey anti-goat (1:500; A-11055), Alexa Fluor 488 donkey anti-rat (1:500; A-21208), Alexa Fluor 568 donkey anti-rabbit (1:500; A-10042), Alexa Fluor 568 donkey anti-mouse (1:500; A-10037), Alexa Fluor 647 donkey anti-goat (1:500; A-21447), and phalloidin (1:200; A30104). Detailed information on the antibodies used is shown in Table 3.

[0202] RNA extraction and real-time qPCR Total RNA was extracted from cells using TRIzol Reagent (15596-026; Invitrogen). Real-time qPCR was performed using SYBR Green PCR Master Mix (4368708; Applied Biosystems) and a StepOnePlus Real-Time PCR System (Applied Biosystems). Fold changes in mRNA expression were determined using the ΔΔCt method with Gapdh as the endogenous control. See Table 4 for the qPCR primers used.

[0203] scRNA-seq sample preparation and dissociation Natural and ETX embryos were transferred to a Falcon tube, washed with PBS, and incubated in TrypLE Express (12604013; Gibco) at 37°C for 15 minutes to dissociate into single cells. If clumps remained, the incubation was extended for an additional 5 minutes at 37°C, and the sample was further pipetted. The sample was filtered to remove large clumps, centrifuged at 200g for 5 minutes, resuspended in PBST (containing 0.02% Tween 20), and processed for encapsulation as previously described. E5.5 embryos were dissociated together in groups of 12 embryos. A total of 15 ETX embryos were dissociated for sequencing. Cultured cells were dissociated into single-cell suspensions using TrypLE Express (12604013; Gibco), multiplexed with MULTI-seq lipid-modified oligos, and then run in two 10X Genomics lanes using the Single Cell 3′ Version 3 reagent as previously described.

[0204] scRNA-seq analysis A previously submitted and filtered scRNA-seq dataset, including ETX and natural embryos, was downloaded from the Gene Expression Omnibus repository (GSE161947). The count matrix was loaded into Seurat version 3, and the percentage of counts mapped to mitochondrial genes was calculated. Objects were then log-normalized to a scale factor of 10,000. The 2,000 most variable genes were calculated, aligned, and regressed on the percentage of mitochondrial counts. Dimensionality reduction was performed using principal component analysis, and the data were projected into a uniform manifold approximation and projection (UMAP) low-dimensional space using 20 principal components. The lineage identities of embryonic endoderm and trophectoderm were pooled from previous annotations and corresponded to clusters with high expression levels of Dnmt3b, Gata4, and Lamb1, and Cdx2 and Gata2, respectively. Mean expression was calculated using the mean expression function, the latter log2-normalized. Cadherin and protocadherin expression levels were then plotted on a heatmap. UMAP plots were plotted directly using a recently described methodology after retaining only ETX and natural embryo samples and recalculating the neighborhood graph (5 neighbors and 30 principal components; code at https: / / github.com / fhlab / scRNAseq_inducedETX).

[0205] Time-lapse imaging For time-lapse imaging, cells were seeded on Gri3D PEG-hydrogel dishes (SUN Bioscience) with glass bottoms and imaged using a spinning disk microscope (3i) equipped with a Zeiss EC Plan-NEOFLUAR 20x / 0.5 objective in a humidified chamber at 37°C and 5% CO2. Constructs were imaged every 5–10 min by collecting image stacks of 10 μm z-planes. Images were processed using SlideBook 5.0 (3i). Raw data were processed using the open-source image analysis software Fiji. Imaris image analysis software (Bitplane) was used for single-cell tracking.

[0206] Quantification and statistical analysis Selection criteria for ETX embryos Egg cylinder structures containing one TS-derived compartment and one ES-derived compartment, covered by an outer XEN-derived visceral endoderm-like monolayer, were considered to be well-sorted ETX embryos for analysis. Structures not meeting these criteria were considered missorted ETX structures. Structures containing all three cell types were collected and counted for quantification.

[0207] Image data acquisition, processing, and quantification Fluorescence images were acquired using a Leica SP8 confocal inverted microscope (LEICA software LAS X; Leica Microsystems) equipped with a Leica FLUOTAR VISIR 25× or 40× objective.

[0208] Images were acquired with a z-separation of 0.5–3.0 μm. To screen the entire structure, we used the tile-scan imaging mode with automated image stitching on an SP8 confocal microscope. All images were analyzed and processed using Fiji software (http: / / fiji.sc). For digital quantification and immunofluorescence signal intensity graphs, the laser power and detector gain were kept constant, allowing for quantitative comparison of different experimental conditions within a single experiment.

[0209] To evaluate cell-cell contact angles in 3D ETX and native embryos, we employed ImSAnE to extract embryo planes from 3D stacks of E-cadherin and unfold them into 2D projections. Different lineages were indicated by different nuclear markers during analysis. Geometric observations and common distortions in projections can also be accurately quantified using correction methods built into MATLAB.

[0210] Numerical simulation CPM Similar to determining the role of cortical stiffness in ETX embryo self-organization, we used CPM to infer predicted distributions of conformations from measurements of cell adhesion from AFM. Adhesion strength was parameterized using the cohesion forces between pairs of cell types measured directly by AFM (Table 6). For each simulation, this distribution was sampled to construct an adhesion (J) matrix. Specifically, for a given element in this matrix, the set of AFM cohesion forces measured between cell type pairs (e.g., ES-ES, ES-TS, etc.) was sampled (with replacement) and run approximately 500 times to establish an ensemble of J-matrix samples. CPM simulations were performed with each J-matrix sample to generate an ensemble distribution of conformations over time. Simulations are developed by stochastic minimization of an energy function (see Equation (1) below) that accounts for both affinity differences and other physical properties of the cells. At each time point in the simulation, each cell type was scored for one of 16 possible sorting configurations (Figure 2I) by determining whether it was surrounding and / or contiguous (see "Modeling" below for details). To investigate the importance of softness in XEN cell externalization, we measured the stiffness parameter (λ) of the XEN cell cortex. XEN ) was varied and the above simulation procedure was repeated.

[0211] Statistics and reproducibility Statistical tests were performed using GraphPad Prism (versions 8.0 and 7.0a) software (except for the analysis of sequencing data). Gaussian-distributed data were analyzed using an unpaired, two-tailed Student's t-test (two groups) or a one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (multiple groups). Significant differences in variance were considered using Welch's correction. Non-Gaussian-distributed data were analyzed using the Mann-Whitney U test (two groups) or the Kruskal-Wallis test with Dunn's multiple comparison test (multiple groups). A minimum of three independent experiments were performed for all quantifications. In vitro cell experiments were not randomized because this was not necessary. In experiments using chemical inhibitors, samples were randomly assigned to control and experimental groups. In in vivo experiments, embryos were randomly assigned to control and experimental groups.

[0212] Data collection and analysis were not blinded to experimental conditions, and no statistical methods were used to predetermine sample size.

[0213] The sample size was determined based on previous experimental experience. The sample sizes used to derive statistics are described herein. No data were excluded from the analysis. Sequencing data were analyzed using standard programs and packages. The significance level is indicated on each graph.

[0214] [Table 3]

[0215] [Table 4]

[0216] [Table 5-1]

[0217] [Table 5-2]

[0218] modeling We used the Cellular Potts Model (CPM) to infer the conformational distribution predicted from AFM cell adhesion measurements and to determine the role of cortical stiffness in the self-organization of ETX embryos. The modeling embodiment used here is described below.

[0219] Model Target The cells were divided into groups according to size (N x ×N y ) occupies a set of contiguous points in a square lattice. Each cell contains the matrix I (N x ×N y ) and is assigned a unique ID recorded in the cell type table. Furthermore, each cell is assigned a cell type (e.g., ES, TS, XEN) and is associated with unique, predefined cell characteristics. The cell type is immutable and is represented by a cell index i and a cell type c. i = 2. Grid points not occupied by cells are given the IDs i = 0 and c0 = 0 to define the medium.

[0220] Energy Function The simulation is developed by stochastic minimization of an energy function that takes into account both the affinity differences and other physical properties of the cells. The energy function is defined as:

number

number

number

[0221] Similarly, the perimeter P of cell i i is the number of lattice points such that: (i) cell i (i.e., ω i ) but (ii) is not itself a member of cell i.

[0222] Bootstrapping Procedure Adhesion forces were parameterized using the cohesive forces between pairs of cell types measured directly by AFM. For each simulation, this distribution was sampled to construct a J matrix. Specifically, for a given element J ij For , the user can enforce symmetry on the J matrix while choosing the cell type c i and c j A set of AFM cohesion forces measured between cells (e.g., ES-ES, ES-TS, ...) can be sampled (with replacement). 0j , J i0) are set to 0. Bootstrap sampling is performed approximately 500 times to establish an ensemble of J-matrix samples. Each J-matrix sample is used to perform a CPM simulation to generate an ensemble distribution of conformations over time.

[0223] Simulation Algorithm The CPM evolves via stochastic minimization. At each Markov chain step (MCS), a random lattice site is selected. Then, one of the four sites in the von Neumann neighborhood is chosen, and the state of the selected site is tentatively reassigned to the states of its neighboring sites. An energy function is then evaluated before and after the swap, defining ΔE. A swap is accepted only if:

number

[0224] As in lattice models, T defines the effective temperature of the system, regulating its tendency to undergo energetically unfavorable swaps. In traditional CPM simulations, the cellular Moore continuity breaks down at high T because the swapping rules are local. As a result, potential state changes that break continuity are universally rejected.

[0225] Automatic conformation scoring To determine the conformation of the simulated structure at a given time point, we established an automated scoring procedure. First, we calculated the cell-cell adjacency matrix (Moore neighborhood) and removed all clusters except the one with the most connected components, thereby eliminating cells that have detached from the main aggregate. Second, we scored each cell type for encirclement. A cell type is defined as encircling if its centroid is not within its own cell type but within a different cell type. Third, we scored the contiguity of cell types by calculating a subgraph of the connectivity matrix that includes only cells of a given type and then determining whether the number of connected components is 1 (i.e., whether they are contiguous). For the three cell types, there were 16 fully screened candidate conformations. These conformations were divided into four categories:

[0226] In category (1) conformations, two cell types sequentially envelop a third. The order of envelopment is determined by the neighboring relationships between cell types. For example, if E envelops X, which envelops T: at least one X must be in contact with T; at least one E must be in contact with T; at least one E must be in contact with the medium; at least one E must be in contact with X; and no E must be in contact with T. Furthermore, the innermost cell types must be contiguous.

[0227] In category (2), one cell type envelops another cell type with a third cell type attached to the periphery; whereas, in category (3), one cell type envelops two other cell types (as in ETX embryos). Both categories must contain two contiguous cell types and a third surrounding cell type. If all cells of the surrounding cell type are in contact with the medium, the conformation is scored as category (3). If any of the cells not in contact with the medium are instead surrounded by a single cell type, the conformation is scored as "unsorted." Alternatively, if any of these cells make precise contact with the other two cell types, the conformation falls into category (2). Variants within category (2) are determined by counting the number of contacts (e.g., if X and E share more contacts than X and T, then X envelops E more than T). Otherwise, the conformation is assigned category (3). Category (4) is assigned when all three cell types are unenclosed and contiguous. If a given structure does not fit into any of these categories, it is classified as "unsorted."

[0228] Furthermore, we define cell externalization as: all cells of that type are either in direct contact with the medium or in contact with cells that are in contact with the medium. More precisely, we define the subgraph of the adjacency matrix containing the rows and columns of a given cell type and medium: if this subgraph has one connected component, then the cell type is externalized.

[0229] JPEG2025532481000013.jpg42166

[0230] [Table 6]

[0231] In at least some of the embodiments described above, one or more elements used in one embodiment may be used interchangeably in another embodiment (unless such substitution is technically feasible). Those skilled in the art will appreciate that various other omissions, additions, and modifications may 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 fall within the scope of the subject matter defined by the appended claims.

[0232] With respect to the use of virtually any plural and / or singular term herein, those of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly provided herein for clarity. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As used herein, references to "or" are intended to encompass "and / or" unless specifically stated otherwise.

[0233] In general, those skilled in the art will understand that the terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that if a specific numerical recitation is intended in the appended claims, such intention will be explicitly set forth in the claims; otherwise, no such intention exists. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitation. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits a particular claim containing such an introduced claim recitation to embodiments containing only one such recitation, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. Moreover, those skilled in the art will recognize that even when a specific number is explicitly stated in an introduced claim recitation, such a recitation should be construed to mean at least the recited number (e.g., the mere recitation "two recitations," without any other modifier, means at least two recitations or more than two recitations).Furthermore, when phrases similar to "at least one of A, B, and C, etc." are used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When phrases similar to "at least one of A, B, or C, etc." are used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms.

[0234] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group.

[0235] As will be understood by those skilled in the art, for all purposes, including in terms of providing written documentation, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully describing and allowing the same range to be divided into at least 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," and "less than" refers to a range that is inclusive of the recited number and can be subsequently divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4 or 5 items, and so on.

[0236] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope and spirit being indicated by the following claims.

Claims

[Claim 1] A method for producing synthetic embryos in vitro: (a) To provide a plurality of manipulated embryonic stem cells (ESCs), wherein the plurality of manipulations At least some of the ESCs overexpress E-cadherin (Cdh1); (b) To provide a plurality of manipulated trophoblast stem cells (TSCs), the plurality of manipulated At least some of the TSCs produced overexpress P-cadherin (Cdh3); (c) To provide multiple extraembryonic (XEN) cells; (d) The plurality of manipulated ESCs, the plurality of manipulated TSCs and the plurality of extraembryonic cells (XEN) cells are brought into contact with a first culture medium to form a co-culture; The plurality of manipulated ESCs and their derivatives, the plurality of manipulated TSCs and their derivatives, and The aforementioned XEN cells and their derivatives organize to form a synthetic embryo. The synthetic embryo contains one compartment derived from the transconjunctival stem cell (TS) and one compartment derived from the transconjunctival stem cell (ES). Furthermore, it is covered with a single layer derived from XEN on the outside.

Images